AJMR - 20 September, 2012 Issue

African Journal of
Microbiology Research
Volume 6 Number 36
ISSN 1996-0808
20 September, 2012
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African
Journal
of Microbiology
Research
International
Journal
of Medicine
and Medical
Sciences
Table of Contents:
Volume 6
Number 36 20 September, 2012
nces
ARTICLES
Research Articles
Conventional and molecular characterization of Trichophyton rubrum
Farzad Aala, Rosimah Nulit, Umi Kalsom Yusuf and Sassan Rezaie
6502
Inhibitory effect of some plant extracts on clinical isolates of Staphylococcus

aureus
Rajaa Milyani and Nahed Ashy
6517
Role of CSE1034 in bacterial lipids and polysaccharides involved in biofilm

formation: a comparison with other drugs
Chaudhary Manu and Anurag Payasi
6525
Analytical specificity and sensitivity determination of 16SrRNA gene based

diagnostic polymerase chain reaction (PCR) for molecular detection of
Coxiella burnetii
Mohammad Soleimani, Keivan Majidzadeh A., Amirhossein Mohseni and
Mohammad Khalili
6532
Effect of Bacillus cereus Br on bacterial community and gossypol content

during fermentation in cottonseed meal
Xin Wang, Jiang-wu Tang, Xiao-hong Yao, Yi-fei Wu, Hong Sun and Yao-xing Xu
6537
Identification and characterization of a fungal strain with lignin and cellulose

hydrolysis activities
Ran Jin, Hongdong Liao, Xuanming Liu, Mang Zheng, Xianqiu Xiong, Xinwu Liu,
Liyong Zhang and Yonghua Zhu
6545
Table of Content:
Volume 6
Number 23 21 June, 2012
Table of Contents:
Volume 6
Number 36 20 September, 2012
nces
ARTICLES
ARTICLES
DNA
viral infections
and transient
bone
marrow
failure
in southern
Iranfor
Influence
of ciprofloxacin
on glioma
cell
line GL26:
A new
application
Kambiz
Mohammad Hossein Karimi, Ramin Yaghobi,
an oldBagheri,
antibiotic
Behnam
Mohammadi,
Mehdi
Dehghani and
Padideh
Ebadi
Abdolreza
Esmaeilzadeh,
Massoumeh
Ebtekar,
Alireza
Biglari and
Zuhair Mohammad Hassan
6551
4891
Survival of microorganisms in high pressure treated minced meat during

chilled
storage and
at pH anddiversity
temperature
mimicking
tract
Identification
of microbial
in caecal
contentgastrointestinal
of broiler chicken
Sami
Bulut G. Dhinakar Raj, A. Rajasekar, D. Vijayalakshmi and T. Devasena
S. Nathiya,
6558
4897
Efficacy
andquality
toxicityofofsome
neutralizers
against
disinfectantsproducts
and antiseptics
Microbial
non-sterile
pharmaceutical
sourced
used
insome
vaccine
production
facility
from
retail
pharmacies
in Lagos, Nigeria
Norhan
S.
Sheraba,
Aymen
S.
Yassin,
Aly Fahmy
and Magdy
A.A.
Amin
Adeola Anifowoshe R., Opara Morrison
I. and Adeleye
Isaac
6565
4903
Effects
of essential
oil extracted
from
Citrullus
colocynthis
(CCT)in
seeds
on
Molecular
detection
of adhesins
genes
and biofilm
formation
methicillin
growth
of phytopathogenic
bacteria
6572
resistant
Staphylococcus aureus
Zahra
Setayesh
Nima
Sanadgol
and LeylaVafadar
Karima
BEKIR,Mehr,
Omayma
HADDAD,
Mohammed
GRISSA,Ghasemi
Kamel CHAIEB,
Amina BAKHROUF and Salem IBRAHIM ELGARSSDI
4908
Development and evaluation of a novel TaqMan fluorescence probe-based
real-time
transcriptase
polymerase
chainBacillus
reaction
assayinfor
Amylasereverse
production
by moderately
halophilic
cereus
solid
detection
and quantification of West Nile virus
state fermentation
Lijun
Shi, HuiqiongD.Yin,
Jingang Zhang,
P. Vijayabaskar,
Jayalakshmi
and T.Zhan-zhong
Shankar Zhao and Gang Li
Microbial
water
qualityand
in the
upper characteristics
Olifants River catchment:
Networking
clusters
sequence
of clusteredImplications
regularly
for
health
interspaced
short palindromic repeats (CRISPR) direct repeats and their
W.evolutionary
J. le Roux, L.comparison
M. Schaeferwith
and B.
Genthe
cas1
genes in lactic acid bacteria
Kaibo Deng, Fei Liu, Chuntao Gu and Guicheng Huo
6576
4918
6580
4927
Partial characterization of a bacteriocin produced by Lactobacillus alivarius

isolated from oral cavity of desert foxes
6589
Aly
E. Abo-Amerscreening
and Mohammed
Y. Shobrak
Antibacterial
of the root,
stem and leaf extracts of Terminalia albida sc.
elliot on selected pathogenic bacteria
S. M. Ayodele, G. Alpheus and O. M. Iruaga
1457
African Journal of Microbiology Research Vol. 6(36), pp. 6502-6516, 20 September, 2012
Available online at http://www.academicjournals.org/AJMR
DOI: 10.5897/AJMR10.736
ISSN 1996-0808 2012 Academic Journals
Full Length Research Paper
Conventional and molecular characterization of

Trichophyton rubrum
Farzad Aala1*, Rosimah Nulit2, Umi Kalsom Yusuf2 and Sassan Rezaie3
1
Department of Medical Mycology and Parasitology, School of Medicine, Kurdistan University of Medical Sciences,
Sanandaj, Iran.
2
Department of Biology, Faculty of Science; Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia.
3
Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical
Sciences, Tehran, Iran.
Accepted 10 September, 2012
Different studies illustrated that Trichophyton rubrum, among all species of Trichophyton, is the most
prevalent and consequently the most important genus. T. rubrum as a worldwide filamentous pathogen
fungus can infect human keratinized tissue (skin, nails and rarely hair), and causes dermatophytosis.
Researchers use two general methods for the identification of dermatophytes namely, conventional
methods on the basis of phenotype variations and molecular methods on the basis of molecular
differences. Due to some limitations in traditional methods, in the recent years, molecular biological
methods are regarded as useful in the exact and rapid recognition of dermatophytes. The present study
identified nine clinical isolates and one ATCC as a control strain of T. rubrum by using both
conventional and molecular methods. The molecular systematics method was used to elucidate genetic
diversity among strains of T. rubrum and within Trichophyton species. Morphological characteristics of
all colonies T. rubrum quite varies among each other; we revealed that that conventional methods are
generally prolonged and may be indecisive. However, molecular studies based on internal transcribed
spacer (ITS) sequencing provides a very accurate result, which is more than 96% the similarity of T.
rubrum among all isolates, and more than 90% similarity within Trichophyton spp.
Key words: Trichophyton rubrum, conventional method, internal transcribed spacer (ITS) regions, identification,
dermatophytes.
INTRODUCTION
Trichophyton rubrum is one of the most commonly
encountered dermatophytes that infect human keratinized
tissue such as skin, nails and possibly hair. This
pathogen causes well-characterized superficial infections,
and also produces skin infections in unusual parts of the
body in immunodepressed patients (Cervelatti et al.,
2004). Nearly 80% of onychomycosis due to T. rubrum
and 90% of the chronic dermatophyte infections are
caused mostly by T. rubrum, this pathogen developed
mechanisms to avoid or suppress cell- mediated
immunity ((Baeza et al., 2006; Baeza et al., 2007).
*Corresponding author. E-mail: farzadaala@yahoo.com. Tel:

+98-9197544944.
Researchers use two general methods for the

laboratory identification of various species of
dermatophytes: a) identification on the basis of
phenotype differences (conventional methods) and b)
Identification on the basis of molecular differences. Faggi
et al. (2001) mentioned that identification of
dermatophyte species by conventional methods requires
the examination of colony, particularly with the method of
slide culture and microscopic morphological structures.
Morphological and physiological features are dynamic. As
a matter of fact, outside factors such as temperature
variation, medium and chemotherapy, can greatly
influence
the
phenotypic
characteristics
and
consequently can make the identification more difficult.
Molecular biological methods, in the recent years, are
regarded as useful in the exact and rapid recognition of
Aala et al.
dermatophytes. Sequencing of the Internal Transcribed

Spacer (ITS) region of the ribosomal DNA, Sequencing of
protein-encoding genes, Restriction Fragment Lenght
Polymorphism (PCR-RFLP) analysis of mitochondrial
DNA, Polymerase Chain Reaction (PCR); Random
Amplification of Polymorphic DNA (RAPD), Arbitrarily
Primed PCR [AP-PCR], and PCR fingerprinting are all
instances of molecular techniques which have brought
prominent advance in differentiating between species and
strains (Faggi et al., 2001; Kanbe et al., 2003; Girgis et
al., 2006; Yoshida et al., 2006; Li et al., 2007). In the
recent years, quite a few molecular studies have been
conducted on the internal transcribed spacer (ITS) region
of the rRNA gene. Sequencing analysis of the ITS
regions is considered as a useful tool for phylogenetic
delineation and the identification of dermatophytes
(Yoshida et al., 2006; Li et al., 2007).
Even though about 80 to 90% of all isolated are T.
rubrum (Brasch and Hipler, 2008), has been isolated to
identify the morphological similarity and the variability
among this species, but only a few study has been done
about the genetic relationship of Trichophyton.
The aim of this work is to identify ten clinical isolates of
T. rubrum by using both conventional methods and
molecular method based on universal fungal primers
which are internal transcribed spacer 1 (ITS1). T. rubrum
(ATCC-10218) was used as a control strain. The
molecular systematics method was used to elucidate
genetic diversity among strains of T. rubrum and within
Trichophyton species.
MATERIALS AND METHODS
Isolates
Nine isolates of T. rubrum which are T. rubrum (1138), T. rubrum
(1059), T. rubrum (1164), T. rubrum (1208), T. rubrum (1160), T.
rubrum (1008), T. rubrum (1298), T. rubrum (1044) and T. rubrum
(2970), were obtained from the culture collection of clinical isolates
preserved at the laboratory of Medical Mycology Department in
Tehran University of Medical Sciences, Iran for study; and T.
rubrum (ATCC-10218) was used as a control strain. All clinical
isolates were kept in sterile saline (0.85%) v/v NaCl at 4C until
required for bioassays.
6503
(PBS) and finally stored at 70C.
DNA extraction
Fungal genomic DNA from T. rubrum was isolated according to
Rezaie et al. (2000) with slight modification. 200 to 300 mg of
mycelia was ground with liquid nitrogen to powder form. 500 l of
DNA extraction buffer (50 mM Tris-HCl pH 8.0), 50 mM EDTA, 25 l
20% SDS, and 10 l of proteinase-K, was added and mixed gently.
Then, incubated at 65C for 60 min and centrifuged at 3000g for
15 min. 25 l Rnase H (10 mg/ml) was added to supernatant and
incubated again at 37C for 30 min. Then mixed with 500 l of
phenol:chloroform:isoamyl alcohol (25:24:1) and and centrifuge at
10000g for 10 min and the supernatant were collected and
transferred to new steril eppendorff tubes. Then mixed again with
500 l of chloroform:isoamyl alcohol (24:1) and centrifuge at
10000g for 10 min, and the supernatant were collected and
transferred to new steril eppendorff tubes. DNA was precipitated by
adding 500 l isopropanol and 30 l 3 M sodium acetate followed
by centrifugation at 15000g for 30 min and the supernatant was
discarded. DNA pellet was rinsed twice or more with 200 l of 70%
cold ethanol and centrifuged at 10000g for 10 min. The pellet was
air-dry and resuspended DNA pellet in 30 l of distilled water at
37C for 60 min and stored at -20C.
PCR amplification
Internal transcribed spacer 1 and 4 (ITS1 and ITS4) (AIT-Biotech,
Singapore) were designed as ITS1 forward primer is 5-TCC GTA
GGT GAA CCT GC-3 and the ITS4 reverse primer 5-TCC TCC
GCT TAT TGA TAT G-3 (Shehata et al., 2008; Yang et al ., 2008 ).
PCR reaction mixtures were prepared in a 25 l volume
containing 2.5 l of 10 reaction buffer, 1.5 l of 25 mM MgCl2, 0.5
l of 10 mM dNTPs, 0.5 l of 0.2 mM of each ITS 1 primer and ITS
4 primer, 0.5 l of genomic DNA and 0.5 l of 1 U Go Taq DNA
polymerase (Promega Corporation, USA), and 18.5 l of distiled
water. PCR reactions were carried out on a thermal cycler (MJ
Research. Inc. USA) with the following conditions: 1 cycle in an
initial step of 94C for 5 min and then subjected to 30 cycles
consisting of denaturation at 94C for 30 s, annealing at 55C for 40
s, and extention at 72C for 40 s. After the last cycle, this was
followed by a final extention step at 72C for 10 min. Then, 5 l of
PCR product was loaded on 1% agarose in 1X TrisAcetic Acid
EDTA buffer and stained with 0.5 mg/ml ethidium bromide at 80 V
for 40 min and visualised with UV transilluminator (Alpha Innotech,
USA), compared with a standard DNA size marker; 100 bp DNA
ladder (Fermenats, USA), and photographed in UV light.
PCR purification
Conventional method
All isolates of T. rubrum were cultured on Sabouraud dextrose agar
media (Difco Laboratories, Detroit, Michigan) at 28C for 14 days.
Then, slide cultures of isolates were prepared and identified under
light microscope (Carl Zeiss, Germany).
DNA PCR products were purified according to the QIAquick PCR

Purification Kit (Qiagen, Germany) and send for sequencing (1st
Laboratories, Seri Kembangan, Malaysia).
RESULTS AND DISCUSSION
Molecular method
Morphological characteristics of colonies T. rubrum
All isolates of T. rubrum maintained on Sabourauds dextrose agar

medium and stored at 4C. Then fungus was cultured in Sabouraud
dextrose broth, and incubated at 28C for 14 days. 200 to 300 mg
of mycelia was harvested and centrifuged at 1600g for 10 min,
then washed twice with ice-cold sterile phosphate buffered saline
This study used both conventional and molecular

methods to diagnose ten isolates of T. rubrum. Studies
revealed that colonies characterization of all isolates
quite varies among each other. Of these isolates, isolate
6504
Afr. J. Microbiol. Res.
numbers 1138 and 1059 are white and cottony or fluffy

but isolates number 1164, 1160, 1008, and 1298 are
cream, flat and downy, but the others are cream with a
carmine and woolly or granular type (isolates numbers
1208, 1044, 2970 and 10218) (Figure 1). The
microscopic features of the isolates also varies, which are
macroconidia and microconidia of isolates numbers,
1138, 1008, 1298, 1044, 2970 and 10218 more abundant
than isolates number 1059, 1164, 1208, and 1160.
However, the shape of the macroconidia and
microconidia of all isolates are almost similar, which is
cyclindrical to cigar shaped (Figure 1).
Isolation,
identification
and
characterization of ITS1 of T. rubrum
molecular
Figure 2 showed that ITS1 of all isolates T. rubrum had

been amplifed and then were isolated and sequenced.
The length of nucleotides sequence of all isolates are not
similar which is T. rubrum (1138) 658 bp, T. rubrum
(1059) 715 bp, T. rubrum (1164) 722 bp, T. rubrum
(2970) 660 bp and T. rubrum (ATCC-10218) 633 bp.
Nucleotide sequence of all isolates of T. rubrum and
ATCC-10218 are shown in Figure 3. Previous studies by
Rakeman et al. (2005) and Shehata et al. (2008) also
revealed that the universal fungal primers amplified the
ITS regions (ITS1-5.8S-ITS2) of the ribosomal DNA
nearly 690 bp for T. rubrum isolates.
Nucleotide sequence of ten isolates of T. rubrum
shown in Figure 3. All nucleotide sequences of T. rubrum
isolates were analyzed using online software CLUSTALW
(www.Pir.geogetown.edu/pirwww/search/multialn.shtm)
to reveal the similarities among isolates. Figure 4 showed
that the similarities among nine isolates of T. rubrum are
higher, which is more than 96% identities.
Nucleotide sequence of isolates T. rubrum were
analyzed using online software CLUSTALW (www.
Pir.geogetown.edu/pirwww/search/multialn.shtm)
to
reveal the similarities among isolates T. rubrum and other
species of Trichophyton which are Trichophyton
raubitschekii strain NOMH 789 (GenBank accession no.
AF170469), T. rubrum strain UAMH 8547 (GenBank
accession no. AF170471), T. kanei (GenBank accession
no. AF170460), T. rubrum strain WM 06.348 (GenBank
accession no. EF568093), T. rubrum strain 05-287-3929
(GenBank accession no. EU200395), T. rubrum 5.8S
rRNA (GenBank accession no. AJ270808), T.
soudanense strain UAMH 8548 (GenBank accession no.
AF170474), T. rubrum strain NCPF 295 (GenBank
accession no. EU181449), T. megninii strain ATCC
12106 (GenBank accession no. AF170464), and T.
rubrum strain ATCC 28188 (GenBank accession no. AF
170472). The similarities of all isolates of T. rubrum and
other species of Trichopthyon is also higher than 90% as

shown in Figure 5, CLUSTAL 2.0.12 multiple sequence
alignment.
DISCUSSION
Traditional method such as investigation of macroscopic
and microscopic features of cultures of fungi had been
applied since early 19th century. However, these methods
seem to be difficult to amplify due to the polymorphic
feature of these characters, besides increased by
differences in media compounds, temperature variations,
and other variables of cultivation. Furthermore, in some
cases, the dermatophytes fail to make reproductive
organization in culture (sterile mycelia) that makes it
impossible for final identification (Malinovschi et al.,
2009). Besides that, conventional method is often difficult
due to abnormal microscopic or macroscopic morphology
(Li et al., 2008). Currently, molecular studies become
crucial and necessary for identification of pathogenic
fungi (Borman et al., 2008; Malinovschi et al., 2009). The
internal transcribed spacer (ITS) regions of the fungal
ribosomal DNA (rDNA) had been used as one of
techniques for species identification becuase it is faster,
accurate species determination, specific, and are less
feasible to be affected by exterior effects such as
temperature changes and chemotherapy (Girgis et al.,
2006; Kong et al., 2008). Studies revealed that
morphological characteristics of colonies of all isolates T.
rubrum are similar to T. rubrum isolated from tinea cruris,
tinea pedis, and tinea capitis of human (Graser et al.,
2000). Colonies of T. rubrum are fluffy to cotonny and
white to cream in colour. Macroconidia are sparse or
abundant and microconidia are present in all isolates.
In this studies, the length of ITS1 of all isolates is about
690 bp, 10 clinical isolates of T. rubrum were collected
from the Clinical Mycology Laboratory at Westmead
Hospital, Sydney, and the Womens and Childrens
Hospital, Adelaide, Australia also have almost the same
length of ITS1, which is 666 bp (Kong et al., 2008).
Consequently, the results of our study are in agreement
with these studies and showed that molecular method
based on ITS sequencing is a reliable and useful method
for the identification of dermatophytes as well as for
confirmation of diagnosis of the conventional methods.
In this study, the molecular method was also used to
clarify genetic diversity among strains of T. rubrum and
within Trichophyton species. The results of this study
regarding nucleotide sequence of isolates of T. rubrum
demonstrated that the similarities among ten isolates of
T. rubrum are more than 96% identities. It also showed
the similarities among ten isolates of T. rubrum and ten
isolates of other genus of Trichophyton are higher than
90%. The results of this study are in agreement with
Graser et al. (2000) who showed that the Trichophyton
species are supported by high similarities with value of
Aala et al.
6505
Figure 1. The colonies and microscopy of 10 isolates of T. rubrum with (macroconidia and microconidia) 400.
more than 86% among isolates of T. rubrum and isolates

of other genus of Trichophyton. Our results are also in
agreement with Li et al. (2008), who revealed that

percentage identity of Trichophyton species with
6506
Figure 2. PCR amplification of isolates of T. rubrum on 1% agarose gel

electrophoresis. T. rubrum ATCC-10218 as positive control strain also showed DNA
amplification at 690 bp.
> T. rubrum (1138)

50
NNNNGGGAGAGCGTAAGTGGGCTGCCACTATAGAGGACCGGACATTCCAT
100
CAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACC
150
TCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTC
200
CGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGA
250
CAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGC
300
AAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATC
350
GATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCG
400
TGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGG
450
GCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGAT
500
GGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCA
550
GTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG
600
CGCCCTCAGGACCGGCCGCCTGGCCCCAATCTTTATATATATATATATC
650
TTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCAT
658
ATCAAAAG
> T. rubrum (1059)

50
NCCAGTAACCGTAGGTGACCTGCGCATATCAATAAGCGGAGGACTCCGTG
100
GGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCTCACC
150
CGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGCG
200
GGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGAC
250
ACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCACAGA
300
CAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGA
350
AGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAAT
400
CATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATG
450
CCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACG
500
ACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGC
550
CAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCC
600
TCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTCA
650
GGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAAT
700
AAGCCGGAGGAAGGGGGGGCCCCCCATAGGGCCCCCCCGCTCTCTTTTTG
715
GGGAAGCAAAATGGG
> T. rubrum (1164)
50
CNNNNNAGACCGTACGTTGGCTGCGCATATCAGATAACCGGACATGACAT
100
CGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCT
150
CACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCC
200
GGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA
250
GACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAA
300
GCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGA
350
TGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTG
400
AATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGC
450
ATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGG
500
ACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGT
550
GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCG
600
CCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTT
650
TTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATAT
700
CAAAAGGGGGGAGGAAGAGGGGGGCCCCCCATAGGGGCCCCCCCCTTTTT
722
TTTTGGGGTAGCGAGAAGGGGG
Figure 3. Nucleotide sequences of 9 isolates of T. rubrum and ATCC10218. Nucleotide sequence numbering is shown on the left.
Aala et al.
> T. rubrum (1208)

50
TNNGCAGACGTACGTGGGCTGCGAATATCAGGAAGCGACATGACTTCGGG
100
GGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCTCACC
150
CGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGCG
200
GGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGACA
250
CCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGCAC
300
AATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGAA
350
GAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAATC
400
ATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATGC
450
CTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACGA
500
CCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGCC
550
AGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCCTC
600
AGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTCAGG
650
TTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAATAA
700
GCCGGGAGGAAGGGGGGGCCCCCCAAAATGCCCCCCCCTCTCTTTTTGGG
713
GGGGAGAGCGGGG
> T. rubrum (1160)
50
NNNNNAAGAATCGTAAGTGACCTGCGCATATCAATAAGCGGAGGATCCGT
100
AGGTGAACCTGCGCGTATCAATAAGCGGAGGACATTCTTGTCTACCTCAC
150
CCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGGC
200
GGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGAC
250
ACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGCA
300
CAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATGA
350
AGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAAT
400
CATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCATG
450
CCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGACG
500
ACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGGC
550
CAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCCC
600
TCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTTC
650
AGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCAA
614
TAAGCGGGGAGGAA
> T. rubrum (1008)

50
NACNAAGAGCCGTAGGTGACCTGCGCATATCAATAAGCGAGAGGACTCCG
100
TAGGTGAACCTGCGTGTATCGGCCGTACGCCCACATTCTTGTCTACCTCA
150
CCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGG
200
CGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGA
250
CACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGC
300
ACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATG
350
AAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAA
400
TCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCAT
450
GCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGAC
500
GACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGG
550
CCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCC
600
CTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTT
650
CAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCA
700
ATAAGCCGGAGGAAGGGGGCCCCGAAGAGGAGCCACCCCCCTCAGGGTGT
719
GTGAAACAAACGGCGGGCC
> T. rubrum (1298)
50
NNACNNAGTATCGTAGGTGACCTGCGCATATCAATAAGCGGAGGATTCCG
100
TAGGTGAACCTGCGCATATCAATAAGCGGAGGATTCCGTTGGTTACCTCG
150
CCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGCCGTCCGG
200
CGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACAGA
250
CACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAGCAAGC
300
ACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCATCGATG
350
AAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCCGTGAA
400
TCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGGGGCAT
450
GCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGATGGAC
500
GACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCAGTGG
550
CCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCGCC
600
CTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCTTTT
650
CAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATATCA
668
ATAAGCGGAGGAA
> T. rubrum (1044)
50
NNNANCGGGACAGCCGTAGTGGGCTGCGCATATCAGATAACGCGGAGATT
100
ACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCT
150
ACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCC
200
GTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGG
250
ACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTTAG
300
CAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGCAT
350
CGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATTCC
400
GTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGGGG
450
GGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGTGA
500
TGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAAGCA
550
GTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG
600
CGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATC
650
TTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCAT
658
ATCAAAAG
Figure 3. Cotnd.
6507
6508
> T. rubrum (2970)

50
ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACT
100
TCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACC
150
TCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTC
200
CGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA
250
300
350
400
450
500
550
GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAG
600
CGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATATATCT
650
TTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAGCATA
660
TCAAAAGCGG
> T. rubrum (ATCC-10218)
50
NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAGGACTT
100
CGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTCTACCT
150
CACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGCCGTCC
200
GGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGAGGACA
250
300
350
400
450
500
550
GGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATTCAGCG
600
CCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATATATCTT
633
GGCAGGTTGACCTCGGATCAGGTAGGGATACGT
Figure 3. Cotnd.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
--NNNNGGGAGAGCGTAAGTGGGCTGCCA-CTAT-AGAGGAC-CGGACAT
---CNNNNNAGACCGTACGTTGGCTGCGC-ATATCAGATAAC-CGGACAT
----TNNGCAGA-CGTACGTGGGCTGCGA-ATATCAGGAAGC---GACAT
NNNANCGGGACAGCCGTAGTGGGCTGCGC-ATATCAGATAACGCGGAGAT
-----NGGGACCGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG
----ANCGGACAGCCGTAGTGGCCTGCGACATATCAGATAACGCGGAGAG
----NCCAGTAACCGTAGGTGACCTGCGC-ATATCAATAAGC----GGAG
--NNNNNAAGAATCGTAAGTGACCTGCGC-ATATCAATAAGC---G-GAG
--NNACNNAGTATCGTAGGTGACCTGCGC-ATATCAATAAGC---G-GAG
---NACNAAGAGCCGTAGGTGACCTGCGC-ATATCAATAAGC---GAGAG
*
**
****
*** *
*
*
50
50
50
50
50
50
50
50
50
50
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
TCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC
GACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC
GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC
TACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC
GACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC
GACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCCCCATTCTTGTC
GACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCCCCATTCTTGTC
GAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGGACATTCTTGTC
GATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGGATTCCGTTGGT
GACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCCACATTCTTGTC
*
***** *
**
*
*
***
100
100
100
100
100
100
100
100
100
100
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
TACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTGCCAGGGAGAGC
TACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTGCCAGGGAGAGC
****** ******************************************
T. rubrum (1138)
CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA
T. rubrum (1164)
T. rubrum (1208)
T. rubrum (1044)
T. rubrum (ATCC-10218) CGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCGCGCCCGCCGGA
T. rubrum (2970)
T. rubrum (1059)
T. rubrum (1160)
T. rubrum (1298)
T. rubrum (1008)
**************************************************
150
150
150
150
150
150
150
150
150
150
200
200
200
200
200
200
200
200
200
200
Figure 4. Comparison of nucleotide sequence between T. rubrum ITS1

orthologues. Nucleotide sequences that are present in all ITS1 are shaded in
blue colour. Nucleotide sequence numbering is shown on the right.
Aala et al.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
GGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAGTCTGAGCGTTT
**************************************************
T. rubrum (1138)
AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC
T. rubrum (1164)
T. rubrum (1208)
T. rubrum (1044)
T. rubrum (ATCC-10218) AGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTCTTGGTTCCGGC
T. rubrum (2970)
T. rubrum (1059)
T. rubrum (1160)
T. rubrum (1298)
T. rubrum (1008)
**************************************************
250
250
250
250
250
250
250
250
250
250
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
350
350
350
350
350
350
350
350
350
350
ATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGAATTGCAGAATT
**************************************************
T. rubrum (1138)
CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG
T. rubrum (1164)
T. rubrum (1208)
T. rubrum (1044)
T. rubrum (ATCC-10218) CCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTCTGGCATTCCGG
T. rubrum (2970)
T. rubrum (1059)
T. rubrum (1298)
T. rubrum (1008)
**************************************************
T.
T.
T.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
400
400
400
400
400
400
400
400
400
GGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGCCCGGCTTGTGT
**************************************************
T. rubrum (1138)
GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA
T. rubrum (1164)
T. rubrum (1208)
T. rubrum (1044)
T. rubrum (ATCC-10218) GATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGACGCGCCCGAAAA
T. rubrum (2970)
T. rubrum (1059)
T. rubrum (1160)
T. rubrum (1298)
T. rubrum (1008)
**************************************************
450
450
450
450
450
450
450
450
450
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
GCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATGGGCAGCCAATT
**************************************************
T. rubrum (1138)
CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATATATATAT
T. rubrum (1164)
T. rubrum (1208)
T. rubrum (1044)
T. rubrum (ATCC-10218) CAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTATATCGCGATAT
T. rubrum (2970)
T. rubrum (1059)
T. rubrum (1160)
T. rubrum (1298)
T. rubrum (1008)
******************************************
****
550
550
550
550
550
550
550
550
550
550
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
650
650
650
650
650
650
650
650
650
650
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1008)
300
300
300
300
300
300
300
300
300
300
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
Figure 4. Contd.
ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG
ATCTTGGCAGGTTGACCTCGGATCAGGTAGGGATACGT-----------ATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCTGAACTTAAG
***** *****************************
500
500
500
500
500
500
500
500
500
500
600
600
600
600
600
600
600
600
600
600
6509
6510
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
CATATCAAAAG--------------------------------------CATATCAAAAGGGGGGAGGAAGAGGGGGGCCCCCCATAGGGGCCCCCCCC
CATATCAATAAGCCGGGAGGAAGGGGGGGCCCCCCA-AAATGCCCCCCCC
CATATCAAAAG---------------------------------------------------------------------------------------CATATCAAAAGCGG-----------------------------------CATATCAATAAGCCGG-AGGAAGGGGGGGCCCCCCATAGGGCCCCCCCGC
CATATCAATAAGCGGGGAGGAA---------------------------CATATCAATAAGCGGAGGAA-----------------------------CATATCAATAAGCCGGAGGAAGGGGGCCCCGAAGAGGAGCCACCCCCCTC
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1138)
(1164)
(1208)
(1044)
(ATCC-10218)
(2970)
(1059)
(1160)
(1298)
(1008)
--------------------------TTTTTTTTTGGGGTAGCGAGAAGGGGG
TCTCTTTTTGGGGGGGAGAGCGGGG-------------------------------------------------------------------------------TCTCTTTTTGGGGAAGCAAAATGGG-----------------------------------------------------AGGGTGTGTGAAACAAACGGCGGGCC-
700
700
700
700
700
700
700
700
700
700
727
727
727
727
727
727
727
727
727
727
Figure 4. Contd.
T. raubitschekii strain NOMH 789

T. megninii strain ATCC 12106
T. rubrum strain UAMH 8547
T. rubrum strain ATCC 28188
T. kanei
T.rubrum 5.8S rRNA gene
T.rubrum strain WM 06.348
T.rubrum strain NCPF 295
T.rubrum strain 05-287-3929
T. rubrum (1138)
T. rubrum (1164)
T. rubrum (1298)
T. rubrum (1008)
T. rubrum (1059)
T. rubrum (1208)
T. rubrum (1264)
T. rubrum (2970)
T. rubrum (ATCC-10218)
T. rubrum (1044)
CGCCCGTCGCTACTACCGATTGAATGGCTCAGTGAGGCCTTCGGACTGGC
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
50
50
50
50
50
50
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
CCAGGGAGGTTGGAAACGACCGCCCAGGGCCGGAAAGTTGGTCAAACTCG
---------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
100
100
100
100
100
100
raubitschekii strain NOMH 789

megninii strain ATCC 12106
saudanese UAMH 8548
rubrum strain UAMH 8547
rubrum strain ATCC 28188
kanei
rubrum 5.8S rRNA gene
rubrum strain WM 06.348
rubrum strain NCPF 295
rubrum strain 05-287-3929
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
T.raubitschekii strain NOMH 789

T. saudanese UAMH 8548
T. kanei
T. rubrum 5.8S rRNA gene
T. rubrum strain WM 06.348
T. rubrum strain NCPF 295
T. rubrum strain 05-287-3929
T. rubrum (1138)
T. rubrum (1164)
T. rubrum (1298)
T. rubrum (1008)
T. rubrum (1059)
T. rubrum (1208)
T. rubrum (1264)
T. rubrum (2970)
T. rubrum (1044)
GTCATTTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTG
--------------------------ACAAGGTTTCCGTAGGTGAACCTG
-----------------------------------------------------------------------------------TCCGTAGGTGAACCTG
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
150
150
150
150
150
150
24
16
Figure
5. Comparison
sequence between T. rubrum ITS1
T. raubitschekii
strain NOMHof789nucleotide
CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G
198
T. saudanese UAMH
8548
orthologues.
Nucleotide
sequences
that are present in all ITS1 are shaded198in
CGGAAGGATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G 198
black
colour.
Nucleotide
numbering is shown on the right.
T. rubrum
strain
UAMH 8547sequence
198
T.
T.
T.
T.
T.
T.
T.
rubrum
kanei
rubrum
rubrum
rubrum
rubrum
rubrum
strain ATCC 28188

5.8S rRNA gene
strain WM 06.348
strain NCPF 295
strain 05-287-3929
(1138)
CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G
------GATCATTAACGCGCAGGCCGGAGGCTGGCCCCC-CACGATAG-G
-------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG
198
198
72
42
64
42
35
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1059)
(1208)
(1264)
(2970)
(ATCC-10218)
(1044)
-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
CGGAAGGATCATTAACGCGCNGGCCGGAGGCTGGCCCCC-CACGATAG-G
-------------NNNNGGGAGAGCGTAAG-TGGGCTGC-CACTATAGAG
-------------NNNNNAAGAATCGTAAGTGACCTGCG-CATATCA-AT
-------------NNACNNAGTATCGTAGGTGACCTGCG-CATATCA-AT
--------------NACNAAGAGCCGTAGGTGACCTGCG-CATATCA-AT
---------------NCCAGTAACCGTAGGTGACCTGCG-CATATCA-AT
----------------TNNGCAGACGTACGTGGGCTGCG-AATATCA-GG
--------------CNNNNNAGACCGTACGTTGGCTGCG-CATATCAGAT
--------------ANCGGACAGCCGTA-GTGGCCTGCGACATATCAGAT
---------------NGGGACCGCCGTA-GTGGCCTGCGACATATCAGAT
----------NNNANCGGGACAGCCGTA-GTGGGCTGCG-CATATCAGAT
** * *
*
*
198
198
198
198
198
198
72
42
64
42
35
35
35
34
33
32
35
35
34
38
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
GA-CCG-ACGTTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC
GA-CCG-ACGTTC-ATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC
GA-CCGGACATTCCATCAGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC
AA-GCG--GAGGAT-CCGTAGGTGAACCTGCGCGTATCAATAAGCGGAGG
AA-GCG--GAGGATTCCGTAGGTGAACCTGCGCATATCAATAAGCGGAGG
AA-GCGA-GAGGACTCCGTAGGTGAACCTGCGTGTATCGGCCGTACGCCC
AA-GCG--GAGGACTCCGTGGGTGAGCATACGTGCGCCGGCCGTACGCCC
AA-GCGA-CATGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC
AA-CCGGACATGACATCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC
AACGCGGAGAGGACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC
AACGCGGAGAGGACTTCGGGGGTGAGCATACGTGCGCCGGCCGTACGCCC
AACGCGGAGATTACTTCGGGGGTGAGCAGACGTGCGCCGGCCGTACGCCC
* **
* ***** * **
*
*
246
246
246
246
246
246
119
90
112
90
84
81
82
82
80
80
84
85
84
88
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG
C-ATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG
ACATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG
ATTCCGTTGGTTACCTCGCCCGGTTGCCTCGGCGGGGCGCGCTCCCCCTG
ACATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG
*** ****** ****************** *************
296
296
296
296
296
296
168
140
162
140
134
131
132
132
130
130
134
135
134
138
CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGAGCCTCGAGCCGGACCG
CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG
CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG
346
346
346
346
346
Aala et al.
Figure 5. Contd.
T.
T.
T.
T.
T.

saudanese UAMH 8548
6511
T. rubrum (2970)
T. rubrum (1044)
6512
CCATTCTTGTCTACCTCACCCGGTTGCCTCGGCGGGCCGCGCTCCCCCTG 135
*** ****** ****************** *************
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1168)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
CCAGAGAGAGCCGTCCGGCGGGCCTCTTCCGGGGGCCTCGAGCCGGACCG
CCAGGGAGAGCCGTCCGGCGGGCCCCTTCTGGGGGCCTCGAGCCGGACCG
**** ******************* **** *** ****************
346
346
346
346
346
346
218
190
212
190
184
181
182
182
180
180
184
185
184
188
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
CGCCCGCCGGAGGACAGACACCAAGAAAAAATTCTCTGAAGAGCTGTCAG
**************************************************
396
396
396
396
396
396
268
240
262
240
234
231
232
232
230
230
234
235
234
238
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
TCTGAGCGTTTAGCAAGCACAATCAGTTAAAACTTTCAACAACGGATCTC
**************************************************
446
446
446
446
446
446
318
290
312
290
284
281
282
282
280
280
284
285
284
288
TTGGTTCCGGCATCGATGAAGAACGCAGCGAAATGCGATAAGTAATGTGA
496
496
496
496
496
Figure 5. Contd.
T.
T.
T.
T.
T.
T.
rubrum
rubrum
rubrum
rubrum
rubrum
rubrum
(1059)
(1208)
(1264)
(2970)
(ATCC-10218)
(1044)
**************************************************
280
280
284
285
284
288

T. kanei
T. rubrum (1138)
T. rubrum (1164)
T. rubrum (1298)
T. rubrum (1008)
T. rubrum (1059)
T. rubrum (1208)
T. rubrum (1264)
T. rubrum (2970)
T. rubrum (1044)
**************************************************
496
496
496
496
496
496
368
340
362
340
334
331
332
332
330
330
334
335
334
338
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
raubitschekii strain NOMH789

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
ATTGCAGAATTCCGTGAATCATCGAATCTTTGAACGCACATTGCGCCCTC
**************************************************
546
546
546
546
546
546
418
390
412
390
384
381
382
382
380
380
384
385
384
388
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
TGGCATTCCGGGGGGCATGCCTGTTCGAGCGTCATTTCAACCCCTCAAGC
**************************************************
596
596
596
596
596
596
468
440
462
440
434
431
432
432
430
430
434
435
434
438
Figure
5. Contd.strain NOMH789
T.
raubitschekii
T. kanei
CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTCCCTTCGGGGGCGGGAC
646
646
646
646
646
646
Aala et al.
6513
6514
T. rubrum (1059)
T. rubrum (1208)
T. rubrum (1264)
T. rubrum
(2970)
Afr. J. Microbiol.
Res.
T. rubrum (1044)
**************************************************
430
430
434
435
434
438
CCGGCTTGTGTGATGGACGACCGTCCGGCCCCTC--TTCGGGGGCGGGAC
********************************** **************
646
646
646
646
646
646
516
490
512
490
484
481
482
482
480
480
484
485
484
488
GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTAGGCGAATG
GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG
GCGCCCGAAAAGCAGTGGCCAGGCCGCGATTCCGGCTTCCTGGGCGAATG
***************************************** ********
raubitschekii strain NOMH789 GGCAGCCAATTCAGCGCCCTCAGG-------------------------megninii strain ATCC 12106 GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA
saudanese UAMH 8548
GGCAGCCAAACCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA
GGCAGCCAATTCAGCGCCCTCAGGACCGGCCGCCCTGGCCCCAATCTTTA
kanei
GGCAGCCAATTCAGCGCCCTCAGGACCGGCNGCCCTGGCCCCAATCTTTA
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
********* *************
696
696
696
696
696
696
566
540
562
540
534
531
532
532
530
530
534
535
534
538
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.

saudanese UAMH 8548
kanei
rubrum (1138)
rubrum (1164)
rubrum (1298)
rubrum (1008)
rubrum (1059)
rubrum (1208)
rubrum (1264)
rubrum (2970)
rubrum (ATCC-10218)
rubrum (1044)
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
T.
Figure 5. Contd.
720
746
746
746
746
746
616
590
612
590
584
581
582
582
580
580
584
585
584
588
Aala et al.
T. raubitschekii strain NOMH789

T. kanei
T. rubrum (1138)
T. rubrum (1164)
T. rubrum (1298)
T. rubrum (1008)
T. rubrum (1059)
T. rubrum (1208)
T. rubrum (1264)
T. rubrum (2970)
T. rubrum (1044)
T. kanei
T. rubrum (1138)
T. rubrum (1164)
T. rubrum (2970)
T. rubrum (1044)
-------------------------------------------------TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT
TATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCGCT
TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG
TATCGCGATATATCTTGGCAGGTTGACCTCGGATCAG------------TATATATATATATCTTTTCAGGTTGACCTCGGATCAGGTAGGGATACCCG
-------------------------------------------------GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC
GAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTGCC
CTGAACTTAAGCATATCAATAAGCGGAGGAAAAGAAACCAACAGGGATTG
CTGAACTTAA---------------------------------------CTGAACTTAAGCATATCAAT-----------------------------CTGAACTTAAGCATATCAATAAGCGG-----------------------CTGAACTTAAGCATATCAAAAG---------------------------CTGAACTTAAGCATATCAATAAGCGGGGAGGAA-----------------------------------------------------------------------------------------------------------------------------------------------------------------
6515
796
796
796
796
796
666
640
662
640
634
631
632
632
630
630
634
635
621
638
846
846
846
846
846
716
650
682
666
656
664
Figure 5. Contd.
reference sequence in GenBank (BLAST search) ranged

from 85.9 to 100%.
Conclusion
By conventional characterization, colonies of all isolates
quite varies; however, the shape of macroconidia and
microconidia are similar. Beside that, molecular
characterization also revealed that all isolates of T.
rubrum show high similarity among them and with other
Trichophyton species.
ACKNOWLEDGEMENT
This study was supported by the Research University
Grants Scheme (RUGS) from University Putra Malaysia.
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DOI: 10.5897/AJMR11.119
Inhibitory effect of some plant extracts on clinical

isolates of Staphylococcus aureus
Rajaa Milyani* and Nahed Ashy
Department of Biological Sciences, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia.
Accepted 30 May, 2011
Accumulating data have been dramatically increasing on multiple drug resistant Staphylococcus
aureus isolates that are incriminated in nosocomial and community acquired infections causing high
mortality and morbidity. Accordingly, 70 resistant isolates were collected from King Fahad General
Hospital and National Guard Hospital, Jeddah, Saudi Arabia in an attempt to find alternative
antimicrobial substances from various plant extracts. The susceptibility pattern was as follows: 50
isolates were resistant to fusidic acid, 28 isolates were resistant to oxacillin, 20 isolates were resistant
to penicillin, 20 isolates were resistant to clindamycin and 21 isolates were resistant to gentamycin.
However, all the isolates were sensitive to vancomycin. The antimicrobial activity of the aqueous
extracts of five medicinal plants namely Canellia sinensis (green tea), Punnica granatum (pomegranate
rind), Psidium guajava Lim (guava leaves), Cinnamomum verum (cinnamon) and Mourus (raspberry)
was tested against the 70 resistant isolates of S. aureus using agar well diffusion assay. Significant
difference was noted in the inhibitory effect between most of the tested extracts. Pomegranate rind
showed the highest activity, followed by green tea, guava leaves, cinnamon bark and raspberry fruits
extract respectively, compared to commercial antibiotics. Interestingly, the inhibitory activity of three
combined extracts: green tea, pomegranate rind and guava leaves was found to be higher on 20
clindamycin resistant isolates compared to each extract alone, indicating synergistic interaction. These
results emphasize the promising role of plant extracts as alternative antibacterial agents against
resistant strains of S. aureus if not other pathogenic bacteria.
Keywords: Staphylococcus aureus, antimicrobial activity, plant extracts, inhibition, methicillin.
INTRODUCTION
Staphylococcus aureus is one of the main causes of
human infections. It can cause diseases ranging from
minor infections such as pimples and boils to serious
systemic fatal infections (Evans and Brachman, 1991).
Many neonates, children and adults may be colonized by
S. aureus and harbor this organism either in the
nasopharynx, skin or any site of their bodies, thus
dispersing this hazardous bacterium. In Saudi Arabia, the
throat carriage rate of S. aureus has been studied and
found to be about 9% among 100 adult and 12% among
150 children (Milyani and Memish, 1987). On the other
*Corresponding author. E-mail: helicobacter2011@hotmail.com
hand, the incidence rate from six sites of the skin in

addition to the anterior nares was 30% in 40 examined
children and 5% in adults (Milyani, 2001). Moreover, S.
aureus predominated among children in both
nasopharynx and ear discharge simultaneously recording
26.7 and 20% respectively (Milyani and Mahfouz, 2004).
Methicillin resistant S. aureus (MRSA) is a bacterium
that has developed resistance to most antibiotics such as
methicillin, gentamycin, fucidic acid and clindamycin that
are commonly used for Staphylococcus infections,
unfortunately, leading to failure of treatment (Shai et al.,
2004). The two major strains of MRSA are known to be
hospital-acquired (HA) MRSA and community-acquired
(CA) MRSA. HA-MRSA includes cases in which the
patient has had a current or recent hospitalization,
6518
receives dialysis, or resides in a long-term care facility.

During the period 1970 to 2010, strains of S. aureus
resistant to multiple antibiotics including methicillin were
increasingly responsible for outbreaks of nosocomial
infections in countries around the world, for example,
Saudi Arabia (Madani et al., 2001), Argentina (Reyes et
al., 2009), South Africa (Shittu et al., 2009), Italy (Soavi
et al., 2010) and the United States (Boyce, 1990). In
many instances, these outbreaks were associated with
individual wards, neonatal, intensive care and burns units
(Liu et al., 2011). Furthermore, increasing incidence of
CA-MRSA has been a growing public health concern
(Mandell et al., 2005; Ma et al., 2007) and has emerged
as the predominant cause of skin infections in the USA
(Stevens et al., 2010).
Only few intervention studies have been published to
explore alternative antimicrobial agents to control and
prevent diseases due to multidrug resistant S. aureus.
Although other practices have been explored such as
bacterial interference therapy (Maibach and Aly, 1981)
and phage therapy (Jikia et al., 2005), medicinal plants
have also been considered by some researchers since
they are frequently used in popular medicine as remedies
for many infectious diseases (Geyid et al., 2005; Mohana
et al., 2008; Rahim et al., 2010). The aim of the present
study was to determine the inhibitory effect of different
plant extracts on the growth of 70 multiple resistant
strains of S. aureus which also includes MRSA obtained
from two main hospitals in Jeddah City, Saudi Arabia.

Media and antibiotics used
Sheep blood agar, nutrient agar, mannitol salt agar (Hi media,
India), Mueller Hinton (Oxoid, England) were used. Commercial
antibiotic discs were obtained from Oxoid, England.
Preparation of extracts
The plants were extracted by dissolving 50 g of the plant powder in
150 ml of sterile distilled water and boiled for 30 min (aqueous
extraction). The extracts were filtered using Whatman filter paper
no. 1, and were stored at 4C until used (Somchit et al., 2003).
Susceptibility test of bacterial isolates to antibacterial agents

Susceptibility pattern of the isolates of S. aureus to commercial
antibacterial agents was determined by the standard paper disc
diffusion method (Baron and Finegold, 1990). According to
Koneman et al. (1997), every strain was tested using the following
panel of antibiotic discs: clindamycin (2 g), gentamicin (10 g),
oxacillin (1 g), penicillin (10 g), fusidic acid (30 g), and
vancomycin (30 g). Susceptibility of strains to each antibiotic was
determined on the basis of diameter of the inhibition zone.
Methicillin-resistant strains were tested by using 1 g of oxacillin
disc. A strain showing inhibition zone of 10 mm diameter was
considered MRSA after 24 h incubation at 35C (Koneman et al.,
1997).
Antibacterial activity of the plant extracts

Agar well diffusion assay was used to determine the antibacterial
activity of the plant extracts. A standard inoculum size of the S.
aureus strains was evenly distributed and streaked onto the surface
of a sterile Mueller Hinton Agar plate using sterile cotton swab. The
density of the suspension was adjusted to approximately 108
CFU/ml by comparing its turbidity to a McFarland 0.5 BaSO4
standard (Koneman et al., 1997). Agar wells were made by using
sterile cork borer (7 mm diameter). Each well was then filled with
200 l of the tested plant extract after which the plates were
incubated at 37C for 24 h. All tests were performed in duplicate
and the antibacterial activity produced by the plant extracts was
expressed as the mean diameter of the inhibition zones (mm). In
addition, combinations of three plant extracts (green tea,
pomegranate and guava) were tested against 20 clindamycin
resistant of S. aureus using 200 l of a mixture of equal volume of
each extract.
Statistical analysis
Bacterial isolates
Seventy S. aureus isolates were obtained from both King Fahad
General Hospital and National Guard Hospital in Jeddah City. They
were isolated from the following clinical specimens: sputum, wound
discharge, blood and cerebrospinal fluid. Identification was
according to Winn et al. (2006). The isolates were preserved in
cooked meat broth (Oxoid) at 4C.
Plant materials
The medicinal plants were obtained from different markets in
Jeddah City, Saudi Arabia. The plants were authenticated at the
Herbarium Unit, Department of Biological Sciences, King Abdulaziz
University, Jeddah. Five medicinal plants were used namely:
Canellia sinensis (green tea leaves), Punnica granatum
(pomegranate rind), Psidium guajava Lim (guava leaves),
Cinnamomum verum (cinnamon bark) and Mourus (raspberry fruit)
was tested. The plant materials were washed and sun dried before
being grinded into powder.
The data were analyzed using SPSS version 10. Analysis of

variance (ANOVA) and Scheffe tests were used to detect any
significant differences among the inhibitory effects of plant extracts
on S. aureus isolates. P< 0.05 was considered significant.

Methicillin-resistant S. aureus (MRSA) is primarily a
nosocomial pathogen that emerged as a major cause of
infection and colonization in hospitalized patients,
whereas, community-associated MRSA infections are
increasing in incidence and said to be severe enough to
cause fatality (Mandell et al., 2005). Strains of MRSA that
cause infections have also developed resistance to
antibiotics commonly used for therapeutic purposes. As
shown in Table 1, the majority of the isolates were
resistant to five of the six antibiotics tested. However, the
Milyani and Ashy
6519
Table 1. The sensitivity of 70 Staphylococcus aureus isolates represented by diameter of inhibition zone (mm) to different antibiotics and five
plant extracts.
Isolate
No.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Fusidic acid
13.0
00.0
11.0
0.00
13.0
12.0
0.0
13.0
12.0
00.0
14.0
30.0
00.0
00.0
12.0
00.0
00.0
12.0
00.0
14.0
00.0
12.0
13.0
00.0
00.0
12.0
14.0
00.0
00.0
00.0
00.0
00.0
00.0
14.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
Antibiotics
Gentamycin Oxacillin
23
20.0
0
00.0
21
14.0
26
14.0
20
15.0
30
17.0
33
17.0
27
21.0
36
17.0
22.0
20.0
31.0
20.0
20.0
20.0
00.0
00.0
22.0
15.0
19.0
16.0
35.0
20.0
27.0
18.0
32.0
19.0
34.0
18.0
27.0
23.0
25.0
15.0
23.0
17.0
32.0
14.0
33.0
20.0
34.0
17.0
32.0
20.0
23.0
19.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
34.0
00.0
20.0
17.0
28.0
00.0
26.0
00.0
20.0
00.0
21.0
00.0
22.0
19.0
Penicillin
25.0
08.0
11.0
00.0
13.0
14.0
17.0
22.0
10.0
11.0
18.0
19.0
00.0
11.0
12.0
16.0
20.0
18.0
19.0
24.0
9.0
10.0
13.0
17.0
00.0
18.0
20.0
8.0
00.0
00.0
00.0
00.0
10.0
00.0
11.0
08.0
08.0
00.0
00.0
08.0
00.0
25.0
00.0
08.0
09.0
00.0
0.0
12.0
Clindamycin
29.0
00.0
26.0
27.0
24.0
32.0
30.0
35.0
36.0
25.0
33.0
00.0
27.0
00.0
26.0
24.0
32.0
29.0
35.0
30.0
33.0
32.0
25.0
36.0
00.0
38.0
38.0
27.0
00.0
00.0
00.0
00.0
00.0
34.0
00.0
00.0
00.0
00.0
33.0
00.0
00.0
35.0
00.0
00.0
00.0
00.0
00.0
24.0
EXT 1
21.0
22.0
23.0
25.0
30.0
20.0
27.0
20.0
19.0
19.0
20.0
21.0
26.0
22.0
20.0
21.0
23.0
20.0
23.0
21.0
19.0
20.0
20.0
22.0
23.0
20.0
31.0
23.0
26.0
22.0
19.0
16.0
22.0
20.0
20.0
27.0
22.0
26.0
29.0
22.0
21.0
24.0
24.0
27.0
23.0
27.0
28.0
25.0
Plant extracts
EXT 2 EXT 3 EXT 4
24.0
11.0
12.0
26.0
14.0
12.0
29.0
17.0
11.0
23.0
16.0
00.0
17.0
15.0
11.0
26.0
13.0
14.0
22.0
15.0
17.0
22.0
20.0
11.0
23.0
13.0
13.0
22.0
18.0
11.0
27.0
17.0
10.0
27.0
14.0
12.0
27.0
17.0
14.0
33.0
16.0
14.0
26.0
14.0
12.0
21.0
00.0
12.0
24.0
11.0
00.0
28.0
08.0
11.0
23.0
08.0
11.0
21.0
10.0
10.0
21.0
15.0
11.0
22.0
19.0
13.0
21.0
15.0
12.0
30.0
16.0
17.0
30.0
16.0
15.0
24.0
19.0
16.0
28.0
15.0
15.0
31.0
30.0
12.0
22.0
13.0
15.0
29.0
13.0
12.0
28.0
16.0
12.0
23.0
00.0
09.0
24.0
19.0
13.0
25.0
12.0
13.0
21.0
11.0
13.0
24.0
16.0
10.0
31.0
13.0
13.0
26.0
16.0
11.0
26.0
20.0
120.0
25.0
18.0
12.0
25.0
19.0
08.0
32.0
00.0
13.0
27.0
16.0
14.0
33.0
16.0
14.0
27.0
19.0
15.0
29.0
00.0
12.0
32.0
00.0
14.0
22.0
13.0
14.0
EXT 5
11.0
14.0
11.0
11.0
13.0
10.0
21.0
10.0
14.0
11.0
10.0
14.0
13.0
14.0
11.0
12.0
00.0
11.0
11.0
09.0
13.0
13.0
10.0
15.0
12.0
15.0
14.0
13.0
14.0
11.0
12.0
09.0
13.0
13.0
11.0
11.0
11.0
09.0
10.0
12.0
16.0
10.0
17.0
15.0
14.0
11.0
11.0
11.0
6520
Table 1 Contd.
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
00.0
20.0
29.0
35.0
17.0
28.0
23.0
15.0
21.0
25.0
27.0
25.0
25.0
29.0
24.0
27.0
23.0
32
27
28
33
31
20.0
28.0
26.0
20.0
21.0
20.0
21.0
22.0
21.0
23.0
20.0
18.0
19.0
22.0
19.0
20.0
21.0
21
24
25
24
24
00.0
13.0
22.0
20.0
19.0
21.0
19.0
25.0
22.0
20.0
24.0
19.0
19.0
24.0
21.0
17.0
19.0
22.0
21.0
22.0
27.0
26.0
12.0
27.0
20.0
20.0
17.0
17.0
18.0
21.0
00.0
00.0
22.0
19.0
21.0
22.0
00.0
13.0
18.0
20.0
19.0
17.0
17.0
21.0
33.0
40.0
25.0
23.0
18.0
21.0
20.0
22.0
21.0
23.0
20.0
18.0
19.0
22.0
19
20.0
21.0
21.0
24.0
25.0
24.0
24.0
23.0
22.0
21.0
22.0
20.0
19.0
17.0
20.0
16.0
17.0
19.0
17.0
20.0
21.0
19.0
19.0
22.0
18.0
28.0
22.0
22.0
21.0
22.0
32.0
16.0
17.0
19.0
18.0
16.0
16.0
17.0
16.0
17.0
17.0
18.0
17.0
17.0
17.0
18.0
29.0
25.0
23.0
27.0
26.0
14.0
18.0
12.0
00.0
00.0
00.0
00.0
00.0
00.0
00.0
08.0
00.0
08.0
08.0
00.0
09.0
00.0
16.0
21.0
19.0
20.0
16.0
17.0
13.0
12.0
13.0
12.0
10.0
14.0
12.0
13.0
12.0
10.0
14.0
12.0
13.0
12.0
12.0
12.0
12.0
13.0
12.0
13.0
14.0
13.0
14.0
11.0
07.0
12.0
13.0
12.0
11.0
12.0
12.0
11.0
11.0
10.0
13.0
12.0
12.0
13.0
00.0
12.0
13.0
12.0
0.0
EXT 1: Green tea, EXT 2: Pomegranate rind, EXT 3: Guava leaves, EXT 4 cinnamon bark, EXT 5: raspberry.
predominant isolates among the 70 studied isolates were

resistant to fusidic acid (50 isolates). 28 isolates were
resistant to oxacillin, 20 isolates were resistant to
penicillin, 20 isolates were resistant to clindamycin and
21 isolates were resistant to gentamycin. In contrast, all
the isolates were sensitive to vancomycin. In accordance
to our results, Zaman and Dibb (1994) isolated 7.5% of
MRSA from wounds, 83% resistant isolates to
gentamycin and 93% resistant isolates to tetracycline in
Jeddah City. On the other hand, Udo and Jacob (2000)
reported 10% of MRSA that was also resistant to fusidic
acid in one of Jeddah hospitals. A total of 587 and 485
isolates of S. aureus were isolated from Abha Maternity
Hospital, Saudi Arabia in 1996 and 1998 respectively. In
both years, 71.0% were methicillin-resistant S aureus
whereas, over 85.0% of all isolates were multi-resistant
(Bilal and Gedebou, 2000). Moreover, 34% of fusidic acid
resistant isolates among 173 MRSA were isolated in
Riyadh City, Saudi Arabia (Al-Digs, 2004). Similarly, high
incidence of fusidic acid resistant strains was reported in
children complaining of impetigo during five years study
at Wales, England (Al-Zimaity et al., 2004). Resistance of
S. aureus strains to fusidic acid should be emphasized
since fucidin cream and/ or ointment are still in use as
topical therapy for boils, sties and superficial skin
infections such as impetigo. Obviously, this might lead to
failure of treatment, disseminating infection and
increasing resistant strains (Nielsen et al., 2009).

Plant products, particularly spices and extracts of
various plant parts have been used extensively as natural
antimicrobials and antioxidants (Iqbal et al., 1998; Oskay
et al., 2009). The sensitivity of 70 resistant isolates of S.
aureus to the aqueous extracts of C. sinensis (green tea),
P. granatum (pomegranate rind), P. guajava (guava), C.
verum (cinnamon) and Mourus (raspberry) has been
studied. These plants belong to different families (Table
2) and the active part of each plant was different.
Although there are different techniques for plant
extraction using organic solvents or water, in the present
study, aqueous extraction was used which gave
satisfactory and reproducible results. This technique was
also utilized by other researchers to investigate the
antimicrobial effect of different plant extracts successfully
(Tomita et al., 1997; Somchit et al., 2002).
Table 1 showed that all the tested S. aureus isolates
were sensitive to green tea and pomegranate extracts
where the diameters of the inhibition zones ranged from
16 to 30 mm. The inhibitory effect of three plant extracts
on S. aureus was shown in Figure 1, where the
antimicrobial activity is measured by the mean diameter
of the inhibition zones. The antimicrobial activity of green
tea may be due to polyphenols and epigallocatechin
gallate which are active substances that possess
excellent bactericidal activity (Toda et al., 1989, 1991;
Milyani and Ashy
6521
Table 2. The common and scientific names and used part of five plant extracts.
Common name
Green tea
Pomegranate
Guava
Cinnamon
Raspberry
Family
Theaceae
Rosaceae
Myrtaceae
Lauraceae
Rosaceae
Scientific name
Canellia sinensis
Punnica granatum
Psidium guajava
Cinnamomum verum
Mourus
Used part of the plant

Leaf
Rind
Leaf
Bark
Fruit
Figure 1. The inhibitory effect of three different plant extracts against resistant Staphylococcus aureus isolate.
(A) Green tea extract, (b) Pomegranate extract, (C) Guava extract.
Shiota et al., 1999; Stapleton et al., 2004). Moreover,

these substances have at least an indirect influence on
biofilm production retarding the formation of dental
plaque (Wolinsky et al., 2000). Similar to our studies,
pomegranate extract has also been found to exhibit
antimicrobial activity against a number of pathogenic
bacteria such as Escherichia coli O157: H7
(Voravuthikunchai et al., 2005) which is one of the
important
emerging
virulent
strain,
Salmonella
typhimurium, Salmonella typhi, Shigella dysenteriae and
Vibrio cholerae (Pradeep et al., 2008). In respect to
guava, raspberry and cinnamon extracts, the numbers of
resistant S. aureus isolates were 23, 4 and 7
respectively. Nonetheless, the diameters of the inhibition
zones of the sensitive S. aureus isolates inhibited by the
three tested extracts ranged from 10 to 20, 10 to 16 and
10 to 17 mm respectively.
Studies to explore the anti-cough activity of guava in

rats and guinea pigs have been carried out by Jaiarj et al.
(1999), thus encouraged them to suggest the use of
guava extracts as anti-tussive product. They also
reported the bactericidal effect of guava extract on S.
aureus and Streptococcus pyogenes strains. In addition,
Vieira et al. (2001) and Anas et al. (2008) have
demonstrated that guava extract had a high inhibitory
effect against S. aureus strains causing food poisoning in
children. Furthermore, using a crude aqueous and water
soluble methanol extract from leaf and bark of guava, a
strong antibacterial activity against multidrug-resistant
Vibrio cholerae O1 was recorded (Rahim et al., 2010), a
fascinating finding that should be further studied to be
applied clinically especially in controlling epidemics of
cholera. On the other hand, cinnamon bark- at the
present work- was inhibitory to S. aureus isolates in
6522
Table 3. Significant differences between the inhibitory

activity of plant extracts under study.
Tested plant
Green tea
Pomegranate
Guava leaves
Cinnamon
Raspberry
p value
0.877
0.00*
0.00*
0.00*
Pomegranate
Guava leaves
Cinnamon
Raspberry
0.00*
0.00*
0.00*
Guava leaves
Cinnamon
Raspberry
0.238
0.349
Cinnamon
Raspberry
1.0
Mean diameter of inhibition

zone (mm)
*: Significant difference at p<0.05.
40
20
0
Extract 1 Extract 2
Extract 3
Extract
1+2+3
Figure 2. The inhibitory effect of three plant extracts and their combination on 20
clindamycin resistant isolates of Staphylococcus aureus. Extract 1: Green tea, Extract
2: Pomegranate rind, Extract 3: Guava leaves.
agreement with the results obtained by Smith-Palmer et

al. (1998) and Chang et al. (2001). Also, Gupta et al.
(2008) stated that cinnamon oil is a potent antimicrobial
with a lowest minimum inhibitory concentration of 1.25%
(v/v) against Bacillus sp., Listeria monocytogenes, E. coli
and Klebsiella sp. Therefore, they suggested cinnamon
extract to be used as food bio-preservative. Moreover,
Mishra et al. (2009) demonstrated that the cinnamon bark
and leaves contain certain constituents exhibiting
potential anti-mould activity against Alternaria solani and
Curvularia lunata.
In addition to our finding on the inhibitory effect of
raspberry on S. aureus, Hipwel and Wilkinson (2003) and
Puupponen-Pimia et al. (2005) have also proved its
antimicrobial activity against a number of pathogenic
bacteria. In Australia, raspberry juice has been used for

the prophylaxis and treatment of gastroenteritis in
livestock, cage birds and humans and was found to
significantly reduce the growth of several species of
bacteria, including Salmonella, Shigella and E. coli (Ryan
et al., 2001).
Comparing the mean diameter of inhibition zone for
each extract, significant differences were apparent
between both green tea and pomegranate with guava
leaves, raspberry and cinnamon bark (Table 3).
Interestingly, the inhibitory effect of the three combined
extracts: green tea, pomegranate rind and guava leaves
on 20 clindamycin resistant isolates was found to be
significantly higher when compared to each extract alone
giving an inhibition zone of 33 mm (Figure 2). The latter
Milyani and Ashy
phenomenon indicates synergistic interaction that could

signify different mode of inhibitory action on the microbial
cell. As a whole, the inhibitory activity of plant extracts is
likely to be due to antimicrobial components present in
plant extracts such as flavonoids, tannins and alkaloids.
In conclusion, this study reveals that multi drug
resistant S. aureus strains might be completely inhibited
with the plant extracts used in the same manner as
antibiotics. Furthermore, combination of two or three
extracts proved to give a powerful antimicrobial activity,
thus giving an extra powerful alternative remedy. Indeed,
plant extracts are preferable because of their non toxic
activity and their environmental friendliness, being simply
available and generally used in our food (Gurib-Fakim,
2006; Reddy et al., 2010; Sangeetha et al., 2010; Opara
and Obani, 2010).
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DOI: 10.5897/AJMR11.1190
Role of CSE1034 in bacterial lipids and

polysaccharides involved in biofilm formation: a
comparison with other drugs
Chaudhary Manu* and Anurag Payasi
Venus Medicine Research Centre, Hill Top Industrial Estate, Bhatoli Kalan, Baddi, H.P. 173205 India.
Accepted 5 December, 2011
In the present investigation minimum inhibitory concentration (MIC) and minimum biofilm eradication
concentration (MBEC) and CSE1034 (Sulbactomax) was compared with piperacilline+tazobactam,
amoxyclave, ceftriaxone, ceftriaxone+sulbactam and cefoperazone+ sulbactam in planktonic and
sessile cells of Acinetobacter baumannii, Enterobacter cloacae and Pseudomonas aeruginosa. MICs
6
were determined by broth microdilution method with a final inoculum size of 10 cfu/ml of plaktonic
cells. MBECs were measured using a calgary biofilm device method to establish a co-relation with
biofilm breaking efficacy of different drugs. The MICs for CSE1034 ranged from 0.5 to 4.0 g/ml and for
other antibacterial drugs ranged from 2 to 32 g/ml. The MBEC for CSE1034 ranged from 8 to 16 g/ml
and for other antibacterial agents, it ranged from 64 to 4096 g/ml. The CSE1034 exhibited
approximately 5 logs reduction in the number of bacteria present in biofilm when compared with other
antibacterial agents. When total lipid and total polysaccharide contents were compared, CSE1034
showed 90 and 84% reduction, respectively. The enhanced efficacy of CSE1034 in the eradication of
biofilm infection is due to presence of EDTA which helps in the destabilizing of the barriers responsible
for the development of biofilm as well as antibiotic resistance. In conclusion, combining of
ceftriaxone+sulbactam with EDTA can significantly reduces the MIC and MBEC values against selected
organisms. Hence, CSE1034 could be one of the best choices to eradicate the biofilm caused by these
organisms.
Key words: Lipopolysaccharide, lipid, log reduction, antibacterial agents.
INTRODUCTION
Biofilms are made of cells and their secreted insoluble
exo-polysaccharide or extracellular polymeric substances
(EPSs). It may account for 50 to 90% of the total organic
carbon of biofilms (Prakash et al., 2003) and can be
considered as the primary matrix. The EPSs are the
major factor influencing the microbial biofilm formation
process (Langille et al., 2000; Liu et al., 2004) and
consisted mainly of polysaccharides (homo and hetero
polysaccharides), proteins, nucleic acids and lipids. EPSs
are also associated with metal ions, divalent cations
*Corresponding author. E- mail: research@venusremedies.com.

Tel: 91-1795-302005. Fax: 91-1795-302133.
(Branda et al., 2005; prakash et al., 2003). They provide

the mechanical stability to biofilms, mediate their
adhesion to surfaces, as a result cells aggregate on the
solid surface (Branda et al., 2005; Langille et al., 2000;
Mattos-Guaral et al., 2000; Miron et al., 2001; Monsan et
al., 2001). EPSs molecule strengthen the interactions
between the microorganisms. The EPS is at least fivefold
higher than extracellular proteins in biofilm (Liu et al.,
2004). Elimination of the exo-polysaccharides layer from
the cells by tri-chloroacetic acid or lysozyme solutions
decreased Bacillus spp. attachment to stainless steel
(Parkar et al., 2001). Biofilms form a gel phase where
microorganisms live inside (Sutherland, 2001, Wingender
et al., 1999).
Different organisms produce differing amounts of EPSs
6526
and the amount of EPSs increase with age of the biofilm.

EPSs also contribute to the antimicrobial resistance
properties of biofilms by impeding the mass transport of
antibiotics through the biofilm, probably by binding
directly to these agents (Donlan, 2008; Hall-Stoodley and
Stoodley, 2004). A number of in vitro experiments have
demonstrated that the bacteria in biofilm are considerably
less susceptible to antibiotic than their planktonic
counterparts (Anwar et al., 1992). In a biofilm, cell
densities are substantially higher than in planktonic
culture (Prigent-Combaret et al., 1999).
The surface of many bacterial species is covered by
polysaccharides. These can be in the form of capsules,
glycoproteins or glycolipids. In Gram-negative bacteria,
the lipopolysaccharide (LPS) also referred to as
endotoxin, covers ca. 40% of the bacterial surface
(Weintraub, 2003). Bacterial biofilms are abundant in the
environment and are involved in several human bacterial
infections (Banin et al., 2006; Darouiche, 2004). The
centers for disease control and prevention estimate and
latter suggest that more than 65% of infections are
caused by bacteria growing in biofilms (Lewis, 2007;
Randall and Garth, 2008).
The treatment of infections caused by bacterial biofilms
are futile with current remedies (Costerton et al., 1999).
Only known possible solution is mechanical removal of
the biofilm from the attached surface, which is costly and
traumatic to the patients. However, biofilm cultures are
much more difficult to eradicate compared to their
planktonic cultures (Brown and Gilbert, 1993). An
alternative approach to controlling biofilm formation could
be to inhibiting adherent process by inhibiting the
production of EPS. The objective of the present work was
to evaluate the in vitro effects of different broad spectrum
antimicrobial agents on planktonic cultures of microbes
and on pre-formed biofilms in co-relation with
polysaccharide and cellular lipid on the sensitivity of
bacteria to antibacterial agents and biofilm breaking
capacity. For this comparative efficacy of broad spectrum
cephalosporin, cephalosporin +beta lactamase inhibitor,
penicillin + beta lactamase inhibitors and a novel
combination of cephalosporin +beta lactamase inhibitor+
EDTA were studied on the biofilm of different microbes.
We first determined the MIC and MBEC of all
antimicrobial agents. We then determined the in vitro
effects of antimicrobial agents on the pre-formed biofilms
of studied microbes and their co-relation with
polysaccharide and cellular lipid contents.
cefoperazone+sulbactam (Ceftop), were procured from Indian

market on behalf of sponsor for the study. All the antibiotics were
reconstituted with the water for injection as stock solutions. Working
solutions were prepared in MHB at a concentration of 0 to 8192
g/ml and from these working solutions serial two fold dilutions
were made in CAMHB in wells of 96-well plate.
Studied organisms
A total of three organisms name A. baumannii, E. cloacae and P.
aeruginosa were used as model microorganisms obtained from
Institute of Microbial technology, Chandigarh, India. Bacterial strains
were maintained as glycerol stocks and stored at -80C. Before
use, bacterial suspensions were spread onto Mueller-Hinton solid
medium (MHSM; Himedia) and incubated at 35C for 24 h. For
each strain, three to five colonies were transferred into 10 ml of
cation adjusted Mueller-Hinton broth (CAMHB) and incubated under
orbital agitation at 150 rpm for 4 h at 35C (Rotary f lask shaker) to
obtain a planktonic culture in exponential growth phase. This
bacterial suspension was used as the inoculum at a concentration
of 106 (cfu/ml).
The CAMHB was also used in the reaction vessels to initiate
biofilm formation. Bacterial counts were done on MHSM. Antibiotic
susceptibility screening and recovery of viable biofilm organisms
were carried out in MHSM.
Minimum inhibitory concentrations (MICs) determination

Susceptibility testing to each drug was performed on planktonic
cultures using the two-fold dilution method according to Clinical and
Laboratory Standards institutes guidelines, 2003. MICs were
performed in 96-well micro plates and results were recorded after
incubation at 37C for 24 h.
Minimum biofilm
determination
eradication
concentrations
(MBECs)
MBECs were performed when pegs contained approximately 107 to

108 bacteria growing as a biofilm described earlier (Ceri et al.,
1999). After development of biofilm on pegs, non-adherent bacteria
on the pegs were washed once by immersion in a microtitre plate
containing 200 l of sterile PBS (pH 7.0). In another microtitre plate,
200 l of each antibiotic of 2-fold dilutions (from 8192 to 2 g/ml)
were prepared. All samples were run in triplicate and one lane
served as a negative control.
The pegged lid with biofilm was then placed onto the microtitre
plate containing antibiotics and incubated, for 24 h at 37C. The lid
with peg was then placed onto a micro plate containing 200 l of
fresh sterile broth medium. The remaining biofilm was removed
from the pegs by ultrasonic disruption for 5 min. This plate was
incubated for 24 h at 37C and the presence of viable bacteria
determined by plate counts or turbidity determined at 650 nm in a
96-well plate reader (Molecular Devices; Fisher Scientific, Nepean,
Ontario). Growth of bacteria in a particular well indicates regrowth
of planktonic bacteria from surviving biofilm. Therefore, the MBEC
value represents the lowest dilution at which bacteria fail to regrow.
Antimicrobial agents
In vitro biofilms formation of all selected strains
Sulbactomax (ceftriaxone : sulbactam:2:1 with 10 mM EDTA) herein

after referred as CSE1034, piperacilline+tazobactam (Zosyn) and
amoxyclave (Augmentin) and ceftriaxone (Rocephin) used in the
study were provided by Sponsor Venus Pharma GmbH, Germany
and combination of ceftriaxone+sulbactam (Oframax forte) and
In vitro biofilm model was developed using Calgary biofilm device

(CBD) (MBEC Biofilm Technologies, Calgary, Alberta) according to
described earlier with slight modifications (Ceri et al., 2009). The
device contains a lid with 96 pegs. The pegs are designed in a way
so that the channels of the bottom component of the reaction vessel
Manu and Payasi
can be fit into the wells of a standard 96-well plate. The bottom of
the vessel serves to channel the flow of medium across the pegs to
create consistent shear force across all pegs, resulting in the
formation of equivalent biofilms at each peg site. Biofilm of each
isolate was developed by taking of 106 cfu/ml bacterial inoculum of
bacterial strain. Biofilm formation was carried out at 37C and 95%
relative humidity on a rocking table such that fluid flowed along the
channels of the CBD, generating the required shear force across all
pegs. Biofilm formation was determined by viable counts on MHA
plates.
Quantification of biofilms
At different time interval, pegs from the lid were removed, placed in
micro centrifuge tubes containing 200 l of MHB and sonicated for
5 min on sonicator. Viable counts were determined on MHSM
plates.
Total polysaccharide estimation

Total polysaccharide content was analyzed by using phenolsulphuric acid method as: to a glass tube, 500 l of above biofilm
suspension and 500 l glacial acetic acid (blank tube) were added.
To each tube 500 l of 5 % phenol (prepared in 0.1 N HCl) and 2.5
ml of concentrated H2SO4 were added and immediately vigorously
mixed on a vortex mixer and left to cool to room temperature. The
optical density at 490 nm was read in a glass cuvette against a
reference tube. The reference curve is composed of dextran
ranging from 2 to 14 g.
Total lipid estimation

Total lipid was estimated in biofilm cells with slight modification
(Izard and limberger, 2003). Briefly, to a stoppered glass tube 100
l of water (reference tube), 100 l of the biofilm suspension was
added. To each tube 1 ml of concentrated H2SO4 were added. The
tubes were incubated in a boiling water bath 20 min and cooled for
5 min in a water bath at room temperature. 2 ml of phosphoric acidvanillin reagent were added to the tubes and incubated at 37C for
15 min. To prepare phosphoric acid-vanillin reagent, 0.120 g of
vanillin was added to 20 ml of water, and the volume adjusted to
100 ml with 85% phosphoric acid. The optical density at 490 nm
was read in a glass cuvette against a reference tube with 100 l
water as sample. The reference curve is composed of triolein
ranging from 10 to 100 g.
Effect of antimicrobial drugs on preformed biofilms

Investigation of the effect of antimicrobial drugs on pre-formed
biofilms was performed on the A. baumannii, E. cloacae and P.
aeruginosa. Assessment of drug activity was performed by three
independent methods: visual observation of growth; crystal violet
staining and enumeration of bacteria before and after treatment of
the biofilm with antibiotics. Biofilms were allowed to form as
mentioned above. The lid with pegs was then removed, rinsed and
placed in contact with various concentration of antimicrobial agents.
The sealed plates were incubated at 37C for 24 h a t 30 rpm in an
orbital shaker. Micro plates were then observed for detection of any
visible growth of bacteria detached from the biofilm through the
cycle of biofilm formation. Staining was done to observe the
persistence of the biofilm. Bacterial count was done after treatment
of different drugs according to Clinical and Laboratory Standards
institutes guidelines, 2003.
6527
RESULTS
MIC and MBEC
The lowest concentration of antibacterial agents
exhibiting no growth of planktonic bacterial cells was
designated as the MIC. Similarly, lowest concentration of
antibacterial agents which exhibits no further growth of
bacterial biofilm was designated as MBEC. MIC and
MBEC observed for the antimicrobial agents against
different microbes are presented in the Table 1. The data
represents the mean resulting from three trials. The
MBEC of CSE1034 for A. baumannii, E. cloacae and P.
aeruginosa biofilms was approximately 16X of MIC,
indicating that 16 times more CSE1034 is required to kill
the biofilms than was required to inhibit planktonic cells.
The MBEC of ceftriaxone, ceftriaxone+sulbactam,
cefoperazone+sulbactam, amoxyclave and piperacilline+
tazobactam was observed in range from 32X to 128X of
MIC indicating that 32 to 128 times more antibacterial
agents were required to kill biofilms than was required to
inhibit planktonic cells. The data in Table 2 shows that
only CSE1034 reduced biofilms of A. baumannii, E.
cloacae and P. aeruginosa and respective log reduction
observed in biofilms are 4.95, 4.59 and 4.25 logs. Other
antibacterial agents reduced the growth of biofilms only
by 0.16 to 1.74 logs (Table 2).
Effect of antimicrobial agents on total lipids and total
polysaccharides of biofilms
The effect of various antimicrobial agents on total lipids
and total polysaccharides of biofilms is depicted in Table
3. The levels of total lipids and total polysaccharides was
found to be decreased after treatment with antibacterial
agents as compared to before treatment of biofilms for all
tested organisms. However maximum decrease in total
lipids and total polysaccharides was noted with CSE1034
compared to other antibacterial agents. The decrease in
cellular lipid resulted in an increase of the susceptibility of
bacteria to antibacterial agents.
DISCUSSION
Bacterial biofilm play an important role in the
development and persistence of various chronic
infectious diseases. Biofilms are composed primarily of
microbial cells and EPSs. It is commonly accepted that
biofilms are more resistant to antibiotics than are
planktonic cells (Costerton et al., 1999; Donlan, 2000;
Costerton and Stewart, 2001; Qian et al., 1999). The
mechanism for enhanced antimicrobial resistance is
believed to involve alterations in gene expression leading
to a phenotype difference between the planktonic and
sessile forms. The sessile forms are more resistant as
they produce exo-polysaccharide, have different growth
6528
Table 1. MIC and MBEC of antimicrobial agents against planktonic and biofilms cells.
CSE1034
Piperacilline+
Tazobactam
0.5
16
16
MBEC
1024
128
256
256
512
MIC
32
64
16
MBEC
64
1024
512
1024
2048
1024
MIC
64
MBEC
32
1024
256
512
4096
128
Name of organisms
MIC
Ceftriaxone+ Cefoperazone+
Amoxyclave Ceftriaxone
Sulbactam
Sulbactam
A. baumannii
E. cloacae
P. aeruginosa
MIC: minimum inhibitory concentration; MBEC: minimum biofilm eradication concentration. All are presented in g/ml.
Table 2. Effect of antibacterial agents on biofilms.
Name of organism
Time (h)
48
48
Name of drugs
CSE1034
Piperacilline+Tazobactam
Biofilm concentration
(cfu/peg) before
treatment
(A)
Biofilm concentration
Log reduction
(cfu/peg) after
treatment
(logA-logB)
(B)
4.710 (7.67)
5.310 (3.72)
4.95
4.210 (7.63)
1.410 (6.14)
1.49
1.16
48
Ceftriaxone+sulbactam
2.610 (7.41)
1.810 (6.25)
48
Cefoperazone+sulbactam
6.310 (7.79)
3.810 (6.57)
1.22
Amoxyclave
3.710 (7.56)
0.76
48
Ceftriaxone
3.110 (7.49)
2.1107 (7.33)
0.16
48
CSE1034
4.710 (7.67)
1.210 (3.08)
4.59
6.810 (5.83)
1.74
3.3107(7.51)
4.5106(6.65)
0.86
5.310 (7.72)
7.210 (6.85)
0.87
1.19
A. baumannii
48
48
48
E. cloacae
48
3.810 (7.57)
6.410 (6.80)
48
Amoxyclave
3.810 (7.58)
2.510 (6.39)
48
Ceftriaxone
4.310 (7.63)
6.810 (6.83)
0.8
48
CSE1034
6.110 (7.78)
3.410 (3.53)
4.25
48
48
5.510 (7.74)
4.810 (7.68)
P. aeruginosa
48
48
48
1.610 (6.2)
2.8106 (6.44)
1.54
a
1.24
5.2107 (7.71)
4.4106 (6.64)
1.07
Amoxyclave
6.110 (7.78)
2.510 (6.39)
1.39
Ceftriaxone
1.18
4.810 (7.68)
3.210 (6.50)
Manu and Payasi
6529
Table 3. Total polysaccharide and lipid content in biofilm cells.
Name
of organisms
After
treatment
% reduction
after
treatment
Before treatment
CSE1034
After
% reduction
% reduction
% reduction
After
After
After
treatment
after
after
after
treatment
treatment
treatment
with
treatment
treatment
treatment
Piperacilline+
Tazobactam
Ceftriaxone+Sulbactam
Cefoperazone+
sulbactam
% reduction
after
treatment
Amoxycillin+
clavulanate
After
treatment
% reduction
after
treatment
Ceftriaxone
Total polysaccharide 156.54
28.45
82.0
94.56
39.59
104.45
33.2
134.45
14.11
142.12
9.21
135.65
13.34
Total lipid
38.45
9.34
75.70
22.12
42.47
26.54
31.67
24.78
35.55
30.11
21.70
21.45
44.21
Total polysaccharide
74.20
12.20
83.55
41.45
44.13
61.45
17.18
63.45
14.48
63.78
14.04
65.56
11.64
Total lipid
52.00
5.14
90.0
29.65
43.00
39.45
24.13
42.34
18.57
37.65
27.60
43.45
16.44
Total polysaccharide 138.33
31.10
77.51
85.63
38.09
123.45
10.75
124.45
10.03
120.56
12.84
128.45
7.14
Total lipid
10.43
75.63
20.35
52.45
32.12
24.95
30.45
28.85
32.45
24.18
34.12
20.28
A. baumannii
E. cloacae
P. aeruginosa
42.80
All results are presented in g/ml. The biofilm concentrationused for the estimation were between 4.5 x107 to 6.5x107 cfu/ml. Total lipid includes neutral lipid, glycolipid and phospholipid.
characteristics and take up nutrients and drugs

differently from their planktonic counterpart (Mah
and O'Toole, 2001; Pratt and and Kolter, 1999).
The MIC is used as a standard for determination
of antimicrobial sensitivities for animal and human
pathogenic bacteria (Costerton et al., 1995;
Prescott and Baggott, 1985). In the present study,
among the all tested antibacterial agents,
CSE1034
requires
sixteen
times
higher
concentration of their MIC to show antimicrobial
activity against bacterial biofilm compared to other
antibacterial drugs, indicating CSE1034 at a
concentration of 16 MIC significantly enhanced
the reduction of all bacterial biofilms from 4 to 5
logs in different microbes studied. The observation
indicates that out of different broad spectrum
antimicrobial agents studied only CSE1034 is
effective against the biofilms of A. baumannii, E.
cloacae and P. aeruginosa. The low MBEC of

CSE1034 may be due to the presence of EDTA in
the CSE1034. We hypothesize that EDTA
2+
2+
removes divalent ions (e.g., Mg and Ca ) by
chelating to cations of EPS and outer membrane
of biofilms and enhancing the porocity and making
the bacteria more susceptible to antibiotic.
A significant percentage reduction of total
polysaccharides up to 84% of in vitro biofilms of
microorganisms was achieved by treatment with
CSE1034 parallel to the previous observation
(Kadurugamuwa et al., 1985) reported that
cephalosporins reduced the production of
capsular polysaccharides. It has also been
demonstrated that chelating agents may affect
polysaccharide production either by modulating
enzyme activity or by affecting the functional
integrity (that is, nutrient uptake or translocation)
of the outer membrane (Domenico et al., 1989).

EDTA also causes loss of lipid, lipo protein, fatty
acids and polysaccharide from the cell wall and
plasma membrane (Lieve, 1965). This happens
probably due to PL enzyme activity supported by
presence of EDTA. Two general catalytic
mechanisms have been observed by Garron and
Cygler (2010) among (Polysaccharide lyases)
PLs: (1) metal-assisted neutralization of the acidic
group of the sugar next to the cleaved bond, with,
rather unusually, arginine or lysine playing the role
of Brnsted base and (2) neutralization of the
acidic group on the sugar by a close approach of
an amino or acidic group forcing its protonation
and Tyr or Tyr-His acting as the Brnsted base
and acid. It has been demonstrated that bismuth
compounds inhibited capsular polysaccharide
expression in Gram- negative bacteria (Domenico
6530
et al., 1991). Total polysaccharide production was

significantly reduced in biofilm exposed to bismuth
dimercaprol compared with untreated control (Huang and
stewart, 1999).
We have observed that treatment of bacterial biofilm
with CSE1034 resulted in reduction in lipid content up to
90%. The reduction in lipid may have enhanced the
susceptibility of microorganisms to antibiotics resulting in
enhanced bactericidal activity. It has also been reported
that depletion in bacterial cell lipid led to increase in
sensitivity of microorganisms to antibiotics (Hugo and
Stretton, 1966). Several reports have suggested that
there may be a relation between antibiotic resistance and
the lipid content of the bacterial cell (Benbough and
Orrison, 1967; Huang and stewart, 1999). Differences in
total lipid or fatty acids have been associated with an
increased antibiotic resistance in various bacteria and it
has been suggested that the lipid may be of importance
in preventing the entrance or binding of the antibiotic to
the cell (Norrington and James, 1970; Vaczi and Farkas,
1961; Bishop and Bermingham, 1973). It has also been
suggested that the differences in total lipid or fatty acid
composition, or both, have been associated with an
increased antibiotic resistance in various bacteria, and it
has been suggested that the lipid composition may be of
importance in preventing the entrance or binding of the
antibiotic to the cell (Bishop and Bermingham, 1973).
From the above results, it is concluded that CSE1034
(Ceftriaxone + Sulbactam+ EDTA), is the only product
which has the efficacy to eradicate the biofilm infection
very efficiently as compared to other antibacterial agents.
Use of EDTA along with antibiotics has been reported in
CSE1034. EDTA inhibits DNA relaxases enzyme which
plays a major role in the spreading of antibiotic resistance
among bacterial species. The enhanced efficacy of
CSE1034 in the eradication of biofilm infection is due to
presence of EDTA which helps in the destabilizing of the
barriers responsible for the development of biofilm as well
as antibiotic resistance. Presence of EDTA significantly
reduces polysaccharide content and lipid content in
broken biofims thereby indicating that there is strong corelation of biofilm induced bacterial resistance and EPS
secretion due to which all other broad spectrum
antibacterials failed to respond.
ACKNOWLEDGEMENT
Authors are thankful to sponsor, Venus Pharma GmbH,
AM Bahnhof 1-3, D-59368, Werne, Germany, for
providing assistance to carry out this study.
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DOI: 10.5897/AJMR12.027
Analytical specificity and sensitivity determination of

16SrRNA gene based diagnostic polymerase chain
reaction (PCR) for molecular detection of Coxiella
burnetii
Mohammad Soleimani1, Keivan Majidzadeh A.1, 2*, Amirhossein Mohseni1 and Mohammad
Khalili3
1
Tasnim Biotechnology Research Center (TBRC), AJA University of Medical Sciences, Tehran, Iran.
Breast Cancer Research Center (BCRC), Iranian Center for Breast Cancer (ICBC), Academic Center for Education,
Culture & Research (ACECR), Tehran, Iran.
3
Department of Pathobiology, School of Veterinary Medicine, Shahid Bahonar University of Kerman, Kerman, Iran.
Accepted 7 February, 2012
Coxiella burnetii, the cause of Q-fever, is a widespread zoonosis. Classical methods for its isolation are
time consuming and risky, requiring biosafety level 3 laboratory. However, this limitation is omitted in
molecular detection. The aim of this study was to evaluate the use of 16SrRNA gene in molecular
detection of C. burnetii. For this purpose, specific primers were designed for this gene and a
polymerase chain reaction (PCR) assay was prepared using these primers. To construct a positive
control plasmid, the PCR product was cloned in pTZ57R/T vector. Sensitivity of the assay was
determined using PCR in 10 fold serial dilutions of the positive control plasmid, and then, the last
concentration with positive band was determined as the limit of detection (LOD) of the assay.
Specificity of the test was assessed with PCR in genomic DNA of the negative control bacteria. The
PCR results showed the presence of a 240 bp band as expected. Sensitivity assessment revealed that
the LOD of the assay was 1 ng. No amplification was exhibited in negative control bacteria after PCR.
These data proved the specificity of the procedure. It is concluded from the results of this study that
16SrRNA gene is an appropriated gene for detection of C. burnetii.
Key words: Coxiella burnetii, Q-fever, 16SrRNA gene, polymerase chain reaction (PCR).
INTRODUCTION
Coxiella burnetii, the causative agent of Q-fever in man
and animals, is an obligate intracellular Gram negative
bacterium which belongs to the family Coxiellaceae and
the genus Coxiella. In a relatively recent classification,
this bacterium has been relocated to the order
Legionellaes, class Gamma Proteobacteria (Angelakis
and Raoult, 2009). Ticks are the primary natural
reservoirs of the bacterium. They circulate the infection
*Corresponding author. E-mail: kmajidzadeh@razi.tums.ac.ir.

Tel: +98 2188337928. Fax: +98 2166490764.
among wild animals which transmit it to domestic

animals, but play no role in its transmission to humans.
Cows, sheep, and goats are the major sources of human
infection. Infected animals spread out a large number of
stable and resistant bacteria through urine, feces, milk,
birthing fluids, and fetal placenta. In addition,
transmission by inhalation of infected aerosols or uptake
of raw milk or other dairy products has been reported
(Parisi et al., 2006). Person to person transmission of the
disease is rare; nonetheless, transmission from a patient
to hospital staff has been documented (Jones et al.,
2006). Recent reports have shown that sexual
transmission is also possible in humans (Kruszewska et
Soleimani et al.
Table 1. List of negative control bacteria genomes used for

specificity determination in this study.
Organism name
Shigella sonnei
Klebsiella pneumoniae
Escherichia coli
Bacillus subtilis
Staphylococcus aureus
Enterococcus faecalis
Salmonella Typhi
Streptococcus penumoniea
Neisseria meningitidis
Escherichia coli O157H7
Escherichia coli EPEC
Yersinia enterocolitica
Yersinia pseudotuberculosis
Strain number
ATCC 9290
ATCC 7881
ATCC 25922
ATCC 6051
ATCC 25923
ATCC 29212
ATCC 700931
ATCC 700669
ATCC13060
ATCC 43895
ATCC 43887
ATCC 35669
ATCC 29833
al., 1996) and animals (Maurin and Raoult, 1999; Parisi

et al., 2006). In vulnerable patients with pre-existing
conditions, such as heart diseases or immune deficiency,
Q-fever may turn into chronic months to years after the
first onset (Stein and Raoult, 1993). Due to high
transmission potential of the bacterium which is caused
by its low infectious dose, very wide host range and high
stability of infectious particles, the bacterium has been
classified as B group of bioterrorist agents (Waag, 2007).
C. burnetii can be isolated by classic cultural methods
(Ormsbee, 1952; Williams et al., 1986); however, this is
limited to the laboratories with level 3 of biological safety,
because, the bacterium is highly infectious, and its
isolation can only be achieved in live host or tissue
(monkey kidney cells, laboratory mice, guinea pig or
embryonate hens eggs yolk sac), and is risky, expensive
and time-consuming (Maurin and Raoult, 1999). Since
clinical diagnosis of the disease is difficult, the detection
is usually done by serologic methods such as microagglutination (Fiset et al., 1969; Kazar et al., 1981;
Nguyen et al., 1996), radioimmunoassay, complement
fixation, indirect hemolysis, indirect immunofluorescence,
western immunoblotting, dot immunoblotting, and
enzyme linked immunosorbent assay (ELISA) (Dller et
al., 1984; Herr et al., 1985; Peter et al., 1985; Kovacova
et al., 1987; Blondeau et al., 1990; Tokarevich et al.,
1990; Waag, 2007). These tests altogether have
specificity, sensitivity, and proper significance for positive
prediction and the cost is low, but they are also involved
with considerable disadvantages. Namely, most of them
cannot be used for the detection of infection before 3 to 4
weeks after onset of the disease (Gillespie and Hawkey,
2006). Furthermore, the symptoms of Q-fever are difficult
to distinguish from those of other diseases, such as
influenza and Malta fever. There are reports indicating
that in some cases, the Q-fever was mistaken with the
mentioned diseases (Khalili et al., 2010, 2011). These
6533
constraints reasons highlight the importance of rapid

detection for the bacterium. Molecular techniques such
as PCR are useful methods for rapid detection of the
bacterium in biological samples. The goal of this study
was to evaluate 16SrRNA gene for molecular detection of
C. burnetii.

Genomes and bacterial species
In this study, the genome of C. burnetii (Nine Mile, ATCC 13032)
was kindly provided by the Department of Pathobiology, Faculty of
Veterinary Medicine, Shahid Bahonar University of Kerman
(Kerman, Iran) and was used for the preparation of positive control.
While some Gram positive and Gram negative bacteria were
obtained from the Pasteur Institute of Iran (Tehran, Iran) and were
used as negative control (Table 1). Genomic DNA extraction from
the bacteria was done according to Zhang et al. (1998). Quality and
quantity assessment of the extracted DNA was carried out by
agarose gel electrophoresis and optical absorption measurement at
wavelength of 260 and 280 nm. Moreover, to be sure of the
absence of PCR inhibitors in extracted genomes, the universal
primers were used for amplification of 16SrRNA gene.
Primer design
In this study, C. burnetii 16SrRNA gene (Accession number:
D89799) was chosen as the target. Primer designing was done
using
primer
BLAST
from
NCBI
(http://www.ncbi.nlm.nih.gov/tools/primerblast/index.cgi?LINK_LOC=BlastHome). Thermodynamic appraisal
was done with Gene Runner Software version 3.05
(Hastings Software Inc.,
Hastings,
New
York)
(http://www.generunner.com). The forward primer sequence was 5
ATATCCTTGGGCGTTGACGTTACCC3 and the reverse one was
5 ATCTACGCATTTCACCGCTACACCG 3 . These primers
amplified a sequence of 240 bp. Primer synthesis was done by
Cinnagen Corporation (Tehran, Iran). PCR was performed at a
volume of 25 l including dNTPS (0.2 mM), MgCl2 (1.5 mM), and 0.4
mM from each of the primers, one unit of the Taq DNA polymerase
enzyme and 50 ng of C. burnetii genomic DNA. The negative
control reaction was set as a reaction similar to the aforementioned,
but with deionized water instead of DNA. Thermal conditions of the
PCR consisted of primary denaturation at 95C for 1 min, 40 cycles
of denaturation at 95C for 1 min, annealing at 55C for 45 s,
amplification at 72C for 1.5 min. Following PCR amplification, 7 l
of the PCR product with 1 l of the loading buffer were
electrophoresed on the 1% agarose gel.
Specificity determination of the PCR

For determination of the specificity, PCR reactions were done
according to the aforementioned conditions in the presence of 50
ng of negative control bacterial genomes (Table 1). Also, a positive
control PCR reaction was done using 50 ng of the C. burnetii
bacterial genomic DNA and a negative control PCR reaction was
prepared using double distilled water.
Cloning and preparation of external positive control
For preparing external positive control, the PCR product was first
purified using PCR purification kit (Bioneer, Korea). Ligation
6534
10 pg) were prepared from the pTZ57RT-16S plasmid. PCR was

performed on all these dilutions. Ultimately, the last dilution of the
pTZ57RT-16S plasmid for which the PCR yielded a detectable
band on the agarose gel was assigned as the test LOD.
RESULTS
PCR and specificity determination
After PCR with 16SrRNA gene specific primers of the C.
burnetii bacterium, the 240 bp band was observed on the
1% agarose gel as expected. The results of the negative
control tube showed no amplification which denotes
correctness of the PCR (Figure 1).
Analysis of the agarose gel related to electrophoresis of
the PCR on negative control bacteria genomes using C.
burnetii specific primers led to no amplification. These
results emphasized that the designed primers for
16SrRNA gene were specific and led to no amplification
in all the bacteria, except in C. burnetii. Amplification of
16SrRNA gene in the negative control bacteria genomic
DNA using universal primers showed a 475 bp band. The
presence of this band implied that the PCR was feasible
on these genomes.
Figure 1. The results of the PCR reaction
on C. burnetii genomic DNA, the desired
fragment with the length of 240 bp, is
amplified. M: 100 bp molecular marker; 1:
240 bp related to 16SrRNA gene
amplification; 2: PCR negative control.
reaction between purified sequence and pTZ57R/T vector was

done using T4 DNA ligase (Fermentas, Lithuania) at 22C for 2 h.
Then, the ligated construct was transferred to the competent
Escherichia coli TOP10F cells . The recombinant cells were
cultured on Luria-Bertani Agar (Merck, Germany) including 5bromo-4chloro-3-indole beta di-galactopyranosid (X-Gal; 40 g/ml),
isopropyl beta-D-thiogalacopyranoside (IPTG; 38.4 g/ml),
ampicillin (100 g/ml), and tetracycline (20 g/ml). The mentioned
culture was incubated at 37C for 16 h. Some of the white colonies
and a blue one (as the negative control) were chosen and were
evaluated with colony PCR method. From one of the confirmed
colonies of the previous step, plasmid extraction was done via
AccuPrep Plasmid Mini Extraction Kit (Bioneer, Korea). For final
confirmation of the insert-receiving plasmid (pTZ57R/T-16S), the
restriction map of the plasmid was appraised. For depiction of the
map, 500 ng of pTZ57RT-16S plasmid was digested with 2 units of
the enzyme HindIII (Fermentas, Lithuania). This enzyme has a
restriction site at Multiple Cloning Site (MCS) vector on base pairs
695 and another site on base pairs 84 of the PCR product. The
result of the enzymatic reaction was assessed with electrophoresis
on 1% gel.
Determination of sensitivity (limit of detection, LOD)

In order to determine the sensitivity, first, the concentration of
pTZ57RT-16S plasmid was measured using optical absorption
measurement method at wavelengths of 260 and 280 nm by means
of Picodrop device (Picodrop Limited, Saffron Walden, UK).
Eventually, serial dilutions (1 g, 100 ng, 10 ng, 1 ng, 100 pg, and
External positive control construction

Examination of the 5 white and one blue colony with
colony PCR method revealed the presence of the target
gene in some of the white colonies; however, the blue
one was devoid of it. After plasmid extraction from one of
the approved white colonies and PCR amplification, a
240 bp band was observed on the 1% agarose gel. The
results of the pTZ57RT-16S plasmid digestion with the
enzyme HindIII showed the presence of 2 bands of
approximately 2926 and 200 bp on the agarose gel.
Sensitivity determination
Sensitivity determination showed that the last dilution of
the pTZ57R/T-16S which produced a visible band on the
agarose gel after PCR was the 1 ng dilution. Therefore,
the LOD of the test was determined as 1 ng (Figure 2).
DISCUSSION
Molecular methods, such as PCR provide useful utilities
for rapid detection of C. burnetii in clinical samples (Stein
and Raoult, 1992). The first method to be used was
specific hybridization of the target DNA with probe for
amplification in clinical samples. This method was so
sensitive and specific, but the limitation was the state of
being at hand only in certain laboratories (Frazier et al.,
1990, 1992; Mallavia et al., 1990). The fact that there
were primers for specific genes of C. burnetii facilitated
Soleimani et al.
Figure 2. The results related to PCR sensitivity. According to the

figure, the least pTZ57R/T-16S plasmid concentration that is
detectable after PCR is 1 ng. M: 1 kp molecular marker; 1: PCR
product related to 1 g of pTZ57R/T-16S plasmid; 2: PCR product
related to 100 ng of pTZ57R/T-16S plasmid; 3: PCR product related
to 10 ng of pTZ57R/T-16S plasmid; 4: PCR product related to
pTZ57R/T-16S plasmid with 1 ng concentration; 5: PCR product
related to 100 pg of pTZ57R/T-16S plasmid; 6: PCR product related
to 10 pg of pTZ57R/T-16S plasmid; 7: PCR negative control.
easy detection. Several genes have been used for the

detection. Ibrahim et al. (1997) used 23SrRNA for the
detection and showed that the test was endowed with
high sensitivity and specificity. They showed that it was a
very useful method for C. burnetii detection in clinical
samples (Ibrahim et al., 1997). Stein and their co-workers
(1997) used 16S-23SrRNA internal transcribed spacer;
Stein and Raoult (1992) used superoxide dismutase
gene, while Williams et al. (1993) used QpRS plasmid
and cbbE and htpAB gene for C. burnetii detection (Stein
et al., 1997; Stein and Raoult, 1993; Willems et al.,
1993). In this study, 16SrRNA gene of C. burnetii was
targeted and the specific primers were designed for this
gene. For evaluation of specificity of primers, the primer
BLAST (NCBI) was used. These primers were able to
differentiate Rickettsia-like Coxiella bacteria from true
Rickettsia and do not show any amplification in beta
proteobacteria.
One of the main parts of a diagnostic test is the positive
control being prepared from several ways. Kirkan et al.
(2008) used the genomic DNA as the positive control for
molecular evaluation of C. burnetii infection in herds of
cattle (Kirkan et al., 2008). Studies have demonstrated
that the genomic DNA is very sensitive to temperature
6535
changes due to its size and can be easily damaged. If the

gene is cloned into a plasmid, it will become much more
resistant and stable to the environmental conditions
owing to the smaller size of the plasmid, so it would be a
good candidate for making the control. For preparing a
standard positive control in the present study, the
16SrRNA PCR product was cloned in pTZ57R/T vector.
In the current research, after standard PCR techniques
set up, sensitivity and specificity of the method were
assessed. Ibrahim et al. (1997) prepared serial dilutions
in phosphate buffer saline (PBS) from C. burnetii. After
DNA extraction, the extracted DNA was used as the
templates for determining the sensitivity. They were
3
calculated in 10 bacteria, the LOD of their test (Ibrahim
et al., 1997). One of the barriers to the LOD
determination with this method is the need for live
bacteria which demands level three laboratory safety.
However, if the target gene containing plasmid is used for
the LOD determination, the problem is tackled. The
sensitivity test results of this reaction showed that the last
dilution from the positive control plasmid giving rise to an
apparent band was 1 ng.
The result of specificity determination showed that the
established PCR had high specificity for 16SrRNA gene
of C. burnetii. In this experiment, the genomes of a wide
range of Gram negative and Gram positive bacteria were
used for specificity determination. A posed question
about a molecular techniques specificity determination
(when negative control bacterial genomes are used) is
whether the absence of amplification in the presence of
the negative control bacterial genomes is a consequence
of the method specificity or the presence of PCR
inhibitors in these samples leading to false negative
results.
To reduce concerns prior to utilization of the genomes
for specificity determination, their feasibility for
undergoing PCR was confirmed by the amplification of
the 16SrRNA gene of these agents using corresponding
universal primers. Therefore, it is possible to confidently
trust the method.
Conclusion
The results of this study concluded that 16SrRNA gene of
C. burnetii bacterium is a specific gene for molecular
detection of this bacterium using PCR and this technique
is a reliable test to be clinically used for bacterial
diagnosis of the Q-fever agent.
ACKNOWLEDGEMENTS
The authors would like to acknowledge and appreciate
the Faculty of Medicine, AJA University of Medical
Sciences and Tasnim Biotechnology Research Center
(TBRC), for their support and contribution to this study.
6536
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Effect of Bacillus cereus Br on bacterial community and

gossypol content during fermentation in cottonseed
meal
Xin Wang, Jiang-wu Tang*, Xiao-hong Yao, Yi-fei Wu, Hong Sun and Yao-xing Xu
Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China.
To define new methods for the detoxification of free gossypol (FG) within cotton seed meal (CSM), FG
during CSM fermentation with Bacillus. cereus Br were measured. Four conditions were studied: CSM
inoculated with Br (Br + CSM), CSM without any inocula (CSM), sterilized CSM inoculated with Br (Br +
sCSM), and sterilized CSM without any inocula (sCSM). Samples were taken at various times during the
fermentation and the contents of free gossypol (FG), crude protein (CP), and amino acids were
measured. The polymerase chain reaction and denaturing gradient gel electrophoresis (PCR-DGGE)
method was used to analyze the changes in bacterial community structure during the fermentation.
After the Bacillus. cereus Br was inoculated into CSM, the FG concentrations decreased to the
minimum level of detection at 12 h, while CP and amino acids concentrations reached maxima at 18 h.
The initial content of FG and CP decreased significantly when the CSM was pre-sterilized, and
decreased to minima after 12 h fermentation. The changes in bacterial community structure mirrored
the changes in FG and CP. B. cereus Br was dominant in the CSM + Br group during the first 12 h of
fermentation, after which Br growth was inhibited by native bacteria in CSM. In contrast, B. cereus Br
was always dominant during fermentation of autoclaved CSM. These results suggest that the native
microbes of CSM significantly influenced the detoxification rate, and inhibited B. cereus Br growth
during fermentation. But pre-sterilization of CSM significantly affects the FG content and quality of CSM.
The B. cereus Br was capable of detoxifying FG from CSM, and the optimal time of fermentation was 18
h.
Key words: Bacterial community, free gossypol, detoxification, fermentation, cottonseed meal.
INTRODUCTION
Cottonseed meal (CSM) is a source of high-quality
protein. The use of this nutrient rich resource as animal
feed, however, is hampered by the presence of free
gossypol (FG), a toxic polyphenolic pigment produced in
the seeds. Diets containing FG can negatively effect
animal growth, digestive health, and reproduction (Santos
et al., 2003; Carruthers et al., 2007; Cai et al., 2011; ElSaidy and Saad, 2011; zdoan et al., 2012; Zheng et al.,
2012). Methods have been developed to detoxify FG
from cottonseed, such as solvent extraction (Cherry and
*Corresponding author. E-mail: tangjw@mail.zaas.ac.cn. Tel:

+86-571-86404323. Fax: +86-571-86404323.
Gray, 1981; Rahma and Rao, 1984; Qian et al., 2010;

Saxena et al., 2012), chemical treatment with ferrous
sulfate (Barraza et al., 1991; Tabatabai et al., 2002), and
calcium hydroxide (Nagalakshmi et al., 2002, 2003). All of
these methods are effective in detoxification, but
contamination from residual solvents may be potentially
harmful to animals. Ferrous sulfate can cause feed to
turn black, whereas calcium hydroxide often reduces the
biological activity of vitamins and lowers detoxification
efficiency (Zhang et al., 2007).
Microbial fermentation might be the best detoxification
method, because microorganisms not only reduce the FG,
but also improve the feed value of CSM by enhancing the
content of protein, free amino acids, and secreted
coenzymes such as cellulolytic enzyme, amylase,
6538
protease, and lipolytic enzyme, in addition to some

vitamins and other unknown active substances (Wu and
Chen, 1989; Shi et al., 1998). Some microorganisms,
such as Geotrichum candidum, Candida tropicalis,
Torulopsis candida, Aspergillus flavus, Aspergillus niger
and Aspergillus oryzae have been shown to degrade FG,
and so may reduce FG during solid substrate
fermentation (SSF) (Zhang et al., 2007; Khalaf and
Meleigy, 2008; Sun et al., 2008; Lim and Lee, 2011; Yang
et al., 2011, 2012). In almost all previous studies, the
CSM was pre-sterilized by autoclaving before being used
as the basal substrate in SSF. High temperature
treatment alone would significantly reduce the FG content,
so the rate of detoxification attributable to the
microorganisms is unclear. Furthermore, pre-sterilization
of CSM and maintenance of a sterile environment would
be expensive for mass production. The native microbes in
unsterilized CSM would also undoubtedly influence the
microbial detoxification in SSF. However, there are few
studies examining how this microbial community change
affects the detoxification of CSM during the fermentation
process. In the current study, B. cereus Br was used in
CSM fermentation. We analyzed the composition of the
bacterial communities during fermentation of either
untreated or pre-sterilized CSM, and measured the levels
of crude protein, free amino acids, and FG at specific
times during fermentation.

Basal substrate treatment and microorganisms
The CSM was obtained from Tai-kun Corp. (Xinjiang, China) and
stored at room temperature (25 to 30C) until used. About 2.5 kg
CSM was packaged with brown paper and autoclaved at 121C for
30 min. The strain B. cereus Br was used in this study. It was
isolated from the soil of cotton fields and is capable of utilizing FG
as a carbon source. The inocula were prepared by inoculating B.
cereus Br into a 500 ml Erlenmeyer flask containing 100 ml of
sterile LB medium. The Erlenmeyer flask was incubated on a rotary
shaker at 200 rpm for 16 h at 37C.
Solid substrate fermentation with B. cereus Br and sampling

The CSM was moistened with distilled water at a ratio of 1:0.8 (w/w).
There were 4 treatment groups in the experiment: CSM inoculated
with B. cereus Br (Br + CSM), CSM without inocula (CSM),
sterilized CSM inoculated with Br (Br + sCSM), and sterilized CSM
without any inocula (sCSM). After blending, the mixtures were put
into a sterilized plastic drum, and then incubated at 30C for 48 h
under 85% relative humidity. All treatments were repeated in
triplicate. About 30 g samples were taken at 0, 6, 12, 18, 24 and 48
h during fermentation. All samples were put into sterile bags and
stored at -70C for further treatment and analysis.
Sample processing
Fermented substrates were thawed, and about 1 g of each sample
was placed into sterile 10 ml centrifuge tubes for total DNA
extraction. The rest of the fermented substrates were dried in an
oven at 55C for 48 h and subsequently processed into flour for

other tests.
Total DNA extraction from fermented CSM

Before extraction of DNA, about 5 ml decolor buffer (100 mM Tris-Cl,
50 mM EDTA, 100 mM NaCl, 0.01 mM PVP, 0.02 mM Na2CO3, pH
10) were added into the centrifuge tube containing the fermented
CSM. After vortexing for 2 min, the tubes were placed in a 37C
water bath for 10 min, vortexed again for 2 min, and then
centrifuged at 9000 g for 10 min. The supernatant was gently
removed using a pipette tip and discarded. These steps were
repeated 3 or 4 times until the supernatant was clear. Then 2 ml
DNA extraction buffer (2% w/v) CTAB, 1 mM NaCl, 50 mM EDTA,
50 mM Tris-Cl, pH 8.0) containing lysozyme (4 mg/L) was added,
and the tubes incubated at 37C for 1 h. Next, 2 mg/L proteinase K
(Merck Corp.) and 1% sodium dodecyl sulfate were added and the
mixture incubated at 55C for 2 h. Release and precipitation of
genomic DNA was done using the method of Yeates et al. (1998).
The total DNA extracted from the fermented CSM was purified with
a Spin Column DNA Gel Extraction Kit (Sangon Corp.) using the
manufacturers recommendations. Isolates were stored at -20C
until analyzed.
PCR and denaturing gradient gel electrophoresis (DGGE) of

16S rRNA gene fragments
The DNA extracts were used as templates for PCR amplification of
the bacterial 16S rDNA gene fragment with primers 341F-GC (the
complement of EUB341 with a 40-bp GC clamp 5CGCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGGG
GCCTACG GGAGGCAGCAG-3) and 517R (the complement of
EUB517 5-ATTACCGCGGCTGCTGG-3) (Muyzer et al., 1993). A
touchdown PCR (Don et al., 1991) was used according to the
protocol of Riemann et al. (1999).
The PCR products were purified with a Spin Column DNA Gel
Extraction Kit (Sangon Corp.) and the concentrations of PCR
products determined by NanoDrop 2000 (Thermo Corp.). Equal
amounts of PCR products were loaded on 8% polyacrylamide gels
containing a denaturant gradient of 40 to 60% from top to bottom,
where 100% is defined as 7 M urea and 40% (v/v) formamide.
Denaturing gradient gel electrophoresis was performed with a Dcode universal mutation detection system (Bio-Rad Corp.) at 60C
for 6 h at 150 V in 1TAE buffer. The gels were stained for 20 min in
1TAE containing 0.5 mg/L ethidium bromide and then washed for
15 min in distilled water. Gel band profiles were inspected under UV
illumination.
Dominant bands were excised from DGGE gels, and DNA was
eluted overnight in 50 l TE buffer at 4C. Then, 16s rDNA gene
fragments were re-amplified from excised bands, and analyzed by a
second DGGE in order to ensure that the relevant bands were
resolved. The PCR products were purified using a Spin Column
DNA Gel Extraction Kit (Sangon Corp.) and ligated into the PMD18T vector (Takara). The ligation products were transformed into
Escherichia coli DH5 competent cells and 3 clones with a 200 bp
insert from each band was sent to Sangon Corp. for sequencing.
The sequences were then aligned with closely related 16s rDNA
sequences in GenBank, and a phylogenetic tree was constructed
using MEGA 4.1 software.
The DGGE profiles were analyzed using Quantity One 1-D
software (Bio-Rad Corp.) to determine the position and intensities of
individual bands. The DGGE profiles of bacterial communities were
analyzed by subtracting the background fluorescence from each
lane, and then band intensities were normalized to the total intensity
of all bands in a given lane to give relative band intensities.
Different lanes and their relevant band intensities constituted a
Wang et al.
6539
cereus Br, but the lowest FG concentration appeared at

18 h (215.3 mg/kg) for a maximum detoxification of
52.9%. After autoclave sterilization at 121C for 30 min,
the FG content of the CSM was only 113.7 mg/kg
(detoxification of 75.3%). The lowest FG content of the
inoculated autoclaved CSM (Br + sCMS) appeared at 18
h (34.3 mg/kg DM, 71% detoxification).
Variation of crude protein and amino

concentrations during the CSM fermentation
Figure 1. The FG concentration during the CSM fermentation under

the four conditions. Legend: Br+CSM: treatment of CSM inoculated
with B. cereus Br. CSM: treatment of CSM without inocula. Br +
sCSM: treatment of sterilized CSM inoculated with B. cereus Br.
sCSM: treatment of sterilized CSM without any inocula. Each point
represents meanstandard deviation from three independent
assays. There are not significantly different (P>0.05) between the
results of repeated experiments.
matrix, and Matlab was used to conduct principal component

analysis (PCA) of the change of bacterial species during the
fermentation of CSM.
Related assays
The dry matter (DM) was used for related assays. The FG content
was determined as described by Hron et al. (1996). Crude protein
(CP) assays used the Kjeldahl method (AOAC, 1999). Free amino
acid assays was based on the AOAC method (1999, method
number 994.12) using an Hitachi Model L-8800 automatic amino
acids analyzer (Hitachi Corp.).
The data from each experiment were pooled and then evaluated by
one-way ANOVA (SPSS 13.0 for Windows) and least significant
difference (LSD).
acids
The amounts of crude protein during the CSM

fermentation are shown in Figure 2. The CP content of
the fermented CSM increased from the 6th hour to the
18th hour, after which it began to decrease. The CP
content of the CSM was enhanced by 3.3%, whilst the CP
content of uninoculated sCSM was enhanced by 3.5%.
The increased velocity of CP accumulation in the
fermented CSM inoculated with B. cereus was only
slightly higher than the un-inoculated group during 0 to 12
h. Autoclaving lowered the CP content of the CSM (P <
0.05) in the inoculated CSM at all sample time points.
The consumption of carbohydrate by microbes during the
fermentation, or the release of volatile materials during
treatment, could cause the relative increase in CP
content in the unsterilized fermented CSM. These results
indicate that pre-sterilization before fermentation with Br
leads to a significant loss of nitrogenous materials and
poorer initial quality CSM.
Total amino acid reached a maximum at the 18th hour
in the Br + CSM condition, and then began to decrease
with continued fermentation (Table 1). The essential
amino acids had the same tendency; levels of lysine
increased by 16.4 and 15.3% by the 18th hour in the
CSM and Br + CSM groups respectively. Autoclaving
lowered the total amino acids content of the CSM (as well
as the CP content), and the total amino acid content
continued to decrease, reaching a minimum at 18 h.
However, the total amino acid content of the autoclaved
CSM without any inocula remained statistically
unchanged (P > 0.05).
Change in bacterial community structure during the

CSM fermentation
RESULTS
Variation of FG concentrations during the CSM
fermentation
The FG concentration rapidly decreased from 0 h (463.3
mg/kg) to 12 h (189.7 mg/kg) during the fermentation
after B. cereus Br was inoculated into CSM (Figure 1).
The maximum detoxification was 59.1%. Then the FG
concentration began to increase to 274 mg/kg by the 48th
hour. The FG concentration in CSM without any inocula
approximately mirrored that of CSM inoculated with B.
The PCR-DGGE method was used to analyze the

variation in bacterial community structure, and the results
showed that the bacterial populations changed
significantly in the CSM and Br + CSM groups during
fermentation (Figure 3). In the DGGE profile, the intensity
of bands 5 and 8 increased from 0 to 12 h, and
decreased after 18 h. This suggested that the strains
indicated by bands 5 and 8 were dominant in the
bacterial community. However, the bacteria species
represented by band 5 was always dominant during the
fermentation of sterile CSM with Br (Br + sCSM group,
6540
Figure 2. The crude protein concentrations during the CSM fermentation. Legend: Br +
CSM: treatment of CSM inoculated with B. cereus Br. CSM: treatment of CSM without
inocula. Br + sCSM: treatment of sterilized CSM inoculated with B. cereus Br. sCSM:
treatment of sterilized CSM without any inocula. Each point represents mean
standard deviation from three independent assays. There are not significantly different
(P>0.05) between the results of repeated experiments.
Table 1. The amino acid concentrations (g kg-1 DM) during the CSM fermentationa.
Amino
acids
ASP
THR
SER
GLU
GLY
ALA
CYS
VAL
MET
ILE
LEU
TYR
PHE
LYS
NH3
HIS
ARG
PRO
Total
a
0h
46.8
16.5
22.6
121.5
20.7
20
8.3
21.8
6.0
15.8
31.2
12.9
27.1
18.9
11.0
13.1
59.6
14.0
487.8
Br + CSM
18 h
48.5
17.9
23.1
123.9
21.8
23.6
8.8
23.9
6.3
17.4
33.4
14.0
29.0
22.0
13.5
13.4
54.3
14.9
509.6
24 h
48.4
17.2
23.4
124.9
21.3
20.5
9.0
22.4
5.8
16.0
31.7
13.7
27.6
19.9
11.5
13.4
62.3
14.5
503.1
0h
47.1
16.8
22.8
121.7
20.8
20
9.2
21.9
6.3
15.7
31
13.5
27
18.9
11.1
13.1
60.3
13.9
491.2
CSM
18 h
48.7
17.9
23.4
123.3
21.7
22.5
9.0
24.2
6.2
17.4
33.7
13.9
28.9
21.8
12.6
13.5
53.5
15.1
507.5
24 h
48.1
16.9
23.2
123.5
21.1
20.3
9.3
22.2
6.6
15.8
31.2
13.7
27.4
19.5
11.3
13.3
61.9
14.6
500.1
0h
44.9
16.5
22.0
117.8
20.2
19.8
8.1
21.7
6.0
15.7
30.7
13.1
26.5
19.0
12.1
12.6
54.2
13.7
474.6
Br + sCSM
18 h
44.0
16.5
20.7
108.2
19.4
20.5
7.6
22.2
5.8
16.4
30.9
13.0
25.6
19.2
13.4
12.3
47.6
13.4
456.7
24 h
44.2
16.2
21.8
116.7
19.6
19.5
8.1
21.3
5.4
15.2
30.5
12.7
26.2
18.9
11.5
12.4
54.4
13.8
468.2
0h
45.9
16.0
21.8
116.5
20.2
20.1
8.8
21.9
5.8
15.6
31.1
13.2
26.9
19.3
11.5
12.7
58.5
14.3
480.3
sCSM
18 h
46.7
16.6
20.4
117.8
20.5
20.8
8.6
22.7
5.9
16.1
31.7
14.4
27.4
21.4
12.5
12.6
50.4
15.3
481.7
24 h
46.5
16.2
22.3
118.7
20.4
20.2
8.3
22
6.0
15.8
31.7
13.3
27.4
19.4
11.8
12.9
59.7
14.5
487.1
Values are means of three replicates of each samples got on different time per treatments.
Figure 3).
Fourteen main bands were recovered, cloned and
sequenced. The sequences were aligned with closely
related 16S rDNA sequences from GenBank, and a
phylogenetic tree was constructed (Figure 4). The

bacteria in the fermented CSM were mainly
Acinetobacter sp., Enterobacter sp., and Bacillus sp.
Band 9 was the fragment of eukaryotic chloroplast 16S
Wang et al.
Figure 3. DGGE profile of 16S rDNA gene fragments of bacteria during the CSM fermentation. The group
of Br + CSM, CSM and Br + sCSM was the treatment of CSM inoculated with B. cereus Br, CSM without
inocula and sterilized CSM inoculated with B. cereus Br, respectively. The numbers on the top of each
lane indicate the time of sampling. All marked bands were analyzed by sequence analysis.
Figure 4. Phylogenetic tree of 16S rRNA gene sequences obtained by PCR-DGGE.

Distances were calculated using the maximum-likelihood method, and the tree was
constructed using neighbor-jonining method.
6541
6542
Figure 5. Principal component analysis (PCA) of the DGGE profiles shown in Figure 3. Only
the first principal component (PC1) and the second principal component (PC2) are shown.
The plots (o, *, and squares) indicate the bacterial community structure of samples from
groups Br + CSM, CSM and Br + sCSM respectively. The numbers indicate the time of
sampling.
rRNA gene in the fermented CSM. Based on the

evidence from sequence alignment, the B. cereus Br 16S
rRNA gene product was band 5. The bacteria band 8 was
most similar to Bacillus amyloliquefaciens, the native
autochthonal bacteria in CSM.
After analysis and normalization using Quantity One 1D software, PCA of the DGGE profile was conducted by
Matlab. Two principal components (PCs) explained 79%
of the total variability in bacterial community structure.
Analysis showed that the bacterial communities gradually
and continuously changed during CSM fermentation.
There were four stages of change in the bacterial
structure, rapid changes in stage 1 (0 to 6 h), stage 2 (6
to 12 h), and stage 3 (12 to 18 h). Structures remained
stable after 18 h (stage 4). There were three stages
during fermentation of autoclaved CSM inoculated with B.
cereus Br, a rapidly changing stage 1 (0 to 12 h), a
stationary stage 2 (12 to 24 h), and a moderately
changing stage 3 (24 to 48 h) (Figure 5). The change in
bacterial community structure mirrored the changes in FG
and CP, suggesting that the bacteria were responsible for
the changes in FG and CP during fermentation.
DISCUSSION
Microbial fermentation might be the most efficient method
for detoxification of FG from CSM. In most studies of
microbial fermentation of CSM, to prevent the effect of
native bacteria, the CSM was pre-autoclaved. Zhang et al.

(2006, 2007) reported a detoxification rate of 94.6% when
autoclaved CSM was fermented by C. tropicalis. The
authors proposed that heat treatment facilitated microbial
fermentation and detoxification of FG in CSM. However,
Jia et al. (2009) concluded that detoxification was mainly
a result of CSM sterilization, and that the actual
detoxification rate for Candida sp. was 5.96 to 19.64%.
The current work demonstrated a detoxification rate of
75.3% from autoclaved CSM. The reason that the FG
concentration was significantly reduced by presterilization of CSM might be the formation of stable
bonds between FG and proteins or amino acids in CSM
(Nagalakshmi et al., 2002). Furthermore, the fermentation
conditions used in the studies mentioned above might be
unsuitable for use in mass production because of the
huge additional cost. Microbes native to CSM obviously
influence the fermentation and detoxification, but little
work had been done on how these inherent microbes
affect the process of fermentation for detoxification.
The PCR-DGGE method is a useful molecular tool
for analysis of bacterial diversity and community structure
(Muyzer and Smalla, 1998), and has been used for
bacterial community variation analysis in SSF (Guan et
al., 2012; Lv et al., 2012). In the present study, the DGGE
profile exhibited distinct changes in the bacterial
community during fermentation. B. cereus Br was the
dominant microbe in the fermentation of the autoclaved
CSM, and it was dominant before 12 h in the fermentation
Wang et al.
of un-treated CSM, which suggested B. cereus Br played

key role during the fermentation. However, the native
bacteria of CSM began to grow rapidly after 12 h so that
B. cereus Br was no longer dominant, suggesting that the
growth of B. cereus Br was inhibited by native bacteria.
The bacteria band 8 came from native CSM bacteria and
belonged to Bacillus sp. The species played a similar role
to B. cereus Br during fermentation. The PCA results
showed the variations in B. cereus Br and the bacteria
represented by band 8 were the principal factors that
caused the changes in the constituents measured.
Indeed, the different stages in changing bacterial
community structure mirrored changes in FG and CP,
which suggested that B. cereus Br and the inherent
bacteria were responsible for the fermentation and
detoxification.
It has been reported that some microorganisms are
capable of FG detoxification from CSM, including C.
tropicalis, T. candida, A. flavus and A. niger (Weng and
Sun, 2006; Zhang et al., 2007; Khalaf and Meleigy, 2008;
Lim and Lee, 2011; Yang et al., 2011, 2012), but little
work has been done with bacteria. The mechanism of FG
detoxification by microbial fermentation remains unclear.
It might be caused by transformation of FG to BG (Reiser
and Fu, 1962; Kornegay et al., 1972; Jia et al., 2009), or
FG may be degraded by the microorganism (Weng and
Sun, 2006). In previous studies, it was found that B.
cereus Br could grow well with gossypol serving as the
only carbon source. The FG concentration in the
sterilized CSM decreased after the B. cereus Br was
inoculated, and the detoxification rate in the CSM
inoculated with B. cereus Br was significant higher than
that in the CSM without any inoculates (P < 0.05). These
results provided evidence that B. cereus Br has the
capability to degrade FG in CSM.
Microbial fermentation of CSM provided an economic
and efficient method not only to reduce the FG levels to
an acceptable range, but also to improve the feed value.
Undoubtedly, it was very important to reduce the cost
while this method was used in mass production. In most
of the previous studies, the CSM was pre-autoclaved
before fungi were inoculated for fermentation, and the
fermentation times were all more than 36 h (Weng and
Sun, 2006; Zhang et al., 2007; Khalaf and Meleigy, 2008;
Lim and Lee, 2011). Those methods would obviously
increase energy and time costs. In the current study, B.
cereus Br was used to ferment the unsterilized CSM, the
FG levels were decreasing in 18 h, and the content of CP
and amino acids were increasing. It suggested that the
strain B. cereus Br had great potential application in
mass production of microbial fermentation of CSM.
The interactions between inoculated B. cereus Br and
the native bacteria would cause complex change in
bacterial community during the fermentation in CSM, and
which would affect the detoxification (Figure 1). The CSM
used in this study was obtained from Tai-kun Corp.
(Xinjiang, China);
its native bacteria significantly
6543
influenced detoxification, and inhibited the growth of the

inoculated stain after 12 to 18 h of fermentation (Figure 3).
It could not draw hasty conclusions that the same results
would get while other CSM were used as substrates for
fermentation. However, the present study provided a
direction for the microbial fermentation of CSM. And
further study of changes in bacterial community structure,
and the effect of bacteria community structure on FG
detoxification in unsterilized CSM during fermentation,
might benefit the application of this method for mass
production.
ACKNOWLEDGEMENT
This research was supported by the major projects for
science and technology of Zhejiang province [No.
2006C12097-(1), 2011C12010], and in part by a grant for
innovative research from Zhejiang Academy of
Agricultural Science.
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DOI: 10.5897/AJMR12.476
Identification and characterization of a fungal strain

with lignin and cellulose hydrolysis activities
Ran Jin#, Hongdong Liao#, Xuanming Liu, Mang Zheng, Xianqiu Xiong, Xinwu Liu, Liyong
Zhang and Yonghua Zhu*
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Biology, Hunan University, Changsha, China.
A lignin and cellulose-degrading fungal strain Bio-1 was isolated from the soil of Yuelu Mountain in
China. It was identified as a member of the genus Cladosporium by 18s rDNA, ITS sequences analysis
and morphological characters. Bio-1 could hydrolyze at least 35% of the alkaline lignin in 4 days when
incubated in screening liquid medium. Both laccase and cellulase activities could be detected in the
fermentation liquid and cell homogenate. High extracellular and intracellular laccase activities of 3800
and 5352 U/L were obtained with copper induction, respectively. The extracellular cellulase production
was reached (51 U/Ml). Based on these characteristics, Bio-1 could be well suited as a commercial
fungus applied in degradation of lignocellulose biomass for environment protection.
Key words: Cladosporium, identification, laccase, cellulase, CuSO4.
INTRODUCTION
Paper and pulp, textiles and petrochemical industries
discharge highly colored effluents, which is not only
aesthetically unacceptable but also leads to serious
environment problems in soil, ground or surface water
ecosystem. The color of effluent is mainly due to the
presence of lignocellulose and its derivates. Application of
biological methods based on fungi biodegradation is an
environmentally friendly process to reduce pollution
caused
by
lignocelluloses-containing
material
(Raghukumar et al., 2008).
As an efficient and typical lignin-degrading fungi
(Rajeev et al., 2005), the white-rot basidiomycete attacks
lignin mainly by secreting lignin-degrading isoenzymes,
such as laccase, lignin peroxidase (LiP) and manganese
peroxidase (MnP) (Rajeev et al., 2005). Similarly, four
laccase isoenzymes were synthesized by a Pleurotus
ostreatus strain V-184 (Rajeev et al., 2005). Daedalea
quercina was reported to produce the laccase and Mndependent peroxidase (Rajeev et al., 2005). Laccases
*Corresponding author. E-mail: zyh20@hotmail.com. Tel: +86

731 88821565. Fax: +86 731 88822606.
#
Authors contributed equally to this work.
are the most preferred enzyme and have attracted

increasing scientic attention in recent years due to their
ability to degrade a wide range of lignin related
compounds, which make them very useful for their
application in diverse industrial sectors, including
decolourizing and degrading industrial dyes (RodriguezCouto and Toca-Herrera, 2006).
The cellulases have received tremendous attention
from researchers over the last few decades due to their
importance in several lignocellulose-based agricultural
and waste treatment processes and could be widely used
to improve environmental quality and a sustainable
energy resource supply (Zhang et al., 2006). By far,
Trichoderma
reesei,
Humicola,
Penicillium
and
Aspergillus are generally studied fungi that could convert
native or derived cellulose to glucose (Jahangeer et al.,
2005). Interestingly, white-rot fungi are also a common
fungi that could secrete cellulases such as Phlebia
gigantean (Niranjane et al., 2007) and Ischnoderma
resinosum (John, 1986), which also have the capacity to
remove lignin.
Complex compounds such as cellulose, lignin and
lignocelluloses are main components of colored effluent.
As a result, microorganisms which could degrade both
lignin and cellulose appear to be appropriate in the
biological
treatment
of effluents (Ezeronye and
6546
Table 1. Sequences of the primers.
Primer
18sf
18sr
ITS4
ITS5
Sequence
5-AACCTGGTTGATCCTGCCAGT-3
5-CGACGGGCGGTGTGTAC-3
5-TCCTCCGCTTATTGATATGC-3
5-GGAAGTAAAAGTAACAAGG-3
days and then spores were collected. Conidial suspension at the

concentration of 109 per milliliter was incubated at 28C with
screening liquid medium mentioned above with 7 g/L glucose in it to
induce growth with shaking. To detect the lignin degradation rate,
absorbance measurements at 280 nm were monitored every 2 days
until the tenth day (Lundquist et al., 1977). According to the positive
relationship between the concentration and absorbance, we could
calculate the concentration of the alkaline lignin.
Analysis of the enzyme activity
Okerentugba, 1999) and play an important role in the

cycle of carbon (Sanche, 2009). Several fungal species
were reported on the capacity to reduce chemical oxygen
demand (COD) because of the capacity to degrade highmolecular weight polymers (Prasad and Gupta, 1997).
However, in addition to white-rot fungi (Baldrian, 2004;
Leonowicz et al., 1999), only a few fungi are reported to
secrete both lignin and cellulose degrading enzymes
(Sanche, 2009). In this study, one fungal strain Bio-1
isolated from the soil was found to produce both laccase
and cellulase. CuSO4 could highly improve its laccase
activity.
Considering
the
importance
of
the
lignocelluloses-degrading
process,
Bio-1
has
considerable application potential in the lignocellulose
treating industry.

Screening of the lignocellulose-degrading strain
The lignocellulose-degrading fungus was screened using the
screening medium at 28C for 7 days from the strains which were
isolated from Yuelu mountain soil in Hunan Province, China and
preserved in our laboratory. The screening medium consists of
(g/L): alkaline lignin 2, (NH)2SO4 2, K2HPO4 1, MgSO4 0.2, CaCl2
0.1, MnSO4 0.02, KH2PO4 1, agar 15, and the pH of the medium
was adjusted to 7.0. Then, the mycelia of survivor strains were
incubated on the MEA medium (g/L): malt extract powder 20,
glucose 20, peptone 1, agar 15, to be subcultured.
Identification of the strain

The isolated fungus was maintained on MEA medium plate at 28C
for 4 days and then transferred to liquid MEA medium. After
incubation at 28C for 7 days with shaking, the fruit bodies of the
strains were collected. Genomic DNA was extracted using
conventional methods and was then used as template in PCR with
primers listed in Table 1. 18s rDNA gene was amplified with primers
18sf and 18sr (Daxboeck et al., 2004). ITS sequence was amplified
with primers ITS4 and ITS5. The PCR reaction consisted of 30
cycles: denaturing for 40 s at 90C, annealing at 56C for 40 s,
amplification at 72C for 1 min. An initial denaturing step of 4 min at
94C, and a unique final step of amplification at 72C for 5 min were
both included. The PCR products were analyzed on a 1.0%
agarose gel and were purified using the TIANgel Mini Purification
kit (Qiagen, Beijing) following the manufacturers instructions. The
products were sequenced in Sangong Biotech (Shanghai, China).
The presence of laccase was rapidly detected by laccase detection

medium (LDM) which consists of (g/L): glucose 10, KH 2PO4 2,
MgSO4.7H2O 0.5, CaCl2 0.1, ammonium tartrate 0.5, 2,2-azinobis
(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) 0.1, agar 15 with the
pH 5.0. The fungal mycelia were inoculated in LDM plate and
incubated at 28C. The strain that produced dark-green zone on the
plate indicated the presence of laccase. MnP was detected by LDM
containing 0.1 g/L MnCl2.4H2O instead of ABTS. The formation of
black and dark-brown flecks of manganese oxide (MnO2) indicates
the existence of MnP (Steffen et al., 2000). LiP was detected by
LDM containing 0.1g/L Azure B instead of ABTS. The plate that has
clearance of the blue coloured medium suggests the presence of
LiP (Pointing, 1999).
Laccase activity was determined by measuring the absorbance of
ABTS at 420 nm ( = 3.6 104 cm -1 M-1) at 28C. Conidial
suspension was incubated in Czapek's medium and the
fermentation solution and intracellular solution were taken for
extracellular and intracellular enzyme activities detection,
respectively. The culture fluid was centrifuged at 8000 rpm and the
supernatant was collected as extracellular fermentation solution.
The fruit bodies were ground in a mortar and re-suspended with 0.1
nM Tris-HCl. Then, the suspension was centrifuged at 10000 rpm
and the supernatant was collected as intracellular solution. The
assay mixture in a total volume of 3 mL contains 30 L of 100 mM
ABTS and 100 L aliquots of appropriately diluted culture fluid and
tartaric acid buffer (pH 3.0). One unit of laccase activity is defined
as the amount of enzyme required to oxidize 1 mol ABTS per
minute (Srinivasan et al., 1995). Activities of MnP and LiP were
measured following the method of Gao et al. (2011). The activities
were expressed in U/L.
Cellulase detection medium (CDM) was used for cellulase
determination, which composed of (g/L): MgSO4 0.5, (NH4)SO4 4,
KH2PO4 1, NaCl 1, CMC-Na 1, agar 15. After incubated at 28C for
3 days, the plate was flooded with 0.1% Congo red for 15 min and
then with 1 M NaCl twice (each for 5 min). Stain that produced clear
zone on the plate indicated the presence of cellulase (Catcheside et
al., 2003). The xylanase was detected by CDM containing 1.0%
birchwood xylan instead of CMC-Na.
The fermentation solution and intracellular solution for cellulase
activity detection was obtained using the method above and
assayed in 2.5 mL reaction mixture containing 1% CMC-Na in
citrate buffe, pH 5.0 and appropriately diluted enzyme solution. After
incubation at 50C for 30 min, the reaction was stopped by adding
2.5 mL Dinitorsalycilic acid solution and immersing the tube in
boiling water for exactly 5 min. One unit of enzyme activity was
defined as the amount of enzyme required to liberate 1 mol of
glucose from the appropriate substrate under the standard
conditions (Ghose, 1987). The activity was expressed in U/mL. All
values were the means of at least three replicates.
Analysis of alkaline lignin degradation capability
Identification of the strain
The fungus was maintained on MEA medium plate at 28C for 4
As alkaline lignin is a by-product in the pulp and paper
Jin et al.
6547
compounds. Halaburgi et al. (2011) has purified a

thermostable laccase from a strain of C. cladosporioides.
According to Abrha and Gashe (1992), when grown in
shaking-culture
with
medium
containing
carboxymethylcellulose, a Cladosporium species could
produce cellulase components.
Degradation of the alkaline lignin
Figure 1. Morphological characteristics of Bio-1. (A) Colonies

grown in screening medium at 27C for three days. (B)
Mycelia and fruit bodies under inverted microscope for
magnifications of 40 times.
Colletotrichum
Trichoderma
Mycosspaeralla
Penicillium
Cladosporium EU375523
Figure 2. Phylogenetic tree of 18s rDNA of strain Bio-1. Branch

lengths were drawn to scale, with the scale bar indicating the
amount of divergence.
industry and contains signicant similar structure to

natural lignin, in this paper, the screening medium,
containing alkaline lignin as the sole carbon source, was
used for screening lignin-degrading fungi. One strain,
named Bio-1, could grow well on the screening medium
at 28C. The basic shape of the Bio-1 colony is circular
with entire edge and wrinkled surface, the colour of the
fruit bodies is dark-green (Figure 1).
Total DNA was extracted from Bio-1 and amplified with
the primers of 18 s and ITS, respectively. Two single
bands of 1564 and 538 bp were obtained. Purified PCR
products were sequenced and the results were compared
with sequences in the GeneBank database by using
BLAST (NCBI). The 18s rDNA sequence of Bio-1 has
99% similarity with Cladosporium cladosporioides
EU375523.1. The phylogenetic tree of 18s rDNA was
gotten by neighbor-joining using CLUSTAL X and the
neighbor-joining tree is shown in Figure 2. The same
result (99% similarity) was found from ITS sequence
analysis. The morphological characteristics of Bio-1 were
also similar with genus Cladosporium (Deacon, 1997).
These results showed that Bio-1 belonged to the genus
Cladosporium. The Cladosporium fungus has been found
to have the capacity to biodegrade some aromatic
Based on the fact that lignin consists of large group of

aromatic polymers with a characteristic absorption band
at 280 nm, a simple lignin content detection method was
used to measure the absorbance of medium at 280 nm.
Proteins could contribute to the total amount of the
absorbance value at 280 nm, which interfered with the
results of the measurement. However, the content of the
proteins in the fermentation broth in the first few days
after inoculation was very low (data not shown), and the
concentration of lignin was relatively high. Proteins
contributed little to the absorbance values at 280 nm.
Therefore, decrease of absorbance value at 280 nm
reflected the degradation capacity of Bio-1 to a certain
extent. A decreasing trend of the alkaline lignin was
observed (Figure 3) and the minimum value of the
alkaline lignin concentration was on the fourth day. This
suggested that Bio-1 has secreted lignin-degrading
enzyme after inoculation and 35% of the alkaline lignin
could be hydrolyzed by Bio-1. With the process of lignin
degrading, more aromatic units which were wrapped in
the center of lignin structure were released, and some
carbohydrates also have absorption at 280 nm.
Meanwhile, with the increasing incubation time, proteins
such as lignocelluloses degrading enzymes, which also
had absorbance at 280 nm, were accumulated in the
fermentation broth. These might be reasons why
absorbance at 280 nm was slightly enhanced after the
fourth day.
Rolz and colleagues (1987) showed that twelve tested
white-rot fungi could decrease 38.64% of lignin after
fermentation of sugarcane chips for 5 to 6 weeks at 20C
(Rolz et al., 1987). Considering that Bio-1 decreased
35% of the lignin only in the fourth day after inoculation,
Bio-1 has great development potential in degrading lignin.
Assay of the lignin-degrading enzymes activity

To reflect the degradation capacity of Bio-1 more
accurately, the detection of lignin-degrading enzyme
activities was performed. The presence of laccase, LiP
and MnP were detected to ensure which lignin-degrading
enzyme was secreted by Bio-1. For visual demonstration
of the presence of laccase, a simple rapid assay was
used by incubating the mycelia on the LDM plate at 28C.
A dark-green zone was observed in the medium on the
third day (Figure 4A), which suggested that Bio-1 showed
6548
Incubation time (day)
Figure 3. Lignin degradation curve. Conidial suspension was

cultured at 28C for 10 days and the concentrations of alkaline lignin
were detected by measuring the absorption of medium at 280 nm. All
values were the means of three replications.
Figure 4. Detection of laccase (A) and cellulase (B)

secretion of Bio-1 on the ager plates. (A) The strain
cultured on the LDM at 28C for 3 days; arrow indicates
the dark-green ring. (B) The strain cultured on the CDM at
28C for 3 days; arrow indicates the clear zone.
Figure 5. Activities of the extracellular and intracellular

laccase on the eighth day. Without (1) and with (2) 0.5
mM CuSO4 induction at 28C in Czapek's medium.
laccase activity.
To be sure of the laccase existence, the extracellular and

intracellular laccase activities were detected by ABTS
method. The highest extracellular and intracellular
laccase activities were detected on the eighth day of
incubation, which were 241 and 187 U/L, respectively
(Figure 5). This indicates that the degradation of the lignin
should be continued after the fourth day, the above
experimental results in which absorbance of medium at
280 nm were slightly increased after the fourth day may
be disturbed by the impact of secreted proteins.
Meanwhile, some intermediate products may enhance
lignin degradation and act as the genes inducers, which
promote the degradation of lignin after day 4.
Owing to the ever-increasing demand for laccase in
biotechnological applications, its production process is
required to be economical and further enhanced. Using
inducers may be of benet (Niladevi and Prema, 2008).
Laccase is a multi-copper oxidase and copper could
regulate the synthesis of several laccase isoforms at the
level of gene transcription (Galhaup et al., 2002).
According to Collons and Dobson (1997) results, 0.5 mM
CuSO4 was added to Czapek's medium on the fourth day.
The extracellular and intracellular activities were
enhanced to 3800 and 5352 U/L, respectively. Significant
impact of copper on laccase synthesis in Trametes
versicolor (Niladevi and Prema, 2008), P. ostreatus
(Palmieri et al., 2000) and white rot fungus had been
frequently reported. For the Bio-1, strain copper induced
the extracellular laccase activity by 15 fold and the
intracellular laccase activity by 28 fold (Figure 5). Before
induction by CuSO4, extracellular laccase activity was
higher than the intracellular laccase, while after being
induced, the intracellular laccase activity was much
higher than the extracellular laccase.
Maybe intracellular laccase produced by Bio-1 was
more sensitive to copper. Or, there was no enough time
for the secretion of the enzyme. The effluents often
contain copper component, so the sensitivity to copper of
Bio-1 has large application potential in effluents
treatment.
The highest extracellular laccase activity was 241 U/L,
lower than some classical laccase-producing fungi.
However, 0.5 mM CuSO4 greatly enhanced laccase
production as 3800 U/L, much higher than the same
species, which has been reported earlier (Claus and Filip,
1998). According to Vijiaykumar et al. (2006), a fungus C.
cladosporioides isolated from a coal sample showed
laccase activity with 1413 U/L. Interestingly, only a few
articles have reported that genus Cladosporium strain
could produce intracellular laccase (Tetsch et al., 2006;
Froehner and Eriksson, 1974).
In our case, the intracellular laccase of Bio-1 was
detected and more importantly, it could be greatly
enhanced by CuSO4, even much stronger than
extracellular laccase. So, the strain Bio-1 could be an
attractive source of laccase producer.
We could not detect the MnP and LiP activities, which
Jin et al.
6549
Funds for the Central Universities.

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Figure 6. Extracellular (1) and intracellular (2)

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DOI:10.5897/AJMR12.725
DNA viral infections and transient bone marrow failure

in southern Iran
Kambiz Bagheri1, Mohammad Hossein Karimi2*, Ramin Yaghobi2, Behnam Mohammadi2,
Mehdi Dehghani3 and Padideh Ebadi1
1
Department of Immunology, Faculty of medicine, Kazerun Branch, Islamic Azad University, Kazerun, Fars, Iran.
2
Shiraz Transplant Research Center- Namazee Hospital- Shiraz University of Medical Sciences-Shiraz- Iran.
3
Hematology Research Center and Bone marrow Transplant Unit- Namazee Hospital- Shiraz University of Medical
Sciences-Shiraz- Iran.
Accepted 16 August, 2012
Some viral infections may have roles in decreasing the myeloid and erythroid progenitor cells leading
to hematopoietic crisis and bone marrow failure. In this research, the potential capacity of some DNA
viruses were studied in patients with transient bone marrow failure. In a cross sectional study the
plasma samples of 27 patients with clinically confirmed transient bone marrow failure were collected for
2 years. The genomic DNA of cytomegalovirus, human herpes virus-6, human herpes virus-8, and TT
virus were extracted from collected samples and amplified by different sensitive and specific in-house
semi nested and nested PCR protocols. Human herpes virus-6 and 8 were found in 3(11.1%) and 2 of 27
(7.4%) patients, respectively while, the TT virus was found in only one (3.7%) of 27 patients. However,
cytomegalovirus infection was not detected in plasma samples of studied patients with transient bone
marrow failure. These findings show the pathogenic role of viral infections in studied patients with
transient bone marrow failure.
Key words: Human herpes virus, TT Virus, bone marrow failure.
INTRODUCTION
Inability of bone marrow to produce adequate blood cells
is called transient bone marrow failure (Kurzrock, 2005;
Aslan et al., 2009), which is associated to significant
mortality due to bleeding or infection. The etiologic cause
of the majority of patients with transient bone marrow
failure remains vague. In a subset of these cases
infection may precipitates although it is not clear why only
some individuals are susceptible. Although it occurs in
mostly childhood, the certain age of disease onset may
vary to adult hood (Dokal, 2006; Marsh, 2005; Dokal and
Vulliamy, 2008; Federman and Sakamoto, 2005).
Depending on the cause, degree and duration of failure,
fever, pallor, infection or serious illness may occur. Acute
bone marrow failure is one of the important results of
infection (Kurzrock, 2005; Risitano et al., 2007).
*Corresponding author. E-mail: karimi70@yahoo.com. Tel: +98711-6476331. Fax: +98-711-6476331.
Among infectious agents with inducing role in transient

bone marrow failure, some DNA viruses may important
and also unexplained (Naseem et al., 2011). The marrow is
often cellular, but some cases of bone marrow failure are
due to viral suppression and/or the drug used to control
the viral outcomes (Morales et al., 1990).
Herpes viruses including human herpes virus5(Cytomegalovirus), human herpes virus-6, and human
herpes virus-8 may trigger the pathogenesis pathway in
bone marrow failure. Among them cytomegalovirus
infection is often accompanied by transient neutropenia
and thrombocytopenia based on its related pathogenesis
(Almeida-Porada and Ascensao, 1996).
Human herpes virus-6 activation is frequently observed
in patients who underwent bone marrow transplantation,
and this inhibits marrow engraftment (Rosenfeld et al.,
1995; Knox and Carrigan, 1996). Human herpes virus-8
can express related antigens persistently in bone marrow
cells inducing bone marrow failure (Platt et al., 1999;
Staskus et al., 1997; Rose et al., 1997; Dupin et al.,
6552
Table 1. The primer sequences of in-house PCR protocols.
Viruses
Cytomegalovirus-OF
Cytomegalovirus-OR
Cytomegalovirus-IF
Cytomegalovirus-IR
Human Herpes Virus-8-OF
Human Herpes Virus-8-OR
Human Herpes Virus-8-IF
Human Herpes Virus-8-IR
Human Herpes Virus-6-OF
Human Herpes Virus-6-OR
Human Herpes Virus-6-IF
Human Herpes Virus-6-IR
TT Virus- OF
TT Virus-OR and IR
TT Virus-IF
Primer sequences
5 -TACTGCACGTACGAGCTGTT-3
5 -GCGTACGTGATGAGGCTATAA-3
5 -CCTTCACGTTCATATCACGC-3
5 -GTGGAACTGGAACGTTTGGC-3
5-AT GGGG ACA ACG AGA TTA GC-3
5-CGA CCC GTG CCA GAT TAT G -3
5-TTG GGA AA GAT GGA AGA CG -3
5-AGT CCC CAG GAC CTT GGT TT-3
5-GCT AGA ACG TAT TTG CTG-3
5-ACA ACT GTC TGA CTG GCA- 3
5-TCA CGC ACA TCG GTA TAT 3
5-CTC AAG ATC AAC AAG TTG 3
5-ACA GAC AGA GGA GAA GGC AAC ATG-3
5-GGC AAC ATG TTA TGG ATA GAC TGG-3
5-CTG GCA TTT TAC CAT TTC CAA AGT T-3
1999).
On the other hand, TT virus may be frequently
transmitted through transfusion of blood components. TT
virus may prefer the erythroid and megakaryocyte series
of bone marrow, because thrombocytopaenia and
aplastic anemia have been reported in TTV infected
patients (Tokita et al., 2001; Kamada et al., 2004). TT
virus leads to the maturation arrest and apoptosis of
infected hematopoietic progenitor cells (Dokal, 2006).
Therefore, in this study the prevalence of some human
herpes viruses and TT virus infections were evaluated in
patients with transient bone marrow failure.
Patients and samples
27 patients with transient bone marrow failure, who were admitted
to Namazee Hospital, Shiraz University of Medical Sciences,
Shiraz, Iran, were enrolled in this study. Plasma samples were
collected for two years and stored in -70C till virol ogy tests were
performed. Confirmation of transient bone marrow suppression and
rule out of the other hematological malignancies and abnormalities
were clinically and laboratory done by expert hematologist. The molecular viral assays were performed double blind for elimination of
any possible technologist errors. The genomic DNA of
Cytomegalovirus, human herpes virus-6, human herpes virus-8,
and TT virus were extracted from collected samples and
diagnostically amplified by different sensitive and specific in-house
simple and nested PCR protocols.
Molecular analysis
Extraction of viral genomic DNA

The genomic DNA of Cytomegalovirus, human herpes virus-6,
human herpes virus-8, and TT virus were extracted from the plasma
Length of amplification fragment

600 bp
384 bp
380 bp
167
286 bp
271 bp
of EDTA-treated blood samples of patients with transient bone

marrow failure by DNP kit (CinnaGen, Iran) according to the
manufacturers instruction.
PCR protocols
The sequence of the primers using amplifying and detection of the
genomic DNA of viruses in patients with transient bone marrow
failure are presented in Table 1.
Cytomegalovirus
The genomic DNA of cytomegalovirus was detected in patients with
transient bone marrow failure using in-house nested-PCR protocol.
The outer and inner primer sequences are amplifying fragments of
HCMV-UL55 gB encoding sequence (Table 1).
The simple PCR mixture in a total volume of 50 l contained 5
l of 10X PCR buffer, 1.5 l of MgCl2 (50 mM), 1 l dNTP (10 mM),
1 l of each primer (10 pmol), 0.5 l Taq (2.5 unit), and 5 l of the
sample DNA.
The thermocycling conditions of the simple PCR included 93C
for 3 min (one cycle), 30 cycles of 1 min at 93C, 1 min at 60 C
and 1 min at 72C; and finally, one cycle of 5 min at 72C.
The components of the nested PCR mix were similar to simple
PCR. The thermocycling condition of the nested PCR step
included 94C for 3 min (one cycle), 30 cycles of 1 mi n at 93C, 1
min at 59C and 1 min at 72C; and finally, one cyc le of 5 min at
72C.
TT Virus
The genomic DNA of TT virus was detected in patients with
transient bone marrow failure using in-house semi nested-PCR
protocol. The primer pair sequences used in simple PCR step are
NG059 and NG063 amplifying a 286bp fragment of TT virus
genome located in the N22 open reading frame 1 (ORF1) (Table 1).
In the simple PCR step, the total volume of PCR mix contained 5l
of template DNA, 10 pmol/l of primers, 0.25 mMol of dNTPs, 1U of
Bagheri et al.
Taq DNA polymerase, Tris-HCL 10 mM, KCl 30 mM, and 1.5 mMol
of MgCl2. The primer pair sequences used in the second semi
nested PCR steps were NG061 and NG063 amplifying a 271 bp
fragment of the N22 open reading frame 1 (ORF1) of the TTV
genome (Table 1). The components of the semi nested PCR mix
was similar to simple PCR step. The total volume per reaction in the
two rounds of simple and semi nested PCR steps was 20 l.
The thermocycling condition of simple PCR step was initiated by
a first round at 94C for 10 min followed by a second round of 35
cycles at 94C for 30 s, 60C for 45 s, and 72C for 45 s, finalized
with extension at 72C for 7 min. The semi nested P CR step was
initiated by a first round at 94C for 10 min followe d by a second
round of 30 cycles at 94C for 30 s, 59C for 45 s, and 72C for 45
s finalized by extension at 72C for 7 min.
Human herpes virus-8 infection

The molecular infection of the human herpes virus-8 was evaluated
in patients with transient bone marrow failure. Human herpes virus8 genomic DNA was detected by specific primer pairs amplifying a
380 bp fragment of the LANA gene.
50 l PCR mix Ingredients were the same in both the simple and
nested PCR steps as follow: 5 l of 10X PCR buffer, 1.5 l MgCl2
(50 mM), 1 l dNTP (10 Mm), 1 l of each primer (20 pmol), 0.5 l
Taq (2.5 unit), and 10l of the sample DNA.
Therocycling conditions of the both simple and nested PCR steps
were the same. PCR rounds were initiated with a first step
denaturation in 94C for 5min and followed by 35 cycl es for the
second round at 94C for 30 s, 55C for 60 s, 68C f or 120 s and
terminated by extension at 68C for 5 min.
Human herpes virus-6 infection

The prevalence of human herpes virus-6 infection was studied in
patients with transient bone marrow failure. genomic human herpes
virus-6-DNA was detected using in-house nested PCR protocol by
two specific primer pair amplifying a 167 fragment of IE gene.
The ingredients of 50 l PCR mix were the same in both the
simple and nested PCR steps as follow: 5 l of 10X PCR buffer,
1.5 l MgCl2 (50 mM), 1 l dNTP (10 Mm), 1l of each primer (50
pmol), 0.5 l Taq (2 unit), and 5 l of the sample DNA. The
thermocycling conditions of both simple and nested PCR rounds
were the same. PCR rounds were initiated by a first denaturation
step in 94C for 3 min and followed by 30 cycles for the second
round 94C for 40 s, 51C for 60 s, 72C for 40 s an d terminated by
a final extension step at 72C for 5 min.
6553
The viremia of human herpes virus-8 was found in 2 of

27 (7.4%) patients with transient bone marrow failure.
Acute Myelogenous Leukemia was finally confirmed in
one of these patients with human herpes virus-8 infection
(Table 2).
But the genomic DNA of cytomegalovirus was not
detected in any of studied patients with transient bone
marrow failure (Table 4).
Molecular prevalence of TT virus

The genomic TT virus DNA was detected in only 1 of 27
(3.7%) patients with transient bone marrow failure.
Myelodysplastic Syndrome was finally confirmed in this
patient with TT virus infection (Table 2).
Simultaneous viral DNA infections
Among patients with transient bone marrow failure, none
of them presented simultaneous infection with more than
one studied viruses.
Laboratory and clinical indices in patients with

transient bone marrow failure
Hematology disorders or malignancies were confirmed in
some of the patients with transient bone marrow failure
several months after first evaluation. All of these patients
have been shown pancytopenia (Tables 2 and 4).
3 of 27 (1, 2, and 16) patients that are female, the
transient bone marrow suppression is finally led to Acute
Myelogenous Leukemia (Table 2). In 3 of 27 (10, 11, and
18) patients that two of them are male, the transient bone
marrow failure is finally led to Acute Lymphoblastic
Leukemia (Tables 2). Myelodysplastic Syndrome was
finally diagnosed in 3 of 27 (6, 22, and 26) patients that
all of them are male. Idiopathic Thrombocytopenia
purpura was found in 2 of these patients (1 and 23)
(Table 2).
RESULTS
DISCUSSION
The demographic data, clinical and hematological
abnormalities information, hematological laboratory
indices, and the history of DNA virus infections in 27
studied patients with transient bone marrow failure are
presented in Tables 2-4, respectively.
Molecular prevalence of human herpes viruses
The viremia of human herpes virus-6 was found in 3 of 27
(11.1%) patients with transient bone marrow failure.
Transient bone marrow failure was finally confirmed in
these three patients (8, 13, and 27) (Table 2).
The mechanisms of transient bone marrow failure have

been underestimated in these years (Kaptan et al., 2001).
Several clinical and laboratory results suggested that
some of DNA viral infections are associated to transient
bone marrow failure (Kaptan et al., 2001). Therefore, in
this study the prevalence of some DNA viral infections
was evaluated in patients with transient bone marrow
failure.
Cytomegalovirus infection has the potential to inhibit
hematopoiesis by targeting the functional compartments
of the bone marrow (Kondo et al., 1994).
Cytomegalovirus infection was also detected in human
6554
Table 2. The clinical presentation of patients with transient bone marrow failure.
Patients no.
Age
Sex
Final disease
Symptoms
33
Acute Myelogenous Leukemia
Petechia
29
Idiopathic Thrombocytopenia purpura,

Petechia
Thrombocytopenia,
Hemoglobinopathy
3
4
5
6
39
58
20
57
M
M
F
M
Transient bone marrow suppression

Myelodysplastic Syndrome
Fever, petechia,
Fever, chills, body pain
Fever, body pain
Fever, sleeping disorder
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
22
Fever, abdominal pain,

diarrhea
Pancytopenia
8
9
10
24
38
16
F
F
F

Acute lymphoblastic Leukemia
Fever, malaise, body pain

Fever, malaise, chills
Fever, chills, vomiting
Pancytopenia
Pancytopenia
Pancytopenia
11
37
Acute lymphoblastic Leukemia,

Bone Marrow Transplantation
GVHD, diarrhea, vomiting`
Thrombocytopenia
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
67
25
55
54
15
32
33
48
22
19
35
41
24
26
38
15
M
M
M
F
F
M
M
F
M
M
M
M
F
F
M
M

Acute lymphoblastic Leukemia
Idiopathic Thrombocytopenia purpura
body pain
Weakness, body pain
Fever, chills
Fever, chills
Fever, petechia, vomiting
Fever, diarrhea
Fever, malaise, Chills
Fever, diarrhea
Fever, chills, petechia
Fever, chills
Fever, chills
Fever, chills
Fever, chills
Fever, chills
Fever, chills
Fever, chills
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Pancytopenia
Leukopenia
Pancytopenia
CD34-positive early hematopoietic progenitor cells

(Kondo et al., 1994; Minton et al., 1994). Deficiency in
bone marrow stromal functions inhibits hematopoiesis at
its earliest stage and leading to bone marrow aplasia
(Kondo et al., 1994; Minton et al., 1994).
Cytomegalovirus may alter the function of accessory
cells, decrease the production of hematopoietic factors or
alter the expression of the cell surface adhesion
molecules and or introduce infection in the hematopoietic
progenitor cells (Chen, 2005). But, in this study the
genomic DNA of cytomegalovirus was not detected in
any patients with transient bone marrow failure.
Although the precise mechanisms of human herpes
Hematology diagnosis
Thrombocytopenia,
Hemoglobinopathy
virus-6 in suppression of the bone marrow are still

obscure, frequent isolation of human herpes virus-6 from
bone marrow and the suppressive effect of this viral
infection on the bone marrow cells were reported
(Isomura et al., 1997; Kadakia et al., 1996). These
reports also suggest that direct infection of hematopoietic
precursor cells with human herpes virus-6 may suppress
the bone marrow progenitor cell differentiation. Most of
human herpes virus-6 isolated from transplant patients
are of genotype B (Kadakia et al., 1996). Therefore, pretransplant screening of the patient and donors as well as
serial determination of the copy number of human herpes
virus-6 DNA post-transplantation are necessary to
Bagheri et al.
6555
Table 3. The pattern of hematological indices in patients with transient bone marrow failure.
Patients
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
25
26
27
WBC
4400 (N)
7800 (N)
2500 (L)
2800 (L)
1000 (L)
1000 (L)
3700 (L)
2100 (L)
1800 (L)
1200 (L)
6700 (N)
2600 (L)
3000 (L)
NF
1700 (L)
2650 (L)
2800 (L)
1200 (L)
2000 (L)
1900 (L)
2300 (L)
NF
1800 (L)
NF
PMN
704 (L)
5148 (N)
500 (L)
NF
370 (L)
180 (L)
2220 (N)
861 (L)
108 (L)
240 (L)
3350 (L)
1118 ()
720 (L)
NF
578 (L)
0 (L)
1344 (L)
108 (L)
NF
NF
NF
NF
NF
NF
LYMPH
748 (L)
2418(N)
1700(H)
NF
610(H)
790(H)
1184(N)
1173(H)
108(L)
960(H)
2814(H)
156(L)
2100(H)
NF
826(H)
26(L)
1176(H)
252(L)
NF
NF
NF
NF
NF
NF
MONO
132(L)
156(L)
275(H)
NF
10(L)
0(L)
222(N)
126(N)
54(L)
0(L)
469(N)
26(L)
180(N)
NF
218(H)
0(L)
168(N)
0(L)
NF
NF
NF
NF
NF
NF
EOS
0
78
25
NF
0
0
74
0
0
0
67
0
0
NF
43
0
0
0
NF
NF
NF
NF
NF
NF
PLT
25000 (L)
16000 (L)
3000 (L)
122000 (L)
90000 (L)
130000 (L)
80000 (L)
35000 (L)
70000 (L)
91000 (L)
94000 (L)
60000 (L)
56000 (L)
7000 (L)
117000 (L)
21000 (L)
104000 (L)
334000 (L)
103000 (L)
87000 (L)
137000 (L)
NF
166000 (N)
NF
HB
2.1 (L)
7.4 (L)
14.5 (N)
11.4 (L)
12.7 (N)
3.9 (L)
11.0 (L)
13.0 (N)
12.4 (N)
11.7 (L)
14.1 (N)
NF
8.2 (L)
15.4 (N)
6.8 (L)
7.7 (L)
13.6 (N)
7.3 (L)
10.9 (L)
16.6 (N)
13.2 (N)
NF
9.1 (L)
NF
ESR
93
75
1
6
12
125
45
13
2
60
9
3
21
NF
14
35
27
105
9
2
7
NF
NF
NF
RETIC
0.05
0.26
0.97
0.1
0.3
NF
0.11
0.13
0.12
0.11
NF
NF
0.06
0.3
NF
0.11
NF
0.04
NF
NF
0.23
0.17
0.48
NF
WBC: White blood cell; PMN: polymorph nuclear; Lymph: lymphocyte; MONO: monocyte; EOS: eosinophil; PLT: platelet; HB:
hemoglobin; ESR: erythrocyte sedimentation rate; RETIC: reticulocyte; NF: not found.
interpret Human herpes virus-6 viremia as a main reason

of bone marrow failure (Lagadinou et al., 2010; Koch,
2001).
Transient bone marrow failure has been found as a
frequent complication of the primary infection of the
human herpes virus-6 in infants (Carrigan and Knox,
1995). Moderately, severe suppression of multiple
lineages of the bone marrow cells appears most common
with long term manifestation in bone marrow transplant
patients infected by genotype B of human herpes virus-6.
A variant of human herpes virus-6 has an intrinsically
increased virulence with respect to bone marrow
suppression compared with the B variant (Carrigan and
Knox, 1995). In this study, human herpes virus-6 viremia
was found in 3 of 27 (11.1%) patients with transient bone
marrow failure.
Human herpes virus-8 Late Associated Nuclear Antigen
(LANA) can be expressed latently in immature bone
marrow cells inducing bone marrow failure (Platt et al.,
1999; Staskus et al., 1997; Rose et al., 1997; Dupin et
al., 1999). In a study, the simultaneous occurrence of
disseminated Kaposis sarcoma was found in one renaltransplant
recipient
and bone marrow failure
(Oksenhendler et al., 1998). Human herpes virus-8 can
also cause bone marrow failure, at least in

immunosuppressed transplant patients. The plasma-cell
infiltration in the aplastic bone marrow of transplant
patients may be associated with human herpes virus-8
infection (Oksenhendler et al., 1998; Regamey et al.,
1998). Also in this study the human herpes virus-8
viremia was found in 2 of 27 (7.4%) patients with
transient bone marrow failure.
Some case reports emphasize the possible role of TT
Virus infection in aplastic anemia. For the first time TT
virus was found in a 12 year old Japanese boy suffering
from pancytopenia and acute bone marrow failure
following severe acute hepatitis (Rauff et al., 2011). In
other case study TT virus infection was found in a 17year-old man with severe acute hepatitis that aplastic
anemia was also confirmed in bone marrow analysis
(Miyamoto et al., 2000). Also, among patients with
chronic hepatitis C viral infection or chronic hepatitis, the
platelet count was significantly lower in the patients with
genotype 1 of TT virus coinfection in comparison with
uninfected patients (Tokita et al., 2001; Okamoto et al.,
2000).
In TT virus infected patients with aplastic anemia, the
mechanism of bone marrow suppression and cytopenia
6556
Table 4. Viral infections in patients with transient bone marrow failure.
Patients no.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Cytomegalovirus
-
Human herpes virus -8

+
+
-
started at the peak transaminase levels, but activated

CD8+ T cells were not extremely increased (Ishimura et
al., 2010). The load of TT virus genomic DNA per
mononuclear cells was higher in peripheral blood than in
bone marrow. It might be explained by the fact that
erythroid and myeloid progenitor cells with high TT virus
loads had been already depleted in the hypoplastic bone
marrow cells based on the different cellular tropism
(Ishimura et al., 2010).
However, in other studies there are no available data
regarding TT virus infection in the patients with aplastic
anemia. Also TT viremia was not detected in the patients
with aplastic anemia suggesting that TT virus is not a
major etiologic agent for aplastic anemia (Miyamoto et
al., 2000). On the other hand, no supportive evidence of
higher prevalence of TT viral infection was found in
patients with newly diagnosed aplastic anaemia
comparing normal blood donors (Miyamoto et al., 2000;
Ishimura et al., 2010). Similar to other reports in this
study only one patient with transient bone marrow failure
was infected by TT genomic DNA.
Finally, in this study different molecular frequency of
human herpes virus-6 and 8 and also TT virus was found
Human herpes virus-6

+
+
+
TT Virus
+
-
in patients with transient born marrow failure. These

findings may be related to small sample size or may
emphasize the etiologic role of these viral infections in
patients with transient bone marrow failure. So, it should
be confirmed in completed studies.
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DOI: 10.5897/AJMR12.757
Survival of microorganisms in high pressure treated

minced meat during chilled storage and at pH and
temperature mimicking gastrointestinal tract
Sami Bulut
Food Engineering Department, Faculty of Engineering and Architecture, Trakya University, Edirne, Turkey.
E-mail: samibulut@trakya.edu.tr. Tel: +90 284 226 1217. Fax: +90 284 226 1225.
6
Minced meat samples containing 8.3 10 cfu/g determined by total aerobic count (TAC) were subjected
to pressures between 259 to 540 MPa for 3 to 17 min at 6C. A day after pressure treatment, no surviva l
on Plate Count Agar (PCA) was detected in the samples that were treated at and above 300 MPa for 10
min or more after 24 h incubation. However, survivors were detected in the pressure treated samples,
when the incubation period was extended to 72 h, indicating repair of injured cells. Compared to control
samples, number of surviving microorganisms in pressure treated samples increased significantly after
21 days of storage at 3C, thus implicating repair of injured cells was possible at refrigeration
temperatures. Experiments designed to mimic the temperature and acidity in stomach and lower
gastrointestinal tract revealed that microorganisms not inactivated by pressure treatment could not
recover or show viability at the pH of the stomach (3.0), but recovery and growth could potentially occur
at pH of the lower gastrointestinal tract (6.5). The information could be valuable for the food industry
and could imply more stringent controls for increasing the shelf life of foods by high pressure
processing (HPP).
Key words: High hydrostatic pressure, sublethal injury, chilled storage, gastrointestinal tract, injury recovery,
bacterial spores.
INTRODUCTION
High hydrostatic pressure (HHP) has proven to be
efficient for destruction of vegetative bacteria, viruses,
and yeasts, with bacterial spores being more resistant to
pressure (Nakayama et al., 1996). HHP can inactivate
microorganisms at room temperature or in combination
with a low heat treatment while maintaining the main
nutritional and organoleptic properties of foods (Knorr,
1993) and extend shelf-life. Research on the effect of
high pressure on food was carried out already in the
nineteenth century by Hite (1899) who described an
increase in shelf-life for products such as milk, fruit and
other foods (Rendueles et al., 2011). Intensive research
in this field has taken place in the last three decades and
as a result, a range of pressure treated food products
such as fruit preparations, fruit juices, sauces, rice cakes,
sliced cooked meat products, raw squid, guacamole and
oysters are now on the market in Japan, USA and
Europe. However, due to concerns on safety of utilizing
this process for pasteurization or sterilization of food
products, its commercialization is rather slow. This is due

to the fact that the resistance of microorganisms to HHP
is variable depending on many intrinsic and
environmental factors. The same microorganism can give
different response to HHP in different media. Variation in
pressure sensitivity of microorganisms is common for
different strains of a microorganism (Patterson and
Kilpatrick, 1995). Resistance of microorganisms can be
increased when grown or pressure treated in nutritionally
rich media containing substances which may provide
protection against damage or nutrients essential for
repair (Hoover et al., 1989).
Higher reductions in the number of Salmonella
senftenberg was obtained in phosphate buffer compared
to pressure treatment of S. senftenberg in ground chicken
(Metrick et al., 1989). The pH of the food or media is
another factor influencing the effectiveness of HHP
induced inactivation. A reduction in the pH of the media
could increase the pressure induced inactivation of
Bulut
vegetative microorganisms (Koseki and Yamamoto,

2006). Spore populations of Clostridium sporogenes were
reduced by 2.5 log when exposed to 404 MPa at 25C,
pH 4.0 for 30 min, but the same treatment at pH 7.0
resulted in a less than 0.5 log reduction in spore counts
(Stewart et al., 2000). Other studies reported that
inactivation of spores of Bacillus subtilis was maximally
80% when the spore suspensions were subjected to
pressure treatments at 100 and 600 MPa at 40C over a
pH range from 3 to 8 and was not increased at low pH
(Wuytack and Michiels, 2001). Cells in exponential
growth phase are more sensitive to pressure than those
in stationary phase (Saucedo-Reyes et al., 2009). Water
activity (aw) of the food is also an important parameter
determining the effectiveness of HPP. Goodridge et al.
(2006) demonstrated that inactivation of Salmonella
enteriditis in almonds increased when almonds were
suspended in water and then pressurised. They
suggested that the increased aw at the surface of the
almond allowed the pressure to impart a more destructive
effect on the bacteria. The equipment, pressurization
regime and the pressurizing media can also affect the
efficiency of microbial destruction during HHP application
(Simpson and Gilmour, 1997).
HHP could increase the shelf life of foods through
inactivating and/or injuring the microorganisms present in
food. At standard pressures used in food processing,
spores are not inactivated and a pasteurized product is
obtained, in which surviving spores have no competitors.
In these conditions, the risks posed by pathogenic spores
should not be underestimated (Rendueles et al., 2011).
After pressure treatments at 100 to 600 MPa,
microorganisms could not grow on agar, but still show
metabolic activity. This fraction is often described as
viable but not culturable cells (Ananta et al., 2004). The
presence of spores and non culturable bacteria in food
after pressure treatment might be critical in terms of their
potential for excreting toxic or food spoiling metabolites.
Several studies have reported recovery of HPP treated
pathogenic and spoilage bacteria in nutrient broth (Chen
and Hoover, 2003; Erkmen and Dogan, 2004), milk
(Bozoglu et al., 2004; Bull et al., 2005; De LamoCastellv et al., 2005), phosphate buffer (Koseki and
Yamamoto, 2006) and sliced cooked ham (Aymerich et
al., 2005; Koseki et al., 2007) by storage between 6 h
and 4 weeks at various temperatures.
To the best of our knowledge, there is no information
on how HPP treated microorganisms, not able to repair
themselves and grow at refrigeration temperatures (in a
short time), would function in the human gastrointestinal
(GI) tract, when ingested with food. An assumption is
that, if the microorganisms can endure the low acidity of
the stomach without irrecoverable injury or death, they
can repair themselves and grow in the nutrient rich
environment of the lower GI tract at an optimum
temperature (body) and not as low acidity as in the
stomach.
6559
In the fast state, intragastric pH in healthy subjects is in

the range of 1.3 to 2.5. Eating increases pH to a range of
4.5 to 5.8. About an hour after eating, the pH of the
stomach decreases to less than 3.1 (Malagelada et al.,
1976; Dressman, 1986; Kong and Singh, 2008). It has
been suggested that for bioavailability studies using in
vitro gastrointestinal extraction methods, the pH of the
stomach should be set in the range of 1 to 4 and the
average residence time should be taken as 3 h. Similarly,
to simulate small intestine conditions, the samples should
be subjected to a pH of 4 to 7.5 for approximately 7 h
(Dean and Ma, 2007).
The purpose of this study was to investigate the effect
of pressure and pressurization time on survival of the
natural microbial population in minced meat during 21
days of storage at 3C. Survival and growth of
microorganisms from pressure treated samples at
conditions of stomach and gastrointestinal tracts acidity
and temperature were assessed.
Preparation of minced meat samples for pressure treatment
Minced meat obtained from a local market (Kipa, Edirne, Turkey)
was mixed by using a food processor (Siemens, Slovenia) in order
to homogenize the samples. Using a vacuum packing machine
(MV-20, Lipovak, Gebze, Turkey), homogenized samples (10 g)
were vacuum packed in sterile plastic films made of
Polyamide/Polyethylene
(Saran
Plastik,
Dzce,
Turkey)
impermeable to air and water. The packed samples were vacuum
packed twice to prevent contamination of the pressurization
medium in case of rupture during pressurization. Samples were
prepared in triplicate for each experimental condition and for
untreated control and kept at 3 1C overnight befo re
pressurization. After pressurization, samples were refrigerated at 3
1C until testing on days 1 and 21.
High pressure treatment
A 2 L processing unit (Avure Technologies Incorporated, Kent WA,
USA) was used to pressurize the samples. Water was used as the
pressure medium. Temperature of the pressure transmitting
medium inside the pressure chamber was controlled with a cooling
jacket surrounding the pressure vessel. Iced water was circulated in
the jackets surrounding the pressure chamber to keep the pressure
medium (water) in the chamber at about 6 2C. The pressure was
increased to the test pressure at a rate of 17 MPa/s and
decompression time was less than 5 s. The unit was controlled by a
computer program. In order to monitor the temperatures, 2 K-type
thermocouple was silver-soldered through the center of the top
closure of the pressure chamber and was positioned close to the
sample to monitor temperature during pressure treatments.
Experiments to mimic the gastrointestinal tracts pH and

temperature
In the present study, it was assumed that the average pH of
stomach was 3.0 and the length of time the meat samples spent in
stomach was 3 h. We assumed that the pH in lower GI tract (small
and large intestines) was about 6.5. In order to imitate the worst
6560
Table 1. Experiemntal parameters for HPP and survival of microorganisms in

minced meat after 1 and 21 days chilled storage at 3C. Results are from average
of 3 counts and the standard deviations (StDev) are derived from these 3 counts for
each experiment.
HPP parameters
Pressure (MPa)
Control
259
300
300
400
400
400
400
400
500
500
541
Time (min)
10
10
5
10
10
10
3
17
5
15
10
TAC during chilled storage

Day 1
Day 21
Log cfu/g StDev
Log cfu/g StDev
6.92
0.11
7.94
0.13
4.62
0.08
8.15
0.08
2.42
0.09
8.07
0.07
3.72
0.12
8.12
0.06
2.12
0.10
6.10
0.03
2.18
0.08
5.90
0.11
1.92
0.07
5.60
0.12
3.62
0.12
6.80
0.13
1.72
0.07
2.00
0.09
2.02
0.03
3.26
0.06
ND
3.45
0.09
ND
2.40
0.15
ND Not detected.
case scenario, the time meat samples spent in lower GI tract was
taken as 10 h instead of 7 h as suggested by Dean and Ma (2004).
Based on these assumptions, the experimental conditions for
testing the effect of pH and temperature on microorganism in the
pressure treated samples were as following:
Experiment designed to imitate the stomach and Lower GI tracts

acidity were carried out under aseptic conditions. For each set of
experiments, a control sample was tested at the conditions of
stomach and lower GI tract. No contamination was found in any of
the control samples (minimum detection level 50 cfu/g).
Experimental set up for stomachs pH and temperature
Enumeration of microorganisms
Samples of pressure treated meat (ca. 10 g) was opened with a

sterile knife and emptied into a sterile stomacher bag. About 80 g of
a sterile buffer solution at pH 2.6 prepared by adding 57.8 ml of 0.1
M HCl (Merck, Germany) solution into a 1000 ml of 0.1 M
Potassium Hydrogen Phthalate (Merck, Germany) and
homogenized by a Seward Stomacher 400 Circulator (UK) at 150
rpm for 2 min. Finally, the homogenized samples were placed into a
Sanyo MIR-154 Cooled Incubator (Japan) operating at 37C.
During the 3 h of incubation period, the stomacher bags were
removed from the incubator and stomached at 150 rpm for 1 min
every half an hour and then placed back into the incubator.
Control and pressure treated samples (10 g) were mixed with 90 ml

of PBS pH 7.0 (Oxoid, Basingstoke, UK) and homogenized in the
stomacher at 200 rpm for 1 min. A 1 ml sample of suspension from
the stomacher bag was taken into a sterile 1.5 ml Eppendorf tube
and serial dilutions 10 were made using PBS (pH 7). Surviving
cells were enumerated in triplicate on PCA (Biolab, Izmir, Turkey).
The plates were incubated at 37C for 24 to 72 h bef ore counting
the colonies.
The averages of three counts from each dilution were used for
calculation of surviving cells. Microbial reduction was expressed in
terms of logarithmic reduction corresponding to the logarithmic
difference between the initial number of microorganism before
pressure treatment and the number of microorganisms surviving
pressure treatment and storage.
Experimental set up for Lower GI tracts pH and temperature

After holding for 3 h at the pH and temperature of the stomach as
explained earlier, the stomacher bags were removed from
incubator, a 1-ml sample aliquot was taken for microbial analysis.
Then, 10 g of Tryptic Soy Broth (Biolab, Izmir, Turkey) was added
to stomacher bag to provide extra nutrient in order to make the
system more realistic. This is because the lower GI tract could
always hold semi digested nutrients which would be available to
microorganisms. Finally, the pH of the mix was adjusted to 6.5 by
predetermined amount of sterile 2.5 M NaOH solution. After
homogenizing the mix at 150 rpm for 2 min, the stomacher bags
were placed into the incubator and kept at 37C in s tatic condition
for 10 h. The stomacher bags were then taken from incubator and
homogenized before taking a 1-ml sample of aliquot for microbial
analysis.
The effect of two main variables; pressure from 200 to 500 MPa,
holding time from 5 to 15 min and their interactions on the
inactivation of microorganisms were designed and studied by the
surface response methodology employing Design-Expert v8
Software (Stat-Ease, Inc. Minneapolis, USA) using central
composite design. Assessment of error was derived from three
times repetition of the centre point of experimental design, which
was set as 400 MPa and 10 min. The parameters of the
experimental design are shown in Table 1.
In order to understand the effect of pressure, pressurization time
and their interactions on inactivation of microorganisms, statistical
Bulut
6561
Log Reduction (cfu/g)
7.0
6.0
5.0
4.0
3.0
2.0
15
500
13
450
11
400
Time (min)
350
7
5
Pressure (MPa)
300
Figure 1. Reduction in the number of microorganisms in HHP treated minced meat samples
after 1 day of storage at 3C as a function of press ure and time. Response surface
constructed based on counts obtained on PCA after 72 h of incubation at 37C.
analyses was conducted on survival data obtained from samples a

day after the pressure treatment and incubated for 72 h at 37C. By
using a linear model, numbers of surviving cells determined by TAC
a day after HHP treatment were statistically analyzed by DesignExpert software v8. Level of significance was set for P < 0.05 and
the significance of each response variable was assessed by F-test
statistical analysis.

Microbial load of minced meat samples
The average number of microorganisms in untreated
6
control samples was 8.3 10 cfu/g and increased to 8.7
7
10 cfu/g after 21 days of storage at 3C.
Effect of pressure and pressurization time on

survival of microorganisms
A linear model was fitted to data and analysis of variance
(ANOVA) showed that pressure (P = 0.0116) and time (P
= 0.0444) were significant parameters. The model Pvalue (0.0166) and R-squared value (0.7448) showed
that the model was representing the data well. Response
surface model produced the following equation for
prediction of log reduction in the pressure treated
samples a day after pressure treatment:
Log reduction (cfu/g) = 4.63 + 1.03A + 0.64B
Where, A is pressure in MPa and B is temperature in C.
Survival of microorganisms was reduced by increased

pressure at all pressurization times tested. In general, the
positive effect of pressure and pressurization time on
inactivation of vegetative cells is well documented in the
literature (Kalchayanand et al., 1998; Ritz et al., 2000;
Dogan and Erkmen, 2004). Increased pressure resulted
in higher reduction in number of survivals a day after
pressure treatment. There was no growth on PCA after
24 h of incubation at 37C at pressures above 300 M Pa
regardless of the pressurization time, which corresponds
to a minimum of 5.2 log reduction based on a 50 cfu/g
minimum detection level. However, extending incubation
time of PCAs to 72 h resulted in at least 1 colony count
on all the samples, except treatments at 500 MPa for 15
min and 541 MPa for 10 min. Response surface showing
log reductions in the samples a day after pressure
treatment (Figure 1) was constructed based on the
counts obtained on PCA after 72 h of incubation at 37C,
as it was not possible to obtain a response surface by
only a few survival data after 24 h of incubation at 37C.
It could be noted from Figure 1 that the increased
pressurizing time increases log reduction which is in line
with the literature (Chen and Hoover, 2003; Gao and
Jiang, 2005; Bover-Cid et al., 2012).
Change in total aerobic counts during chilled storage
In pressure treated samples, the number of surviving
microorganisms significantly increased during the storage
at 3C. After 21 days at 3C, survival of microorga nism in
samples that were pressure treated at 300 MPa or less
6562
were not statistically different than untreated control

samples (P = 0.502), averaging to 8.04 log unit with a
standard deviation of 0.092. Survival of microorganisms
detected in the samples that were pressure treated at
300 MPa or less were increased by an average of 4.6 log
unit after 21 days of storage at 3C whereas, the n umber
of microorganisms in control samples increased by an
average of 1.0 log unit, which suggests that the increase
in number of microorganisms in pressure treated minced
meat samples were mainly due to the recovery of injured
cells during storage at 3C. The extent of recovery was
higher at moderate pressures up to 300 MPa whereas,
the recovery of microorganisms were significantly
delayed at samples treated at higher pressures as seen
in Table 1. A day after the pressure treatment, no survival
of microorganisms was detected in samples that were
treated at 500 MPa for 15 min and 541 MPa for 10 min.
However, on day 21, the number of microorganisms
detected in the samples that were treated at 500 MPa 15
3
2
min and 541 MPa 10 min, were 2.8 10 and 2.5 10
cfu/g, respectively.
Compared to control samples, the significant increase
in number of survival (2.4 to 5.6 log unit) in pressure
treated samples during 21 days of storage at 3C co uld
be considered as an evidence of injury due to pressure
treatment. Also, when the incubation time was increased
from 24 to 72 h, the number of colonies counted on PCA
plates were increased significantly, which supports the
presence of injury in pressure treated samples.
Pressure is known to inflict sublethal injury in
vegetative cells of bacteria (Alpas et al., 2000; Ritz et al.,
2001; Ananta et al., 2004; Yuste et al., 2004; Kilimann et
al., 2006; Koseki et al., 2008) and sublethally injured
bacteria could recover in nutrient rich medium. Bozoglu et
al. (2004) stated that primary and secondary injuries
could occur after HHP treatment and injured cells may
not form visible colonies on selective or non-selective
agar, however colonies could first form on non-selective
agar and later on selective agar during prolonged
storage. Other studies that support the recovery of
sublethally injured cells during storage after a pressure
treatment of various types of foods are: minced beef
(Carlez et al., 1993), sliced cooked ham (Aymerich et al.,
2005), poultry meat (Yuste et al., 1999) and smoked
salmon (Erkan et al., 2011).
In the literature, there are variations in degree of
recovery of microorganisms in pressure treated food
products and model food systems during storage
however, existence of some degree of recovery
depending on the parameters of pressure treatment, type
of food and temperature of storage is evident.
Our view is that the increase in number of microbial
counts during chilled storage is due mainly to the
recovery and subsequently growth of the injured cells;
however, possibility of germination
and growth of
bacterial spores should also be noted. It has been
reported that refrigeration at 4C was sufficient t o prevent
germination and outgrowth of Clostridium perfringens

spores on vacuum-packaged beef surfaces whereas,
storage at 37C for 1 day resulted in spore germina tion,
outgrowth, and rapid proliferation to levels exceeding 7
log cfu/g (Novak and Yuan, 2004).
Survival of
temperature
microorganisms
at
GI
acidity
and
There was no growth in any of the 19 days old HHP

treated samples when they were kept at pH 3.0 0.1 and
37C for 3 h with stomaching every an hour. It co uld be
assumed that the pressure treated samples may contain:
i) completely inactivated vegetative cells; ii) injured
vegetative cells (Alpas et al., 2000; Ritz et al., 2001;
Ananta et al., 2004; Bozoglu et al., 2004; Yuste et al.,
2004; Kilimann et al., 2006; Koseki et al., 2008); iii)
germinated spores (Gould and Sale, 1970; Wuytack et
al., 2000; Aouadhi et al., 2012; Vercammen et al., 2012);
and iv) spores. Injured vegetative cells and newly
germinated spores may be stressed by low pH of TSB
(pH 3.0) and therefore may not be able show viability on
PCA. On the other hand, aerobic and facultative
anaerobic bacterial spores could not germinate and/or
grow during the 3 h of exposure to low pH of TSB and
subsequently on solid surface of PCA during incubation
for 18 to 24 h. Inability of bacterial spores germinating at
low pHs could be supported by the results of Ciarciaglini
et al. (2000) who studied the effects of preservative
treatments on germination of Bacillus subtilis spores in
nutrient broth with 10 mM L-alanine and observed that
the germination was completely inhibited at pH 4.0 when
incubated at 30C for up to 6 h.
Survival of microorganisms were observed in the HHP
treated samples when the pH of the mix was brought to
6.5 by addition of NaOH and 10 g TSB and then
incubated 10 h at 37C following the exposure to
stomach acidity and temperature for 3 h. Average
survival of microorganisms in the samples treated at 300,
400 and 541 MPa were 5.8 (0.48 SD), 5.1 (0.30 SD)
and 4.8 (0.43 SD) log units, respectively. Significant
number of viable cells observed in the samples from the
simulation of lower GI tracts acidity and temperature
could be explained by either the recovery of the injured
vegetative cells or germination of spores in nutritionally
rich (due to added TSB and minced meat itself) liquid
media and optimum pH (6.5) and temperature (37C).
Wheeldon et al. (2008) reported that the optimum pH
range for germination of Clostridium difficile spores was
6.5 to 7.5 and the extent of germination decreased at pH
5.5.
Our results suggests that vegetative microorganisms
that are not completely inactivated during the HHP
treatment may not be able to repair the injuries at the
simulated conditions of stomach pH and temperature to
be able to show viability on PCA. Similarly, bacterial
Bulut
spores that are not inactivated by pressure treatment

could not germinate at the conditions mimicking the
stomach pH and temperature and show viability on PCA.
However, vegetative microorganism could recover from
their injuries and bacterial spores could germinate at the
conditions of lower GI. In addition, the growth of the
recovered microorganisms and germinated spores could
also be possible. To the best of our knowledge, there is
no study on injury recovery of vegetative cells in GI tract,
however there are studies on fate of bacterial spores in
GI tract that are supporting our view on possible
germination and/or outgrowth of bacterial spores at the
conditions mimicking the pH and temperature of lower GI
tract. In characterizing spore probiotics, Hoa et al. (2001)
studied the persistence and dissemination of B. subtilis
spores given orally to mice. Their results showed that the
number of spores excreted in the feces of mice was, in
some experiments, larger than the original inoculum. This
was explained by germination of a proportion of the spore
inoculum in the intestinal tract, followed by limited rounds
of cell growth and then sporulation again. Using a murine
model, Casula and Cutting (2002) showed that spores of
Bacillus species can germinate in significant numbers in
the jejunum and ileum and may colonize the small
intestine. Cartman et al. (2008) showed that orally
administered spores of B. subtilis germinated in GI tracts
of chicks and 20 h after spores were administered,
vegetative cells outnumbered spores throughout the GI
tract.
It should be noted that our conclusions are not taking
into account the complicated processes and interactions,
such as effect of digestive enzymes, competitive
microflora of the guts, interactions of nutrients, etc. into
account. However, our results point out a possible risk of
recovery
and
growth
of
injured
pathogenic
microorganisms and germination and/or growth of
bacterial spores in GI tract, if the pathogens and spores
are not completely inactivated during the HHP treatment.
Conclusions
Our results showed that despite the absence of growth
immediately after a pressure treatment in a nutrient rich
environment such as, minced meat, a substantial
recovery could be possible during storage. The
undetected microbial growth immediately after HHP
treatment could be due to injury rather than death of the
microorganisms. It is assumed that the injured
microorganisms can recover in the GI tract and
grow/multiply.
Our
results
suggest
that
the
microorganisms could not recover from injury or grow at
stomach acidity and temperature. However, recovery and
growth could potentially occur in lower GI tract where the
acidity is low. This could have important implications in
terms of the safety of HHP treated foods especially when
they are consumed by immunologically impaired. It
6563
should be noted that our findings are based on a

simplified approach which does not take the complicated
processes and interactions, such as effect of digestion
enzymes, bile and pancreatic juices, competitive
microflora of the guts, interactions of nutrients, etc. into
account. Therefore, the degree of recovery and/or growth
of microorganisms could be different in vivo. However,
the possibility of the risk for recovery of pathogenic
microorganisms in GI tract could not be ignored, if the
pathogens are not killed but injured by HHP treatment.
Further in vitro and in vivo studies are required to better
understand the possibility of the recovery of pressure
injured microorganisms and differentiate the extent of
recovery and growth of injured cells from the extent of
germination and growth of bacterial spores in GI tract.
ACKNOWLEDGEMENTS
This work was supported by Republic of Turkeys Small
and Medium Size Enterprises Development Organization
(Grant no: 02229). The author thanks Dr. G. A.
Evrendilek and A. Yaman for letting them use HHP
equipment and for their assistance. Special thanks to N.
Oner who assisted with the experimental work throughout
the study.
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DOI: 10.5897/AJMR12.934
Efficacy and toxicity of neutralizers against

disinfectants and antiseptics used in vaccine
production facility
Norhan S. Sheraba1, Aymen S. Yassin2*, Aly Fahmy1 and Magdy A. Amin2
1
VACSERA, the Holding Company for Biological Products and Vaccines, Giza, 22311, Egypt.
Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, 11562, Egypt.
Effective neutralization of chemical biocide is the first step in the accurate evaluation of antiseptics and
disinfectants to avoid overestimation of the biocide activity. The aim of this study is to screen novel
neutralizers (or neutralizer combinations) against different antiseptics and disinfectants that are highly
used in the principle vaccine production facility in Egypt. Neutralizer efficacy (NE) ratios were
determined by comparing the recovery of the challenging index microorganisms from the neutralizing
solution in the presence, or the absence, of the biocide. Neutralizer toxicity (NT) ratios were determined
by comparing the recovery of viable microorganisms in the neutralizer exposed population and the
viability population. An effective and non-toxic neutralizer was initially identified by NE and NT ratios of
0.75 (75% recovery). The most potent neutralizers were 1% physiological peptone water, 0.1 % Tween
80 in 1% physiological peptone, 0.6% sodium thiosulphate and Dey-Engley. Due to the absence of a
universal neutralizer, the evaluation of NE and toxicity is an essential step in testing any new biocide.
The evaluation of neutralizer activity should be carried out separately for each index microorganism
and neutralizer pair to identify the most potent compound in each case.
Key words: Antiseptics, disinfectants, neutralizers.
INTRODUCTION
The appropriate use of effective antiseptics and
disinfectants is crucial to prevent transmission of
pathogenic bacteria or viruses carried in the air, on the
ground or on the hands of workers in clean rooms and
aseptic areas, common in vaccine development facilities
and hospitals (Murtough et al., 2002; Kampfa et al.,
2003). Antiseptics are chemical agents that inhibit or kill
microbial growth and are non-toxic when applied to living
tissues; they are used for antisepsis of skin, mucous
membranes as well as wounds (Kramer, 2000).
Disinfectants are chemical and/or physical agents used to
destroy or irreversibly inactivate many or all of the
pathogenic microorganisms but not necessarily spores
*Corresponding author. E-mail: aymen.yassin@live.com
and not all viruses on inanimate objects (Madigan et

al.,2002). Sometimes, the same compound is used as
antiseptic in low concentration and disinfectant in high
concentration. Traditional agents, such as alcohols,
quaternary ammonium compounds (QAC), phenol and
amphoteric surfactants are effective against bacteria in
their vegetative state but useless against spores (Rutala
and Weber, 1998). Biocides that have sporocidal activity
include aldehydes, hypochlorites and hydrogen peroxide
(Rutala and Weber, 1997). When disinfectants and
antiseptics are used; more than one type should be
employed to avoid the development of resistant bacterial
strains (Gorman and Scott, 2004). Effective neutralization
of a chemical biocide is critically important to the quality
of the data derived from any assay of biocidal efficacy to
avoid carry-over of active biocide to the recovery media,
which, in turn, may result in biostasis of the organism,
6566
this biostasis would in turn lead to an overestimation of

the biocides efficacy (Russell, 1998). Neutralization is
intended to prevent the inhibitory concentration of
antimicrobial agent from being transferred to the recovery
medium which is essential for the biocidal assay.
Neutralization can be achieved by several methods:
diluting the biocide to such a level that it ceases to have
any inhibitory effect in the recovery medium, chemically
neutralizing the biocide by means of an appropriate
neutralizing agent (antidote) that is itself non-toxic,
effective and fast acting or removing the biocide by
membrane filtration (Russel, 1998; Sutton et al, 2002).
Consequently, the experimental design used to
establish the efficacy of biocide neutralization has a
major impact on the estimation of antimicrobial efficacy
(Sutton et al., 1991). Many references have been made
to the lack of "universal neutralizer"; therefore, the quality
and properties of the recovery medium are of critical
importance to the test (Espigares et al., 2003). The
neutralizing /recovery medium must possess two
properties. First, it must adequately neutralize the
disinfecting agent to allow unrestrained microbial growth;
this is known as the neutralizing efficacy (NE) of the
medium. NE is determined by comparing growth
supported by the neutralizing media in the presence and
the absence of the specified amounts of the disinfectant
solutions. The ability of the neutralizing medium to
promote growth is a second important consideration
termed neutralizer toxicity (NT), which is determined by
comparing growth in the neutralizing medium without the
disinfectant with growth in a rich medium [Tryptone Soy
Agar (TSA) or Sabouraud Dextrose Agar (SDA)]. An
effective and non-toxic neutralizer is initially identified by
NE and NT ratios of 0.75 (75 %) (Sutton et al., 2002).
The US pharmacopeia recommends the application of
the neutralization assays against four indicator
microorganisms: Staphylococcus aureus, Pseudomonas
aeruginosa,
Aspergillus
brasiliensis
(previously
Aspergillus niger) and Bacillus subtilis. In a previous
study carried out by the same authors in the same facility,
the Staphylococci represented the most common isolate
that was detected from an environmental monitoring
programme with Staphylococcus hominis representing
51% of all Staphylococcci isolates (Sheraba et al., 2010).
Given the absence of a universal neutralizer, and as a
part of a general environmental monitoring program, the
goal of this study is to evaluate the efficacy and toxicity of
different neutralizing solutions for use in antiseptic and
disinfectant efficacy assays applied at the main vaccine
production facility in Egypt, against the most common
index microorganisms in addition to the most common
environmental isolate identified.
METHODOLOGY
A list of all disinfectants and antiseptics used with their composition
and dilutions is shown in Table 1. A list of all neutralizers tested
with their exact composition is shown in Table 2. All culture media
were from Bacto, France.

Bacterial and fungal strains
S. aureus ATCC 6538, P. aeruginosa ATCC 9027, A. niger ATCC
16404 and B. subtilis ATCC 6633 were selected as index
organisms representing Gram-positive and Gram-negative bacteria,
fungi, and spore forming bacteria respectively. S. hominis isolated
from an environmental monitoring program previously carried out in
the same facility was also used in the tests.
Preparation of working cultures of bacterial, fungal and spore

suspension
According to European standard EN 12353 (Jette et al., 1995), test
organisms were subcultured from the stock culture by streaking into
TSA and incubated at 36C for bacteria or into SDA and i ncubated
at 20 to 25C for fungi (in the case of Aspergillus). After 24 h, a
second subculture was prepared from the first one in the same way
and incubated again. The test organisms of the second subculture
were washed off with 10 ml of a diluent containing 0.1% (w/v)
tryptone and 0.85 % (w/v) sodium chloride; samples were washed
twice with phosphate buffer saline (PBS) and adjusted to
approximately 100 to 115 colony forming unit (CFU)/ml. For A.
niger, a subculture was grown on SDA and incubated at 20 to 25C
for 7 to 14 days then mycelial mats were harvested from the agar
surface of the working culture, homogenized with sterile glass
beads in 1% physiological peptone and filtered through sterile
cotton gauze to remove hyphae then suspended in 1%
physiological peptone with 0.1% Tween 80. Samples were washed
twice with PBS and then standardized to approximately 100 to 115
CFU /ml. Spore cultures were developed for a period of 3 to 8
weeks in a sporulation medium (Peptone: 15g/L, yeast extract: 3
g/L, NaCl: 6.0 g/L, D (+)-glucose: 1.0 g/L, Manganese sulphate: 0.1
g/L) at 37C then they were harvested, centrifuged (4 times at
1935g/ 30 min), heat-shocked 80C/10 min and samples were
washed twice in PBS and then standardized to approximately 100
to 115 CFU/ml.
Quantitative method to evaluate neutralizer efficiency and

toxicity
This method was achieved as previously described by Sutton et al.
(1991, 2002), Espigares et al. (2003) and follows three treatment
populations for comparison. One (1) ml of each of the biocide or
PBS solution was added to tubes containing 9 ml of neutralizing
broth. These tubes represented the neutralizer and biocide and
neutralizer exposed populations, respectively. These suspensions
were then incubated for 10 min at room temperature. A third tube,
containing 10 ml PBS, was prepared and served as the viability
control. Each of these solutions was inoculated to a final
concentration of ~100 CFU/ml. Each inoculated suspension was
incubated for an additional 10 min at room temperature. Recovery
of all organisms was performed by plating 0.1 ml on TSA
supplemented with 0.5% glucose TSAG and incubating at 30 to
35C for 3 days for bacteria and on SDA at 20 to 25C for 7 days
for fungi. All plates were examined for recovery of CFUs, and the
populations analyzed are also described. The neutralizer efficacy
(NE) and toxicity of the entire test was determined using all
organisms of the test, where NE was estimated by comparing the
recovery of the challenge organisms in the neutralizer exposed
population and the neutralizer with biocide population, while, NT
was estimated by comparing the recovery of the specific challenge
organism in the neutralizer exposed population and the viability
population. Acceptable NE and NT ratios are defined as 0.75
Sheraba et al.
6567
Table 1. Antiseptics and disinfectants used and their dilution and composition.
S/No.
Biocidal
solution
Purpose
Use
dilution
Sanigel
Antiseptic
75%
Ethanol 62%, deionized water, glycerin, perfume oil, PEG-9 Dimethicone, PEG-9, Isostearate and
acrylic polymer (Formulin, Egypt).
Hospidermin
Antiseptic
75%
42.6 g Ethanol 96%, 3 g potassium thiocyanate, 0.1 g 5-Chloro-2-hydroxy-benzoicacid, Butan-2-one,

colouring additives: E 124 and E 110 and Purified water, in 100g (Lysoform, Berlin).
3
4
5
6
Alcohol
AHD 2000
Sanipine
Sanismell
Antiseptic
Antiseptic
Disinfectant
Disinfectant
75%
75%
1:20
1:20
95% Ethanol (United Company for Chemicals, Egypt).

79.6% Ethanol, lactic acid, fragrance, water, macrogolglycerolcocoate (Lysoform, Berlin).
Sodium lauryl sulphate, QAC, solvents with pine smell (Formulin, Egypt).
Sodium lauryl sulphate, QAC, solvents with fresh smell (Formulin, Egypt).
Lysoformin
3000
Disinfectant
1%
7.5 g Glyoxal, 9.5 g glutaraldehyde, 9.6 g didecyl dimethyl ammonium chloride, in 100 g (Lysoform,
Berlin).
Trichlorol
Disinfectant
0.75%
9
10
H2O2 25%
Chlorax
Disinfectant
Disinfectant
3%
1:16
Composition
80 g Sodium tosylchloramide. 3H2O, sodium lauryl Sulphate, sodium chloride, fragrance, in 100 g
(Lysoform, Berlin).
Hydrogen peroxide 25% (Roam Chene, Belgium, United Co.).
Sodium hypochlorite, surfactant (Chlorax, Egypt).
(75% recovery) (Sutton et al., 2002). All antiseptics and

disinfectants were used at their highest available concentrations in
the market to ensure that the neutralization achieved is applicable
at any equal or lesser concentration.
RESULTS
All experiments were performed for all biocides shown in
Table 1, in use concentration with all neutralizing
solutions shown in Table 2, for each index microorganism
but data shown is for only 75% recovery in the
presumptive test for both the NE and NT comparisons.
Each test was repeated three times and the average
result was calculated and used for evaluation (Tables 3 to
7). Generally, all neutralizers examined showed some
degree of effectiveness as neutralizing agents and no
detectable toxicity. Physiological peptone water (1%) was
a suitable neutralizer for alcohol, AHD 2000,
Hospidermin, Sanigel, Sanipine and Sanismell for all
challenge organisms except B. subtilis. However, 0.1%
Tween 80 in 1% physiological peptone was the most
effective neutralizer to Sanipine and Sanismell when the
challenge organism was B. subtilis. Sodium thiosulphate
(0.6%) was the most effective neutralizer to Clorox
against all challenge organisms. It was also the most
efficient neutralizer to Trichlorol against all organisms
except P. aeruginosa and B. subtilis. Instead, Dey-Engley
was the neutralizer of choice to Trichlorol against P.
aeruginosa and B. subtilis. Dey-Engley was also the
neutralizer of choice to Lysoformin 3000 for all challenge
organisms. PBS was the effective neutralizer to hydrogen
peroxide against all challenge organisms. S. hominis

showed nearly the same results as S. aureus ATCC 6538
(Index microorganism for Gram-positive bacteria) which
revealed that there is no resistant approach for
environmental isolates (Table 7). Consequently, no
neutralizing broth was suitable for all organisms and
biocides. It is important, therefore, to determine NT and
NE separately for each index organism and neutralizer
combination.
DISCUSSION
This study is a part of an environmental monitoring
program aimed at identification of the most common
environmental isolates present in the main vaccine
production facility to be followed by the most appropriate
corrective plane. The proper use of disinfectant and
antiseptics and evaluating their biocidal activity is the first
step in the correction program as stated in USP 34
<1172>. Choice of an ideal neutralizer is not always a
trivial task due to the lack of a universal neutralizer.
Many substances like organic matter, QAC, surface
active agents and heavy metals can interfere with the
activity of antimicrobial agents like antiseptics and
disinfectants. Such interference is of great importance to
be taken into consideration when evaluating the
antimicrobial activity and efficacy. A neutralizer is usually
a mixture of interfering substances to which the antiseptic
or disinfectant is sensitive.
In the present study, Lysoformin 3000 showed to be the
6568
Table 2. List of neutralizers tested.
Neutralizer
A- 1% Physiological peptone water
Peptone
Sodium chloride
Disodium phosphate
Monopotassium phosphate
0.9% Saline
Composition
10.0 g
5.0 g
3.5 g
1.5 g
1000 ml
B- 0.6 % Sodium thiosulphate in 1% physiological peptone water

C- Phosphate buffer saline
D- 0.1% Tween 80 in 1% physiological peptone water.
E- Dey-Engley broth
Sodium thioglycolate
Sodium thiosulfate
Sodium bisulfite
Polysorbate 80
Lecithin (soybean)
Tryptone
Yeast extract
Glucose
Distilled water
1 g/L
6 g/L
2.5 g/L
5 g/L
7 g/L
5 g/L
2.5 g/L
10 g/L
1000 ml
Table 3. Efficacy and toxicity of used neutralizers for disinfectants and antiseptics on S. aureus ATCC 6538 (Index microorganism for Gram-positive bacteria).
S/No.
1
2
3
4
5
6
7
8
Chemical agent name
Neutralizer
Sanigel
Hospidermin
Alcohol
AHD 2000
Sanipine
Sanismell
Lysoformin 3000
Chlorax
1% Physiological peptone
Dey-Engley
0.6% Na2S2O3
Neutralizer exposed
population (CFU)
108
104
107
106
104
103
108
105
Efficacy
Neutralizer with
biocide (CFU)
100
96
97
100
99
98
100
100
Neutralizer
efficiency ratio
0.93
0.92
0.91
0.94
0.95
0.95
0.93
0.95
Neutralizer exposed
population (CFU)
108
104
107
106
104
103
108
105
Toxicity
Viability
population (CFU)
97
88
99
98
100
100
96
94
Recovery
percentage ratio
0.90
0.85
0.93
0.92
0.96
0.97
0.89
0.90
Sheraba et al.
Table 3. Contd.
9
10
Trichlorol
H2O2 25%
0.6% Na2S2O3
PBS
103
108
99
89
0.96
0.82
103
108
93
96
0.90
0.89
Table 4. Efficacy and toxicity of used neutralizers for disinfectants and antiseptics on P. aeruginosa ATCC 9027 (Index microorganism for Gram-negative bacteria).
S/No.
Chemical agent name
Neutralizer
1
2
3
4
5
6
7
8
9
10
Sanigel
Hospidermin
Alcohol
AHD 2000
Sanipine
Sanismell
Lysoformin 3000
Chlorax
Trichlorol
H2O2 25%
Dey-Engley
0.6% Na2S2O3
0.6% Na2S2O3
PBS
Neutralizer exposed
population (CFU)
108
105
107
108
103
102
103
105
102
105
Efficacy
Neutralizer with
biocide (CFU)
99
96
100
99
96
88
92
100
97
98
Neutralizer
efficiency ratio
0.92
0.91
0.93
0.92
0.93
0.86
0.89
0.95
0.95
0.93
Neutralizer exposed
population (CFU)
108
105
107
108
103
102
103
105
102
105
Toxicity
Viability
population (CFU)
113
113
113
113
113
113
118
118
118
118
Recovery
Percentage ratio
1.05
1.08
1.06
1.05
1.1
1.11
1.15
1.12
1.16
1.12
Table 5. Efficacy and toxicity of used neutralizers for disinfectants and antiseptics on A. niger ATCC 16404 (Index microorganism for fungi).
S/No.
1
2
3
4
5
6
7
8
9
10
Chemical agent name
Neutralizer
Sanigel
Hospidermin
Alcohol
AHD 2000
Sanipine
Sanismell
Lysoformin 3000
Chlorax
Trichlorol
H2O2 25%
Dey-Engley
0.6% Na2S2O3
0.6% Na2S2O3
PBS
Neutralizer exposed
population (CFU)
108
108
108
108
108
108
106
107
107
109
Eficacy
Neutralizer with
biocide (CFU)
96
97
93
92
95
91
93
94
97
98
Neutralizer
efficiency ratio
0.89
0.90
0.86
0.85
0.88
0.84
0.88
0.88
0.91
0.90
Neutralizer exposed
population (CFU)
108
108
108
108
108
108
106
107
107
109
Toxicity
Viability
population (CFU)
113
113
113
113
113
113
113
113
113
113
Recovery
percentage ratio
1.05
1.05
1.05
1.05
1.05
1.05
1.07
1.06
1.06
1.04
6569
6570
Table 6. Efficacy and toxicity of used neutralizers for disinfectants and antiseptics on B. subtilis ATCC 6633 (Index microorganism for spores).
S/No.
1
2
3
4
5
6
7
8
9
10
Chemical
agent name
Neutralizer
Sanigel
Hospidermin
Alcohol
AHD 2000
Sanipine
Sanismell
Lysoformin 3000
Chlorax
Trichlorol
H2O2 25%
0.1% Tween in 1% physiological peptone
0.1% Tween in 1% physiological peptone
Dey-Engley
0.6% Na2S2O3
0.6% Na2S2O3
PBS
Neutralizer exposed
population (CFU)
104
104
104
104
105
105
107
108
106
103
Efficacy
Neutralizer with
biocide (CFU)
97
98
95
96
92
97
96
94
92
98
Neutralizer
efficiency ratio
0.93
0.94
0.91
0.92
0.88
0.92
0.90
0.87
0.87
0.95
Neutralizer exposed
population (CFU)
104
104
104
104
105
105
107
108
106
103
Toxicity
Viability population
(CFU)
98
92
94
96
97
98
99
100
100
96
Recovery
Percentage ratio
0.94
0.88
0.90
0.92
0.92
0.93
0.93
0.93
0.94
0.93
Table 7. Efficacy and toxicity of used neutralizers for disinfectants and antiseptics on S. hominis (Index microorganism for environmental isolate program).
S/No.
Chemical agent
name
Neutralizer
1
2
3
4
5
6
7
8
9
10
Sanigel
Hospidermin
Alcohol
AHD 2000
Sanipine
Sanismell
Lysoformin 3000
Chlorax
Trichlorol
H2O2 25%
Dey-Engley
0.6% Na2S2O3
0.6% Na2S2O3
PBS
Neutralizer exposed
population (CFU)
105
104
106
109
105
106
107
105
102
104
most difficult disinfectant to be neutralized and

was neutralized with Dey-Engley broth against
each test bacterium. This finding agrees with
(Sutton et al., 1991; Buck and Rosenthal, 1996)
who reported that Dey-Engley broth was an
effective medium for neutralization of all
Efficacy
Neutralizer with biocide
(CFU)
98
95
92
98
100
93
94
100
97
98
Neutralizer
efficiency ratio
0.93
0.91
0.88
0.90
0.95
0.88
0.88
0.95
0.95
0.94
Neutralizer exposed
population (CFU)
105
104
106
109
105
106
107
105
102
104
disinfectant solutions with the exception of 3%

hydrogen peroxide. Dilution in PBS containing
0.2% sodium thiosulphate (wt/vol) was found to be
an effective neutralizer to hydrogen peroxide; this
finding agrees with (Elkins et al., 1999) who
reported that PBS containing 0.2% sodium
Toxicity
Viability population
(CFU)
118
94
100
113
118
113
113
118
118
92
Recovery
percentage ratio
1.12
0.90
94
1.04
1.12
1.07
1.06
1.12
1.16
88
thiosulphate (wt/vol) was used to neutralize

hydrogen peroxide against catalase found in P.
aeruginosa biofilm. Conversely, Sutton et al.
(1991), Holton et al. (1995) and Coates (1996)
found that 5% thiosulphate and 0.025% catalase
were effective for neutralization of hydrogen
Sheraba et al.
peroxide. 0.5% sodium thiosulphate was observed to be

effective neutralizer for chlorox and trichlorol against all
challenge organisms except P. aeruginosa and B.
subtilis, in that case, Dey-Engley was the best
neutralizer, in agreement with Coates (1996) and Traore
et al. (2002) who reported that saline with 0.5% sodium
thiosulphate was found to be effective neutralizer for
chlorine based disinfectants.
Another finding of this study is that dilution with 1%
physiological peptone was the most effective neutralizer
for alcohol, Hospidermin, AHD 2000 and Sanigel
(alcohol-based gel) against all test bacteria, nearly similar
to results reported by Traore et al. (2002) who stated that
dilution with buffered saline was used because of its
known effectiveness as a neutralizer for alcohol based
hand-wash agents, on the other hand, Adams et al.
(2005) reported that mixture of 0.2% Tween 80, 1.17%
Lecithin, 0.1% Triton and 0.5% sodium thiosulphate in
distilled water was effective neutralizer for alcohol based
gel.
This study showed that dilution with 1% physiological
peptone was the most effective neutralizer for Sanipane
and Sanismel against each test bacterium except B.
subtilis, where in that case it was observed that 0.1%
Tween 80 in 1% physiological peptone is the most
efficient neutralizer, this might be in contrast to Mohamed
(2004) who found Letheen broth to be an effective
neutralizer for QAC.
Conclusion
Evaluation of NE and toxicity should be performed prior
to testing of a new biocide. We have determined the most
effective neutralizers for the most common disinfectants
and antiseptics used in the main vaccine production
facility in Cairo, Egypt as a part in an environmental
monitoring program. Based on our findings, 1%
physiological peptone water, 0.1% Tween 80 in 1%
physiological peptone, 0.6% sodium thiosulphate and
Dey-Engley were the most effective. The efficacy of
neutralizers should be determined using several index
test organisms and environmental isolates if detected.
ACKNOWLEDGEMENTS
The authors would like to thank Dr. Hamdalla Hafez
Zedan, chairman of the Egyptian Company for Production
of Vaccines, Sera and Drugs (EGYVAC) for his
continuous support and help in carrying out the study, Dr.
Mohamed Rabei, Chairman of the Holding Company for
Production of Vaccines, Sera and Drugs (VACSERA) for
his continuous encouragement and Dr. Ramy Karam
Aziz, Associate professor at Cairo University for help in
editing the manuscript.
6571
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DOI: 10.5897/AJMR12.981
Effects of essential oil extracted from Citrullus

colocynthis (CCT) seeds on growth of
phytopathogenic bacteria
Zahra Setayesh Mehr, Nima Sanadgol* and LeylaVafadar Ghasemi
Department of Biology, Faculty of Science, Zabol University, P.O.Box 1568, Zabol, Iran.
Accepted 14 July, 2012
Citrullus colocynthis (CCT) is a non-hardy, herbaceous perennial vine, branched from the base.
Originally from Tropical Asia and Africa, it is now widely distributed in the Sistan phytogeographic
region of Iran. In a search of alternative ways to control plant disease, essential oil from seeds of CCT
was examined for antibacterial properties. The seeds are edible and have a high oil content with a large
proportion of linoleic acid (C18:2) which is important for human nutrition and an essential fatty acid
also contains only traces of linolenic acid (C18:3). Antibacterial activity of oil separated from the seeds
was tested against Xanthomonas campestris, Burkholderia cenocepacia, Pseudomonas syringae and
Agrobacterium tumefaciens. The agar disc diffusion method was used to assess inhibitory effect by
measuring the inhibition zone against the test microorganisms. Antibacterial activity of the seeds oil
was confirmed for all bacterial, but with different ranges. This activity was observed to be doseindependent. X. campestris was the most sensitive bacterium tested. A weak inhibitory effect was found
against Pseudomonas syringae. These results offer a scientific basis for the use of C. colocynthis seed
oil to prevent diseases caused by these bacteria.
Key words: Citrullus colocynthis, phytopathogens, agar disc diffusion.
INTRODUCTION
Bacterial pathogens and their control are a serious
problem in agriculture practice. Many of the currently
available antimicrobial agents for agriculture are highly
toxic, non-biodegradable, and cause extended environmental pollution (Vyvyan, 2002). Diseases caused by
pathogens including bacteria and fungi significantly
contribute to the overall loss in crop yields worldwide
(Savary et al., 2006). Despite the existence of plant
defense mechanisms, a major difficulty encountered is
the lack of effective control agents against some severe
plant bacterial diseases. On the other hand, application of
chemical derivatives has effectively controlled the plants
from bacterial disease but this threatens to contaminate
the environment, hindering the management of diseases
in crops and agricultural products (Burhan, 2009). The
search for agents to cure infectious diseases began
*Corresponding
author.
E-mail:
n.sanadgol@uoz.ac.ir,
sanadgol.n@gmail.com. Tel: +98-915-7444696. Fax: +98-5422240696
long before people were aware of the existence of

microbes. Herbal medicine represents one of the most
important fields of traditional medicine all over the world
(Hamil, 2003). Nowadays, medicinal plants receive
attention to research centers because of their special
importance in safety of communities (Mona, 2002).
The curative properties of medicinal plants are mainly
due to the presence of various complex chemical
substances of different composition which occur as
secondary metabolites (Karthikeyan, 2009). Plant based
natural constituents can be derived from any part of the
plant like bark, leaves, flowers, roots, fruits, seeds, etc
(Gordon and David, 2001). Medicinal and aromatic
plants form a large group of economically important
plants that provide the basic raw materials for indigenous pharmaceuticals, perfumery, flavor and cosmetic
industries. Citrullus colocynthis (Linn.) Schrad.,
(colocynth, wild-gourd or bitter-apple) is an important
medicinal plant belonging to the family Cucurbitaceae. It
is a well-recognized plant in the traditional medicine and
was used by people in rural areas as purgative, antidiabetic
Mehr et al.
and insecticide. Various oils are biocides against a broad

range of organisms such as bacteria, fungi, viruses,
protozoa, insects and plants (Dung et al., 2008;
Gurudeeban et al., 2010). Recent researches are showed
that essential oils of many plants possess antimicrobial
activities and maybe used for the treatment of
different diseases in the near future (Elaissi et al., 2011;
Erkan et al., 2012; Vairappan et al., 2012; Sivasothy et
al., 2012; Makhloufi et al., 2012; Abdelhady and Aly,
2012). There is vast diversity among aromatic and
medicinal plants and different chemotypes of the same
species may grow in the same place and produce
different oils with different activity (Darokar et al., 1998).
The current work presents an evaluation of antibacterial
activity of essential oil from Iranian C. colocynthis and
their inhibitory effect against the growth of some
phytopathogenic bacteria.
6573
(NCCLS, 1993; CLSI, 2009). Bacteria were cultured overnight at

30C. The test samples of oil were dissolved in 5% DMSO.
Dilutions were prepared in a 96-well microtiter plates to get final
concentrations ranging from 0 to 4 g/ml. Finally, 20 l of inoculum
(106 107 CFU/ml) was inoculated onto the microplates and the
tests were performed in a volume of 200 l. Plates were incubated
at 30C for 24 h. The standard reference drug, ampici llin, was used
as a positive control for the tested plant pathogenic bacteria. The
lowest concentrations of tested samples, which did not show any
visual growth after macroscopic evaluation, were determined as
MICs, which were expressed in g/ml. Using the results of the MIC
assay, the concentrations showing complete absence of visual
growth of bacteria were identified and 50 l of each culture broth
was transferred onto the agar plates and incubated for the specified
time and temperature as mentioned above. The complete absence
of growth on the agar surface in the lowest concentration of sample
was defined as the MBC. Each assay in this experiment was
replicated three times.

Oil isolation and extraction
Fresh fruits were collected from south-eastern of Iran during
2010/2011, especially Sistan region in large quantities. The seeds
were generally collected after fruit ripening, between September
and October. Dried seeds were powder and hydrodistilled for 5 h
using a Clevenger apparatus with a water-cooled oil receiver. The
oil was dried over anhydrous Na2SO4 and preserved in a sealed vial
at 4C in the dark until further analysis (yield 0.88 %, w/w).
Test microorganisms
The test organisms used in this study (reference strains) were
Xanthomonas
campestris
pv.
Campestris
(ATCC33913),
Pseudomonas syringae pv. Syringae (B728a), Burkholderia
cenocepacia (HI2424) and Agrobacterium tumefaciens (str. C58).
The stock cultures were maintained in nutrient agar (NA) slant at
4C and sub-cultured monthly. Working cultures were pre pared by
inoculating a loopful of each test microorganism in 3 ml of nutrient
broth (NB) from NA slants. Broths were incubated at 37C for 12 h.
The suspension was diluted with sterile distilled water to obtain
approximately 106 CFU/ml.
Antibacterial testing
The seeds oil was tested for antibacterial activity by the disc agar
diffusion method (Murray et al., 1995). Disk diffusion: 5 mm of sterile
disks were incorporated in 100 l of plant extracts (5 mg/disk). The
disk (6 mm in diameter, Whatman No. 1) was completely saturated
with the extract and allowed to dry. Mueller Hinton (MH) agar plates
were swabbed with test bacteria and six extract disks with one of
the standard positive control disks (streptomycin) was placed on the
MH agar plate. Dimethyl sulfoxide (DMSO) was taken as the
negative control (10% DMSO did not show any antibacterial
activity). The plates were incubated at 37C for 24 h and the
diameter of the inhibition zones were measured in mm.
Minimum inhibitory and minimum bactericidal concentrations
Micro-dilution susceptibility assay was performed using the NCCLS
and CLSI methods for the determination of minimum inhibitory
concentration (MIC) and minimum bactericidal concentration (MBC)
The data obtained for antibacterial activity of essential oil and

various extracts were statistically analyzed and mean values were
calculated. A Students t test was computed for the statistical
significance of the results at p<0.05. All experiments were
performed at least, three times (unless indicated otherwise) and
were highly reproducible.

The oil obtained from the seeds of CCT revealed
relatively potential antibacterial effects at the
concentrations utilized against all selected plant pathogenic bacterial (Table 1). Xanthomonas campestris pv.
Campestris (ATCC33913) was found most sus-ceptible
pathogenic bacteria to the oil of CCT seeds. The
diameter of the inhibition zones of the oil against the
tested strains of Xanthomonas were in the range of 14-20
mm. On the other hand, standard streptomycin showed
both lower antibacterial (Xanthomonas) and comparable
(Burkholderia) effect as compared to the seeds oil
dependent of bacterial species (Table 1). The minimum
inhibitory concentration of the extracts varied between
35.0 - 81.86 g/ml while the minimum bactericidal concentration was between 59.0-123.0 g/ml (Table 1). As
shown in Table 1, the minimum concentrations of seeds
oil were found more susceptible to the tested plant
pathogenic bacteria of X. campestris pv. Campestris
(ATCC33913) as compared to the other bacteria. The
seeds oil had a detrimental effect on Xanthomonas. The
seeds oil displayed remarkable antibacterial activity
against tested strains such as X. campestris pv.
Campestris (ATCC33913), Burkholderia cenocepacia
(HI2424) and Agrobacterium tumefaciens (str. C58) with
MIC and MBC values of 35.26-70.1, 62.5-250 and 62.5250 g/ml, respectively. On the other hand, the seeds oil
displayed better antibacterial effect against the tested
bacterial pathogens as MIC values as compared to
standard streptomycin (MIC: 300-500 g/ml). However, in
some cases, the extracts had a higher antibacterial effect
6574
Table 1. Antibacterial activity, Minimum inhibitory concentrations (MIC) and minimum bactericidal concentration (MBC) of seeds oil
of CCT against selected plant pathogenic bacterial.
Bacterial pathogen
Xanthomonas campestris pv. Campestris (ATCC33913)
Pseudomonas syringae pv. Syringae (B728a)
Burkholderia cenocepacia (HI2424)
Agrobacterium tumefaciens (str. C58)
Seeds oil
c
IZ
*
15.00.0
*
8.00.0
14.00.0
*
12.00.0
MIC
35.0
65.2
37.00
81.86
MBC
70.0
65.0
59.0
123.0
Standard
IZ
11.00.0
*
13.00.0
14.00.0
*
15.00.0
MIC
500
350
400
300
oil used at 1,000 g/disc; bStandard: streptomycin (20 g/ml, all values are in g/ml); * P<0.05 significant, cData are expressed as the
diameter of inhibition zones (IZ) in mm; Values are given as an average of triplicate experiments.
as compared to the standard antibiotic which might be

due to the presence of highly bioactive compounds in
seeds oil. The increased awareness of the environmental
problems associated with conventional non-biodegradable
agrochemicals has led to the search for non-conventional
chemicals of biological origin for the management of
post-harvest disease in fruits and vegetables (Abhay et
al., 2012). Bioactive compounds are naturally produced in
the plants and among of them essential oil are important
for the physiology of plants contributing properties confer
resistance against microorganisms, other organisms and
even antibacterial activities (Abhay et al., 2012; Cantore
et al., 2009; Kotan et al., 2010). The observed antibacterial properties of CCT essential oil show its potential
for the practical use of the essential oil towards plant
pathogenic bacteria as a natural bactericide and it was
similar with previous studies (Marzouk et al., 2010). The
preliminary qualitative phytochemical screening of CCT
was reported in previous paper (Najafi et al., 2010;
Gurudeeban et al., 2010). Analysis of CCT fatty acid
methyl esters showed the presence of palmitic, stearic,
oleic, linoleic and linolenic acids in appreciable quantities
(Kulkarni et al., 2012). The obtained results suggest that
the use of CCT oil as antibacterial agent may be
judiciously applied to prevent the decay of fruits and
vegetables due to bacteria. The isolation and purification
of the phytochemical followed by a detailed study might
result in identification of lead compound and thus a
potential cure for the diseases caused by this bacteria.
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DOI: 10.5897/AJMR12.1054
Development and evaluation of a novel TaqMan

fluorescence probe-based real-time reverse
transcriptase polymerase chain reaction assay for
detection and quantification of West Nile virus
Lijun Shi1*, Huiqiong Yin2, Jingang Zhang2, Zhan-zhong Zhao1 and Gang Li1
1
Institute of Animal Science and Veterinary Medicine, Chinese Academy of Agriculture Sciences, Beijing 100193, China.
2
Institute of Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
In order to improve and accelerate the detection of West Nile virus (WNV), a rapid and specific real-time
reverse transcription polymerase chain reaction (rtRT-PCR) was established. Primers and probe were
designed according to the conservative sequence of capsid protein gene of WNV. Tenfold successive
dilutions of positive WNV DNA were used to measure the sensitivity of rtRT-PCR. The amplifying curve
1
showed that this method could successfully amplify 10 copies/l WNV gene, while reference to
Japanese encephalitis virus (JEV) and blank control were all negative. The assay system showed high
reproducibility with coefficient of variation (CV) < 2%. The detection of WNV can be completed within 2
to 3 h. By detecting cDNA samples (n = 55) with rtRT-PCR and the conventional PCR assay, the
established rtRT-PCR showed 96.36% (37 + 16/55) coincidence rate with the conventional PCR. All the
results showed that the newly established rtRT-PCR assay was shown to be a rapid, sensitive and
specific test for detecting WNV.
Key words: West Nile virus, capsid protein gene, real-time RT-PCR.
INTRODUCTION
West Nile virus (WNV) is an arbovirus (genus Flavivirus;
family Flaviviridae) that was first isolated in a fever
womans blood in Uganda in 1937. WNV has a wide
geographical range that includes portions of Europe,
Asia, Africa, Australia (Kunjin virus) and North, Central
and South America. According to the Centers for Disease
Control and Prevention of USA, from 1999 to 2010 there
have been over 30,622 human cases of confirmed
symptomatic WNV infection in the USA with over 1163
fatalities (De Filette et al., 2012). In the recent years, it
also was separated from migratory birds in some regions
of Xinjiang Uigur autonomous region of China (Petersen
and Roehrig, 2001; Sambol et al., 2009). WNV is
*Corresponding author. E-mail: ljshi2008@yahoo.com.cn. Tel:

+86-10-62819634. Fax: +86-10-62819364.
transmitted to vertebrates by infected mosquitoes or tick

vectors. Mosquitoes of the genus Culex are the main
vectors of WNV (Esteves et al., 2005; Figuerola et al.,
2007; Lanciotti et al., 2000). Wild birds are the reservoir
host of WNV (Brault et al., 2007; Komar et al., 2003;
Wodak et al., 2011). WNV can disseminate through blood
transfusion, which brought huge threat towards health
safety of humankind (Nicolle et al., 2004).
Neither a vaccine nor an effective therapy has been
developed against WNV infection in humans. WNV
diagnosis is achieved routinely by serological assays;
however, there is cross-reactivity with other flaviviruses.
For this reason, plaque reduction neutralization tests
(PRNTS) must be done as the reference assay for
specific diagnosis. PRNT is a laborious and timeconsuming technique that has to be carried out in biosafety level 3(BSL-3) facilities with the consequent risks
for the personnel involved in live virus manipulation
Shi et al.
(Alonso-Padilla et al., 2010; Dauphin and Zientara, 2007).

So development of an effective and safe assaying
method is essential for the prevention of WNV. For forecasting the infection and epidemiology of WNV in
humans, horses, and other animals, we need to establish
a monitoring system (Bakonyi et al., 2005; Bakonyi et al.,
2006).
This study developed a real-time reverse transcription
PCR with a novel TaqMan probe. The assay should be
economical for large-scale routine test of clinical samples
and blood products in China.
Virus strains, virus gene, reagents and sample
The WNV NY99 (AF 202541) strain and WNV cDNA sample were
kindly provided by the Department of Haematology of the University
of Cambridge. The live vaccine of JEV (Japanese encephalitis
virus) was maintained in our lab. The Premis Ex Taq Hot Start
version (TaKaRa, Dalian, China) was used in all real-time PCRs.
The AMV system, plasmid kit, gel extraction kit and pGEMT-easy
vector were all bought from the Promega Company (Madison,
Wisconsin, USA); the DNA/RNA nucleic acid extraction kit was
bought from AXYGEN Company (San Francisco, California, USA).
Primers and probe design

Specific real-time primers and probe were designed according to
the conservative region of capsid gene of WNV. The oligonucleotide
sequences of the primers and the TaqMan fluorescence probe were
as follows: Forward Primer (WP1): 5'-CAAGAGCC GGGCTGTCAATA-3'; Reverse Primer (WP2): 5'-CTTCAGTCCAATCAAGGACAA3'; TaqMan probe (WP): FAM-ATGCTAAAACGCGGAATGCCCCGCGTGTT-TAMRA. The primers and probe were synthesized by
Beijing Sun-bio Company.
Preparation of standard templates of WNV

The RNA extraction of WNV was accorded to the manual of
AXYGEN RNA extraction kit. Total RNA was extracted directly from
200 l of the supernatant of infected cell cultures. The amplified
segments were subcloned into pGEM-T easy vectors. Sequencing
of the amplified segment was carried out by Beijing Sun-bio
Company. Ten-fold successive dilutions of positive WNV DNA (10 7
to 103 copies/l) plasmids were used as standard templates of
WNV.
Real-time amplification
The standard templates (107 to 103 copies/l) were amplified by
real-time. Real-time PCR was performed in a final volume of 20 l
containing 2 l 10PCR Buffer, 4mM MgCl2, 0.25 mM dNTPs, 2 M
CP1, 2 M CP2, 2 l WNV plasmid DNA templates, 5U TaqTM, 1 M
TaqMan probe. The RT-PCR mixtures were followed by
denaturation at 94C for 5 min and 40 cycles of 94C for 30 s, 60C
for 30 s and 68C for 30 s.
Specificity, sensitivity and reproducibility of the real-time PCR

assay
The specificity of assaying system was determined by amplified
6577
WNV gene segments and JEV gene segments with the same
primers, probe and reaction system of WNV. To measure the
sensitivity of real-time assay for WNV, the assay was performed
using serially diluted standard templates. The reproducibility of intra
assay and inter assay was evaluated by Coefficient of Variance
(CV).
Comparison between the conventional PCR and real-time PCR

in human samples detection
Mimic cDNA samples extracted from human serum were detected
with the procedure, and detected with the Conventional PCR assay
(Yeh et al., 2010) to confirm the coincidence. 55 cDNA samples and
39 positive cDNA samples from humans infected with WNV were
kindly provided by the Department of Haematology of the University
of Cambridge, and 16 negative cDNA samples were extracted from
human serum maintained in our lab.
RESULTS
Establishment of real-time PCR standard curve
Ten-fold serial dilutions of the standard templates were
used in the real-time PCR assay. The Ct values for each
dilution were measured in duplicate (Figure 1).
The log-linear regression plot generated from the
collected data showed a strong linear relationship (Figure
2). The regressive equation: Y = -2.225X + 46.375; r =
0.967. By using the regressive equation, we were able to
quantify the amount of unknown samples.
Specificity of the real-time assay

To determine assay specificity, JEV live vaccine strains
and WNV gene were tested in the real-time PCR assay.
1
The detection limit of the assay was determined to be 10
copies/l. The result indicated that the assaying system
could not amplify the JEV gene segments. Thus, the
results indicated a high specificity for assaying WNV.
Reproducibility of the real-time assay

The reproducibility of inter assay was assessed by testing
the standard templates in different days. The
reproducibility of intra assay was assessed by testing the
standard templates in 6 repeats. The assay system
showed high reproducibility with CV < 2%.
Detection specimens with the real-time PCR and the

conventional PCR
By the real-time PCR and the conventional PCR assay,
the cDNA samples (n = 55) were tested respectively. The
established real-time PCR showed 96.36% (37+16/55)
coincidence rate with the conventional PCR (Table 1).
6578
Figure 1. Establishment of the real-time PCR standard curve. 1, 1 107 copies/l; 2, 1 106 copies /l; 3, 1 105 copies/l; 4, 1
104 copies /l; 5, 1 103 copies /l.
Figure 2. Standard amplification regression curve.
Table 1. Coincidence rate between the real-time PCR and

conventional RT-PCR.
Conventional RT-PCR
Positive
Negative
Total
Real-time PCR
Positive
Negative
37
0
2
16
39
16
Total
37
18
55
DISCUSSION
Although virus isolation is still considered as the gold
standard, it needs high-security laboratories and many
days to obtain results. The novel real-time RT-PCR
assays for the detection of WNV, which are faster and
more sensitive than virus isolation, have been developed

in recent years. Real time fluorescence quantitative PCR
was developed based on conventional PCR, and it has
several advantages over conventional PCR. Real-time is
more sensitive and yields more information. Detection,
classification and quantitation take place within one tube.
Real-time have been extensively used for detection of
many kinds of pathogens (Wacharapluesadee et al.,
2011; Jansen et al., 2011; Bennett et al., 2011).
TaqMan probe method is to use the 5' end with
fluorescent substances (such as: FAM), 3'-end with
fluorescence quenching the material (such as: TAMRA).
The fluorescent probes in TaqMan assay are known to be
target specific and sensitive to mismatches. The real-time
PCR assay permits the simultaneous detection and
quantification of DNA. It is useful for understanding the
pathogenesis of the disease and the mechanisms of virus
Shi et al.
transmission by enabling the investigation of viral

dynamics (Yang et al., 2009; Mackay et al., 2002).
Compared to the conventional RT-PCR assay, this real1
time RT-PCR was more sensitive and could detect 10
copies/l WNV gene whereas the conventional RT-PCR
3
had a detection limit of 10 copies/l WNV gene. Lanciotti
et al. (2000) developed a TaqMan qRT-PCR for rapid
detection of WNV in human clinical specimens, this
TaqMan RT-PCR could detect less than 1 PFU of virus.
Compared with others, the first limitation of this present
study is that the number of samples is small. However,
even with this small number of samples, it also revealed
that the developed real-time RT-PCR assay could be
useful for rapid and sensitive detection of WNV.
Secondly, restricted by policy of China, we only detected
the cDNA samples. Finally, this study only detected WNV
in human samples. Further evaluation of this assay to
detect WNV in mosquitoes, birds and horses is needed.
With the increase of human movement, the migration of
migratory birds, and the input of foreign blood products,
the risk of WNV emergence in China is eminent. So, it
was necessary to establish an assay method to detect
WNV in China. The amplified segments in this study
chose the most conservative capsid area of WNV genes.
The development of the real-time RT-PCR assay
described earlier represents a new step towards the
control of exotic infectious diseases in China. The realtime RT-PCR described here for detection and
quantitation of WNV has been shown to be sensitive and
specific. It could be used for monitoring in WNV outbreak
areas or as a screening method for WNV eradication
strategies.
ACKNOWLEDGEMENT
This study was supported by the Central Public-interest
Scientific Institution Basal Research Funds (2010 JS-2)
of China.
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DOI: 10.5897/AJMR12.1204
Microbial water quality in the upper Olifants River

catchment: Implications for health
W. J. le Roux1*, L. M. Schaefer1 and B. Genthe2
1
Natural Resources and the Environment, CSIR, Pretoria, South Africa.

Natural Resources and the Environment, CSIR, Stellenbosch, South Africa.
The Olifants River has been described as one of the most environmentally polluted rivers in Southern
Africa, particularly in the upper catchment. Untreated water from the catchment may be used for many
purposes including irrigation, recreation, consumption, and domestic purposes. As poor quality water
remains a leading cause of morbidity and mortality, it is essential to understand the health risks that the
water users in the Olifants catchment are exposed to. The aim of this research was to identify the extent
of the microbiological pollution in the upper Olifants River catchment and determine the sources of the
microbial contaminants by monitoring faecal indicator levels and selected water-borne pathogens. It
was shown that sections of the upper Olifants River catchment are highly contaminated with faecal
indicator bacteria and pathogenic micro-organisms. A quantitative microbial risk assessment was also
performed and it showed that the polluted waters pose an unacceptably high risk to water users within
this catchment. In all regions where extreme levels of faecal pollution were observed, the source could
be traced back to inadequate wastewater treatment. The microbial water quality in the upper Olifants
River catchment could therefore be remediated to a large extent by ensuring proper functioning of
wastewater treatment works.
Key words: Faecal indicator organisms, Olifants river catchment, wastewater, pathogens, quantitative microbial
risk assessment, microbial water quality.
INTRODUCTION
In South Africa where water is a scarce resource, the
water quality of natural resources is of paramount
importance. Apart from serving as a source of drinking
water (treated or untreated), water needs to be fit for a
variety of other domestic and economic activities. The
Olifants River is presently one of the most threatened
river systems in South Africa (Ballance et al., 2001; Van
Vuuren, 2009). There is an intense demand for natural
resources in the catchment, and rigorous mining activities
occur in most of the nine secondary catchments. The
South African Department of Water Affairs State of the
Rivers Report on the Olifants River system, states that
river ecosystems in this area are generally in a poor to
fair condition. Mining-related disturbances were seen as
*Corresponding author.
(+27)12 841 2189.
E-mail:
wleroux@csir.co.za.
Tel:
the main cause of impairment of river health in the upper

parts of the catchment, with the exception of the lower
reaches of the Olifants River, which are protected by
conservation activities (Ballance et al., 2001). However,
most of the water quality parameters currently routinely
measured in the Olifants River catchment by the National
Chemical Monitoring Programme have little relevance to
human health (de Villiers and Mkwelo, 2009).
Micro-organisms may adversely affect water quality, as
microbial pathogens present in water can cause serious
human disease. According to the World Health
Organization diarrhoeal disease linked to unsafe water
supplies remains a major contributor to mortalities in
developing countries (WHO, 2004). The health risks are
of
particular
concern
for
immunocompromised
individuals, who are more susceptible to infection and
disease than healthy individuals. Water resources that
are contaminated by pathogenic micro-organisms would
in most instances be unsuitable for use in untreated form,
Roux et al.
6581
Figure 1. The upper Olifants River catchment study area. Routine sampling sites, numbered from
1 to 12, are indicated.
and in addition pose a health risk to water users in

applications that do not necessarily involve consumption,
such as bathing, washing of clothes, and gardening.
Catchments can also be regarded as the first potential
barrier to pathogen hazards in the water supply system
(Ferguson et al., 2007). Reducing pathogen loads
exported from environmental sources to drinking water
reservoirs is thus an important priority in applying a riskbased approach to managing water supplies.
Two approaches can be used to determine the
microbial quality of a water resource; monitoring for the
presence of pathogenic micro-organisms or alternatively
assessing the levels of faecal indicator organisms.
Indicator organisms (like Escherichia coli) are valuable
tools to measure the occurrence and extent of faecal
contamination in water sources.
These organisms act as indicators of faecal pollution,
and have similar fate, transport, and survival
characteristics to the related bacterial pathogens that
they represent (Wu et al., 2011). Methodologies to
ascertain the presence of pathogens, and their
concentrations, are generally expensive and timeconsuming, but provide valuable information that can be
used to perform quantitative microbial risk assessments.
In contrast, the monitoring of faecal indicator organism
levels is relatively rapid and inexpensive, but relies on the
assumption that the chosen indicator is an accurate
surrogate for pathogen occurrence. Various South
African and international guidelines exist that state the
acceptable limits of faecal indicator organisms in water,

the most applicable being the South African Water
Quality Guidelines (DWAF, 1996). Other standards such
as the Health Guidelines for the Use of Wastewater in
Agriculture and Aquaculture (WHO, 1989), also provide
good guidance.
In order to manage and mitigate health risks,
monitoring of environmental waters for the presence of
water-borne pathogens and high levels of faecal
indicators is required. In this study faecal indicator levels
and selected water-borne pathogens were monitored in
the upper Olifants River catchment.
The data was used to perform a quantitative microbial
risk assessment, and was also used to identify possible
sources of microbial contaminants. Insights gained can
be used to shape and guide mitigation strategies for the
remediation of microbial water quality problems within
this catchment.
METHODOLOGY
Study area
The Olifants River catchment is located in the Mpumalanga
province of South Africa and covers 54, 570 km2. The Wilge,
Bronkhorstspruit, and Klein Olifants Rivers are tributaries of the
Olifants River that, together with the Olifants River, originate in the
Highveld grasslands (Ballance et al., 2001). The study was
conducted in the upper section of the Olifants River catchment
(Figure 1).
6582
Table 1. Formulae used to calculate daily risk of infection.
Description
Formulae
Reference
Equation 1
Pi 1 1
(2 1)
N 50
Equation 2
Pi 1 (1 d / )^
Equation 3
Pi 1 exp( rN )
WHO
(2001)
Haas et al.
(1999)
Haas et al.
(1996)
Pi: Probability (risk) of infection, d: dose or exposure (number of organism

ingested based on consumption of 100 ml per day resulting from
accidental or unintentional ingestion), : parameter characterised by
dose-response relationship, : parameter characterised by doseresponse relationship, N50: median infectious dose, r: parameter
characterised by dose-response relationship.
Microbial monitoring
Microbial water quality was monitored over a two year period.
During the first year, faecal indicator counts (E. coli) levels were
monitored bi-monthly at eleven sampling sites (Figure 1). Sampling
at site 7 (included initially) was discontinued as the stream was
contaminated with industrial pollution to a point where no viable
bacteria could be detected. Pathogens (Salmonella species,
Shigella species, Vibrio cholerae, Giardia species, Cryptosporidium
species, Norovirus and Enterovirus) were tested for (bi-monthly) at
the sites that exhibited high E. coli counts (Table 2). Faecal
indicator counts were also measured at 12 additional sites during
the year to aid in identifying sources of microbial pollution, and on
average, these sites were monitored on three different occasions.
During the second year, 86 sample sites (Global Positioning
System (GPS) coordinates available on request) were monitored for
E. coli during low flow conditions using a once-off sampling
approach.
Quantification of E. coli was determined using the Colilert Most
Probable Number (MPN) method (IDEXX, USA). This was carried
out according to the manufacturers protocol. For detection of
Salmonella spp., one litre water samples were filtered and the
filtrate incubated overnight in 30 ml of Selenite Broth (Merck,
Germany). Cells were harvested from the broth and the DNA was
extracted using InstaGene Matrix (Biorad Technologies, USA).
Salmonella was detected by real-time polymerase chain reaction
(PCR) targeting the invA gene (Malorny et al., 2003). Bacteria
belonging to the genus Shigella were detected by concentrating
one litre water samples (by filtration), followed by overnight
incubation of the filtrate in 30 ml of Peptone Broth (Biolab, South
Africa). Cells were harvested from the broth and DNA was extracted
using InstaGene Matrix (Biorad Technologies, USA). Shigella
(virulent types) and/or enteroinvasive E. coli (EIEC) were detected
by real-time PCR targeting the ipaH (invasion plasmid antigen)
gene (Theron et al., 2001).
For the detection of V. cholerae, one litre water samples were
filtered and the filtrate was incubated overnight in 30 ml Alkaline
Peptone Water (APW) (Merck, Germany). Cells were harvested
from the broth and DNA was extracted using InstaGene Matrix
(Biorad Technologies, USA). For the detection of V. Cholerae (all
strains) the gene coding for the Outer Membrane Protein (ompW)
was targeted using real-time PCR (Nandi et al., 2000; le Roux and
van Blerk, 2011). For the detection of enterotoxigenic strains, the
ctxAB genes were targeted in a real-time PCR amplifying a section
of the cholera toxin A and B sub-unit (Goel et al., 2005; le Roux and
van Blerk, 2011).
The water was analysed for the presence of Enterovirus by

concentrating 10 L (using ultra-filtration) of water per sampling site.
Genetic material was extracted from the concentrate, and
quantitative real-time PCR was performed using TaqMan
technology and primers and probes as described by Fuhrman et al.
(2005). The same process was used to test for the presence of
Norovirus, but with the real-time PCR assay making use of primers
and probes as described by Loisy et al. (2005) and da Silva et al.
(2007).
Protozoan parasite (Giardia and Cryptosporidium) analysis was
carried out according to the EPA 1623 method (EPA, 2010). Briefly,
parasites were concentrated (from 10 L of water) using 1.2 m
membranes filters, separated from the filtrate by immuno-magnetic
separation (Invitrogen, Norway), labelled with fluorescent speciesspecific antibodies (Cellabs, Netherlands), and counts were
obtained by fluorescence microscopy.
Quantitative microbial risk assessment (QMRA)

The average counts and/or detection data for indicator organisms,
pathogenic bacteria, viruses and parasites were used to perform a
quantitative microbial risk assessment. Doses were calculated
based on the volume used for assessment (assuming, based on
observations, that a conservative volume of 100 ml of untreated
river water was consumed). The formulae used to calculate the
daily risk of infection in the quantitative microbial risk assessment is
given in Table 1.
Pathogen specific parameters used in the probability of infection
formulae are given as follows. Equation 1: Salmonella spp. N50 =
23600 = 0.21 (Haas et al., 1999), Shigella spp. N50 = 1120 =
0.16 (Haas et al., 1999), and Vibrio spp. N50 = 243 = 0.25 (Haas
et al., 1999). Equation 2: Norovirus N50 = 50 = 0.022 (Fankem,
2008), Enterovirus (Poliovirus 1) = 0.097 = 13020 (Haas et al.,
1993), and E. coli = 0.395 = 2.473 (Strachan et al., 2005).
Equation 3: Giardia spp. r = 0.0198 (Teunis et al., 1996) and
Cryptosporidium spp. r = 0.00419 (Teunis et al., 1996).
For the QMRA making use of quantitative pathogen data, the
average concentrations per 100 ml were calculated from the results
of five sampling rounds and used in the risk calculations as
described earlier. Where only qualitative data was available
(presence/absence), namely for Salmonella, Vibrio, and Shigella,
the number of times the organisms were detected was used as a
conservative input for the dose calculation. E. coli concentrations
were adjusted to represent pathogenic bacteria numbers. To
achieve this, a ratio was calculated based on the lowest
concentration of E. coli detected when pathogens were detected.
The ratio used for the E. coli pathogenic to non-pathogenic in this
study was 200.
RESULTS
Eleven sites (Figure 1) within the upper Olifants River
catchment were monitored on five occasions (bi-monthly)
over one year for faecal indicator levels. Six of these sites
(located in the Olifants-, Wilge- and Klip River, and
tributaries of the Klein Olifants River) were also
monitored for the presence and/or levels of seven waterborne pathogens. A summary of the monitoring data is
provided in Table 2.
High levels of E. coli were recorded at three sites (Sites
6, 8, and 9), and these sampling points were all located in
urban areas and were directly downstream of wastewater
treatment works (WWTWs). A fourth sampling point (Site
Roux et al.
6583
Table 2. Summary of pathogen and indicator organism detection results for five sampling rounds.
Site
Site 1
Site 2
Site 3
Site 4
Site 5
Site 6
Site 8
Site 9
Site 10
Site 11
Site 12
E. coli (average)
most probable
number/100 ml
34
264
441
364
611
44,110
15,999
13,632
2,319
723
163
Norovirus
(average)
virions/10 L
ND
ns
ns
ns
ns
162
48
828
ND
ND
ns
Mean counts
Enterovirus
(average)
virions/10 L
ND
ns
ns
ns
ns
1620
ND
2760
ND
ND
ns
Giardia
(average)
cysts/10 L
0.5
ns
ns
ns
ns
ND
ND
0.25
7.5
1
ns
Cryptosporidium
(average)
cysts/10 L
ND
ns
ns
ns
ns
ND
ND
0.25
1.2
0.25
ns
Salmonella (%
detection out of
5 samples)
40
ns
ns
ns
ns
80
80
80
40
40
ns
Percentage detection
Shigella (%
Vibrio cholerae
(% detection out
detection out of 5
of 5 samples)
samples)
ND
ND
ns
ns
ns
ns
ns
ns
ns
ns
20
60
40
60
100
80
60
ND
60
ND
ns
ns
ns: Not scheduled, ND: not detected. Site 7 is not included in the table as no viable micro-organisms were detected in early sampling efforts, and sampling at this site was discontinued.
10, situated downstream of a cattle feedlot) also

exhibited elevated E. coli levels. The sites with the
highest levels of E. coli also harboured the most
water-borne pathogens. A site in the Wilge River
(Site 1) had low indicator bacteria levels and
similarly few water-borne pathogens were
detected. Bacterial pathogens were present at
many of the tested sites. Salmonella spp. and V.
cholerae (non-enterotoxigenic) were highly
prevalent, with Shigella spp. detected at fewer
sites. It is noteworthy that the two protozoan
parasites (Giardia and Cryptosporidium) were
more prevalent at sites 10 and 11 as these sites
were directly down-stream of cattle feed-lots.
Giardia was detected more often than
Cryptosporidium.
The data presented in Table 2 was used to
perform a quantitative microbial risk assessment.
Figure 2 shows the combined probability of
infection (risk of infection of all pathogens
summed) for the seven pathogens that were
monitored for during the study, the pathogens

were: Salmonella spp., Shigella, V. cholerae (nonenterotoxigenic), Cryptosporidium spp., Giardia
spp., Norovirus and Enterovirus. The risk was
based on a conservative estimate of a once-off
consumption of 100 ml of untreated water.
Water from site 9 (a tributary of the KleinOlifants River) resulted in the highest risk of
infection (Pi = 26%), followed by site 10 (a
tributary of the Klein-Olifants), site 8 (Olifants
River), and site 6 (Brugspruit/Klip River). The risks
ranged from 26% probability to 12% probability
based on very conservative single event exposure
consumptions. In all sites, with the exception of
site 10, the high risk was due to the presence of
Norovirus followed by the bacterial pathogens, V.
cholerae and Shigella. The high risk of infection
calculated for site 10 was due to the presence of
Giardia spp.
The seven pathogens represented in Figure 2
are only a small fraction of the total pool of water-
borne pathogens that may be present in

contaminated waters. In an effort to provide a
representative risk, E. coli was used as a
surrogate for pathogens in calculating the
probability of infection as described earlier (Figure
3).
Understandably the sites with the highest levels
of E. coli (sites 6, 8, 9, and 10) had the highest
probability of
infection. Once again the risk assessment was
based on the consumption of 100 ml of untreated
water. The probability of infection at these sites
ranged between 70 and 82%.
In order to gain insight on the sources of
microbial pathogens, 86 additional sample sites
were sampled in the upper Olifants River
catchment for faecal indicator levels. All major
tributaries of the Wilge, Klein Olifants, and Olifants
Rivers were sampled during low flow conditions
using a once-off sampling approach. A summary
of the results is given subsequently.
Percentage
(%)
6584
Percentage
(%)
Figure 2. Combined probability of infection (Pi) for the seven water-borne pathogens monitored in this
study.
Figure 3. Risk of infection calculated using E. coli as a surrogate for water-borne pathogens.
The Wilge River had many sites where moderate E.

Coli counts were measured (100 to 1000 MPN/100 ml),
with only one site exceeding 1000 MPN/100 ml. In the
Olifants River, eight sites had E. coli numbers above

1000 MPN/100 ml. Six of these eight sites were located
directly downstream of WWTWs, one was directly down-
Roux et al.
stream of an urban area and another was situated close

to an intensive mining area. From a microbiology
perspective the Klein Olifants River had fairly good
quality water. However, the water quality in the vicinity of
urban and intensive mining areas was found to be poor at
certain sites. Elevated faecal indicator numbers were
recorded in the Klein Olifants as it flowed through
Middelburg. The microbial quality improved downstream
of the WWTW indicating that this facility may not be the
source of contaminants. Further upstream in the Klein
Olifants River three sites had intermediate faecal
indicator counts (100 to 1000 MPN/100 ml), and this area
is characterized by intensive mining. The only other
sampling site to show very high numbers of faecal
indicator bacteria was located directly downstream of a
WTWW on the outskirts of the town of Hendrina.
Of the 86 sites sampled, 12 sites had E. coli levels
exceeding 1000 MPN/100 ml. Of the 12 sites, 7 were
located directly downstream of WWTWs, two sites were
directly downstream of urban areas, one was located in
an area characterized by intensive mining activities, and
another two sites were located in agriculture regions. On
average, the E. coli levels were an order of a magnitude
higher for sites linked to WWTWs as compared to nonpoint sources like agriculture and other intensive land
uses (average E. coli counts were 40,000 MPN/100 ml
and 2700 MPN/100 ml, respectively). Where possible
water samples were taken from the stream or river
directly upstream and downstream of the effluent
discharge point to confirm that a specific treatment works
was the cause of the contamination.
In other cases streams only existed as a result of
effluent discharge and had no other source of flow. In this
regard, in four of the seven sites linked to WWTWs, the
faecal contamination could only be accounted for by
below par WWTW functioning. Indications are that the
three remaining sites located close to WWTWs also
receive their contaminants from inadequate wastewater
treatment facilities.
DISCUSSION
Faecal indicator levels
Many sampling sites had high counts of E. coli, indicating
that sections of the upper Olifants River system were
heavily contaminated by faecally derived microbes (Table
2). Sites that exhibited lower numbers of E. coli were
generally located in regions of lower population density,
whereas sampling sites located closer to settlements and
towns generally had higher counts. The exceptions to this
rule were two sites located in tributaries of the Klein
Olifants River. The periodically high counts observed
here could be explained by runoff from nearby cattle
feedlots. Indicator counts for sites that were not directly
6585
downstream of WWTWs were higher during the high

rainfall months of January and March than the low rainfall
months of July and September. Surface water runoff from
polluted areas is known to carry high loads of microbial
contaminants (Doran and Linn, 1979; Curriero et al.,
2001; Tate et al., 2006). Therefore, the most likely
explanation for the increase in E. coli counts is the high
rainfall that preceded the sampling days. The sites that
receive inflow from WWTWs did not show a decrease in
faecal indicator counts during the dry months, and
instead showed a general increase. This is most likely
due to the effluent being more concentrated in the
absence of rain water runoff.
Pathogen monitoring
Bacterial pathogens were readily detected at the sampled
sites, with Salmonella being the most prevalent (Table 2).
Salmonella causes diseases such as typhoid and
paratyphoid. The widespread occurrence of this organism
in waters from the upper Olifants River catchment is a
concern. This bacteria remains an important pathogen,
with an estimated 216,000 deaths per year (globally)
attributed to typhoid (Crump et al., 2004). Another
devastating disease, dysentery, causes an estimated 1
million deaths per year globally (Niyogi, 2005). Dysentery
is caused by the bacteria of the genus Shigella, and was
readily detected at three sampling sites in the upper
Olifants River catchment (Table 2). The causative agent
of cholera is the bacterium V. cholerae, and more
specifically enterotoxigenic V. cholerae sub-types.
Although, V. cholerae was widely detected in water from
the upper Olifants River (Table 2), none of these strains
were enterotoxigenic. The detected strains cannot cause
epidemic cholera, but may cause sporadic disease as
non-toxigenic strains may cause opportunistic infections
(Bubshait et al., 2000). Predictably, the sites that had the
highest faecal indicator (E. coli) counts also harboured
microbial pathogens more often (Table 2), highlighting
the value of indicator bacteria during monitoring (Wu et
al., 2011).
The protozoan parasites (Giardia and Cryptosporidium)
were detected at two sampling sites; sites 10 and 11
(Table 2), situated in close proximity and directly
downstream
of
cattle
feedlots.
Giardia
and
Cryptosporidium are associated with livestock farming
and domesticated animals (Olsona et al., 1997; Traub,
2008). It is highly likely that the parasites detected
originate from the cattle feedlots. Rainfall events, and the
associated run-off, can cause higher loads of the
pathogens to be present in regional waters. Giardia and
Cryptosporidium can survive for extended periods as
(oo)cysts, and have low infectious doses (Adam, 2001;
DuPont et al., 1995; Guillot and Loret, 2010). In addition,
disease caused by Cryptosporidium is difficult to treat,
and is a concern for immuno-compromised individuals
6586
(Guillot and Loret, 2010).
Investigation of sources of microbial pollution

Initial monitoring efforts were directed at 12 sampling
sites within the upper Olifants River catchment during the
first phase of this project (Figure 1). Repeat sampling
was performed over a period of one year and provided
microbiological base-line data. However, due to the
limited number of sites that were investigated, a complete
picture was still lacking. To address the knowledge gaps,
more sample sites were investigated during a second
monitoring phase. These sample sites were chosen in
such a way that they could provide information on how
each tributary and contributing stream affected water
quality, both locally and to the main stem of each river.
The Wilge, Klein Olifants, and Olifants Rivers were
sampled during separate sampling trips, all during low
flow. Each river, and all sites linked to it, were sampled
within the shortest possible time frame (usually within two
days) in order to minimize time-related variances. For this
reason the data may have limited value for comparing
microbial quality between the three river systems, but are
more suited to infer what the drivers and sources of
microbial contaminants within a specific system were at a
given time.
Sites were focussed on where indicator levels were
found to exceed 1000 E. coli MPN/100 ml of water. This
value was chosen because WWTWs should not release
effluent that exceeds this value and South African
irrigation guidelines also advise against using water that
exceeds this value for agricultural purposes (assuming
that no purification steps are employed) (Department of
Water Affairs and Forestry, South Africa, 1996). In
addition, the recommended water quality guidelines for
wastewater use in agriculture for the irrigation of crops
likely to be eaten uncooked, public parks and sports
fields is 1000 faecal coliforms per 100 ml of water
(WHO, 1989). It should be noted that water containing
faecal indicators at this level is deemed to carry a
significant health risk to water users. Twelve of the 86
sites sampled were found to contain E. coli at levels
exceeding this value, with the Olifants River and its
tributaries accounting for eight of the sites. Of the twelve
sites, seven were directly downstream of WWTWs, two
sites were directly downstream of urban areas, one was
located in an area characterized by intensive mining
activities, and another two sites were located in
agricultural regions. Intensive land uses are known to
increase faecal indicator loads to rivers and streams, but
these non-point sources would in many cases only be
sporadic contamination events (Loague and Corwin,
2005).
It should be noted that not all WWTWs in the upper
Olifants River catchment are dysfunctional, because the
data suggested that a treatment works on the northern
side of Middelburg was operating within specification.

Another treatment works on the south side of Middelburg
was recorded to repeatedly contaminate a tributary of the
Klein Olifants River with extremely high levels of faecal
indicator organisms and human pathogens. However, a
drastic improvement in this streams microbial quality was
observed during the latter part of the monitoring.
The data show that small microbial water quality gains
can be made by improving agricultural and industrial
practises. However, this would entail a paradigm shift in
the way that many small, and some large enterprises
function; all of this to achieve a slight improvement in
microbial water quality. In contrast, large gains can be
achieved by ensuring the proper functioning of a few
WWTWs within the upper Olifants River catchment.
Quantitative microbial risk assessment

Untreated water may be used for many purposes in the
upper Olifants River catchment. These include irrigation,
recreation, consumption, and domestic purposes. Poor
quality water remains a leading cause of morbidity and
mortality on the global scene (WHO, 2004), and it is vital
to understand the risks that the water users in the
Olifants catchment are exposed to.
Various sites within the upper Olifants River catchment
have been shown to be highly contaminated with faecal
pollutants. These sites harbour diverse human pathogens
that pose an unacceptably high health risk to water users.
The data show that an individual consuming 100 ml of
untreated water from such a water source could expect to
have up to a 26% chance of falling ill (from any of the
seven pathogens that were monitored for in this study)
(Figure 2). Important water-borne pathogens as they may
be, these organisms are but a fraction of the total
potential pathogen pool expected to occur in poor quality
waters, and the true risk water users face would in fact
be much higher. Using E. coli as an indicator for
pathogens, the risk of infection at some sites was
calculated to be in the region of 80% based on a single
exposure event (Figure 3). It is important to note that the
probability of infection, resulting from exposure to water,
makes use of dose-response models. These doseresponse models typically make use of studies involving
healthy volunteers, thereby not taking into account the
sensitive subgroups, such as the young, elderly, and
immune-compromised. Not all infected individuals would
develop clinical symptoms. The percentage of infected
people that would develop clinical illness would depend
on a number of issues, such as the immunity of the
person and the virulence of the pathogen. For this reason
more illness may be expected (as opposed to infection)
among the relatively high proportion of HIV positive
individuals in this region.
Ideally, these contaminated waters should not be used
for human consumption, but research data indicate that
Roux et al.
consumption of untreated water by humans is not

uncommon in the study area (personal communication,
C. Wright.). According to the WHO, the acceptable
annual risk of infection (from drinking water) is
considered to be 1 in 10,000 (WHO, 2001), however, a
higher risk of 1 in 1,000 is now being considered as more
appropriate. Regardless of which of these
should be
considered as acceptable, the risk of infection in this
study resulting from possible exposure to pathogens is
significantly higher.
Priority areas
From the results, it is clear that sections of the Wilge,
Klein Olifants, and Olifants River systems are highly
contaminated with pathogenic micro-organisms. It is also
clear that these organisms, at the level that they are
present, pose a serious health risk to water users in this
catchment. However, some polluted areas may be of low
importance due to the absence of significant exposure
mechanisms. For instance, poor quality river water in a
region of low population density, where other sources of
drinking water are readily available, will have less of an
impact than poor quality water in a densely populated
region with no formal service delivery. Some of the sites
that exhibited poor water quality during the second
monitoring phase of this study are located in low
population density areas, even though the most likely
cause of their poor state lies in densely populated areas
(that is, WWTWs).
Two priority areas were identified where poor quality
water co-existed with high population densities, the most
prominent being Emalahleni and the surrounding areas.
The Klip River, Blesbokspruit and sections of the Olifants
River posed unacceptably high risks to water users within
this area. The Klein Olifants in the vicinity of Middelburg
was also highly contaminated with faecally derived
microbes.
Conclusion
It was shown that sections of the upper Olifants River
catchment are highly contaminated with faecal indicator
bacteria and pathogenic micro-organisms. Data from the
quantitative microbial risk assessment also showed that
the polluted waters pose an unacceptably high risk to
water users within this catchment. Making use of E. coli
data as a surrogate for the possibility of other pathogens
in the quantitative microbial risk assessment resulted in
very similar probabilities of infection, illustrating the value
of E. coli in a quantitative microbial risk assessment.
Diffuse sources may be responsible for a portion of the
observed faecal pollution, but in most cases the
contamination associated with diffuse sources (such as
informal and formal housing areas) was a few orders of
magnitude lower than that of the point sources identified.
6587
Extreme levels of faecal pollution could in most instances

be traced back to inadequate wastewater treatment.
In order to mitigate water-borne risks in the upper
Olifants River, wastewater treatment works need to be
maintained and operated in such a way that sewage
effluent meets effluent discharge criteria at all times. In
addition service delivery in developed and developing
areas should be maintained and expanded where
necessary to provide hygienic living conditions, with safe
drinking water provision and wastewater infrastructure
being paramount. Until the current short comings are
addressed, water users at certain locations within this
catchment will continue to be at risk from water-borne
infections.
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DOI: 10.5897/AJMR12.1505
Partial characterization of a bacteriocin produced by

Lactobacillus salivarius isolated from oral cavity of
desert foxes
Aly E. Abo-Amer1,2* and Mohammed Y. Shobrak3
1
Division of Microbiology, Department of Biology, Faculty of Science, University of Taif, P.O. Box 888, Taif, Saudi
Arabia.
2
Division of Microbiology, Department of Botany, Faculty of Science, Sohag University, Sohag (82524), Egypt.
3
Division of Zoology, Department of Biology, Faculty of Science, University of Taif, P.O. Box 888, Taif, Saudi Arabia.
Bacteriocin production by animal-associated bacteria is considered as a natural defense against

pathogens. So, the objectives of this work were to isolate, characterize, and screen lactic acid bacteria
producing antimicrobial compounds from oral cavity of desert foxes. Forty one isolates of lactic acid
bacteria were isolated and screened for their ability to produce antimicrobial compounds. Only four
isolates showed inhibition of indicator bacteria. Among these isolates, one isolate called AA13 showed
a wide range of inhibition of pathogens and therefore it was selected for further investigations. This
isolate was identified morphologically, biochemically, and confirmed by API 50 and 16S rRNA as
Lactobacillus salivarius. Cell free supernatant of AA13 strain inhibited the growth of many pathogens.
Antibacterial activities of crude extract of AA13 strain were abolished by proteolytic enzymes, indicative
proteinaceous nature. Substance AA13 was resistance to heat at 121C for 20 min and a wide range of
pH (3 to 10). The substance AA13 was produced at log phase and had a bactericidal effect on Listeria
monocytogenes cells. Therefore, the substance AA13 was characterized as a novel bacteriocin and
designed salivaricin AA13. Moreover, the strain AA13 was resistant to 2% (w/v) bile salt and acidic pH
(3.0) indicating a potential probiotic strain. Therefore, the bacteriocin produce by L. salivarius AA13
could be used as an antibiotic alternative and a food preservative.
Key words: Lactobacillus salivarius, 16S rRNA, oral cavity, foxes, bacteriocin, probiotic, antibiotic.
INTRODUCTION
Bacteriocins produced by animal-associated bacteria
might be one of the most potent weapons of these
animals to fight against infectious diseases. In addition,
bacteriocinogenic bacteria may prevent pathogen
dissemination by occupying the same ecological niche.
Bacteriocinogenic bacteria associated with animals are a
relevant source for isolation of probiotics. At higher
temperatures as climate change, numerous bacteria
exhibit greater virulence because of reduced resistance
*Corresponding author. E-mail: abo-amer@lycos.com.
and increased virulence and transmission (Marcogliese,

2008). At the same time, use of antibiotics is harmful to
aquatic and terrestrial environments, animal, and human
health (Zhou et al., 2009). That is the reason why the
European authority has preferred to limit antibiotic use as
medicinal agents. Therefore, scientists are looking for
friendly alternatives such as antibiotic substitutes
(Dorrington and Gomez-Chiarri, 2008) or use of probiotic
(Kesarcodi-Watson et al., 2008). Bacteriocin producing
bacteria appear to be an excellent candidate for a friendly
alternative as bacteriocin might be used as an antibiotic
substitute (Joerger, 2003), while bacteria might be a
potential probiotic (Gillor et al., 2008).
6590
Bacteriocins are proteinaceous substances and toxic to

bacteria closely related to the producing bacteria (Gillor
et al., 2005). Bacteriocins may serve as anti-competitor
compounds enabling an invasion of a strain or species
inan established microbial community (Riley and Wertz,
2002). Nevertheless, using pure bacteriocins is not
practical since it has no economic basis. One way to
substitute antibiotics smartly and sustainably will be the
selection of bacteriocinogenic and antipathogenic strains
from animal-associated bacteria for use as probiotics.
Bacteriocins have been used by food industry to reduce
the use of chemical preservatives in foods with limited
shelf life, or those foodstuffs that present a high risk for
pathogen contamination (Abee et al., 1995). Bacteriocins
are a diverse family of small, heat stable peptides with
potent antimicrobial activity that are produced by many
bacterial species, including many probiotic strains (AboAmer, 2007, 2011; Messaoudi et al., 2011; Dobson et al.,
2012; O'Shea et al., 2012; Santagati et al., 2012).
Probiotics have a large possibility to affect the host
health positively (Tinh et al., 2008) by competitive
exclusion (Balcazar et al., 2006), by enzymatic role to
digestion (Gillor et al., 2008; Musa et al., 2009) and by
improvement of the immune response (Kelly et al., 2005)
or by production of inhibitory agents (Kesarcodi-Watson
et al., 2008). Production of inhibitory agent is possibly
one of the most studied approaches of probiotic action.
Bacteriocins from lactic acid bacteria have established
their significant potential as therapeutics for veterinary or
medical uses (Tagg and Dierksen, 2003), or as
phytosanitary for plant protection (Holtsmark et al., 2008),
or as food conservatives (Papagianni and Anastasiadou,
2009; Arauz et al., 2009). Many articles reported
bacteriocin production by Lactobacillus salivarius and
none of them is of desert foxes origin (Riboulet-Bisson et
al., 2012; Busarcevic and Dalgalarrondo, 2012; O'Shea et
al., 2011). Rueppell's fox or Sand fox (Vulpes ruepellii) is
found in the desert from Northern Africa to Sudan to the
desert areas in Saudi Arabia, Pakistan and Afghanistan
with six subspecies recognized (Wilson and Mittermeier,
2009). Therefore, this study was amide to screen lactic
acid bacteria isolated from desert foxes for antimicrobial
production, determine their spectrum of inhibition,
including human and animal pathogens, and examine
these bacteria as a probiotic.
Areas, locations, and the method of animal capture
Foxes were trapped from two areas of the east of Taif city, Saudi
Arabia. The first trapping section were carried out at Mahazat asSayd Protected Area, located in the Central West of Saudi Arabia,
170 km North-East of Taif city. The Protected Area is a 2,245 km
fenced area located on the arid plains of Western Saudi Arabia.
Majority of the substrate in the Protected Area is open sandy
gravel. The second area was located in the perimeter of Taif area
into the north-west of the city. The terrain is a more sandy stony
area, with scattered farms.
Trapping was conducted by using Tomahawk live trap, model
208, which have double-door wire trap, about 40 40 108 cm in
size. Thirteen animals were captured and anaesthetized with
mixture of ketamine (10% solution) and xylazine (5% solution) at
ratio 3:2, injected intramuscularly using 1 ml syringes (Kreeger et
al., 1990). Samples were taken from oral cavity of foxes by swabs
and then transported immediately to the laboratory. After taking
samples, the foxes were released to the same area from which they
are captured.
Isolation and identification of lactic acid bacteria

Swaps from oral cavity of foxes were streaked directly onto de Man,
Rogosa and Sharpe (MRS) agar (Merck) and anaerobically
incubated using the BBL Gas-pak system for 48 h at 37C. Different
bacterial colonies were selected from each sample. The colonies
were first characterized morphologically by microscopy, Gram
staining, and detection of catalase activity. Gram-positive isolates
without catalase activity were considered as lactic acid bacteria and
were used in further studies. Bacterial isolates were identified
according to their morphological and biochemical characteristics as
described previously by Krieg (1984) and confirmed by the API 50
CHL system. Lactobacilli isolates were kept at -80C in 40%
glycerol stock cultures.
Molecular characterization of lactic acid bacteria

An aliquot of 2 ml of 24 h culture was centrifuged at 10000 rmp for
5 min. Genomic DNA was extracted from lactic acid bacteria as
described previously by Yu et al. (2009). DNA was resuspended in
50 l of Tris-EDTA (TE) buffer (10 mM Tris-HCl, 1 mM
ethylenediaminetetraacetic acid (EDTA), pH 8). The amount of DNA
obtained was quantified by measuring at 260 nm and its integrity
was visualized by 0.8% (w/v) agarose gel electrophoresis
(Sambrook et al., 2001).
The 16S rRNA gene (~1.5 kb) was polymerase chain reaction
(PCR) amplified using the primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (5'-GGYTACCTTGTTACGACTT-3').
PCR tube (50 l) contained a reaction mix of 10 l 5X buffer, 1.5
mM MgCl2, 200 M of dNTPs (Promega), 0.4 M of each primer
and 2 units Taq DNA polymerase (Promega) and 5 l DNA. The
reaction programme is as follows: 94C for 5 min; 30 cycles of 94C
for 1 min, 55C for 1 min and 72C for 1 min; and a final extension
step at 72C for 7 min. After cycling, the PCR product was
visualized by electrophoresis on a 0.8% w/v agarose gel at 95 V for
30 min, staining with 0.5 g/ml ethidium bromide and visualizing
under ultraviolet (UV) light.
The PCR product of 16S rRNA gene was purified (Wizard PCR
SV Gel & PCR Clean-Up System kit, Promega) and sequenced.
The sequence was compared with the sequences deposited in the
GenBank database using the BLAST. Also, selected sequences of
other microorganisms with the greatest similarity to the 16S rRNA
sequences of the bacterial isolate were extracted and aligned using
CLUSTAL W (1.81) Multiple Sequence Alignment and N.J. plot
generating a phylogenetic tree. The 16S rRNA gene sequence of
the isolate AA13 reported in this study was deposited in the
DDBJ/EMBL/GenBank with an accession number of AB731447.
Preparation of cell-free supernatant

Isolates of lactic acid bacteria were grown in MRS broth at 37C for
Abo-Amer and Shobrak
6591
Table 1. Spectrum of growth inhibition by antimicrobial compounds produced by Lactobacilli strains.
Organism
S. faecalis ATCC 19433
S. pyogenes ATCC 19615
S. mutans, Lab. isolate
S. pneumoniae, Lab. isolate
E. coli ATCC 11229
E. faecalis, Lab. isolate
L. monocytogenes S39, Lab isolate
S. aureus ATCC 9664
S. epidermidis ATTC 12228
Micrococcus luteus A102, Lab. isolate
Micrococcus roseus R76, Lab. isolate
B. cereus ATCC1 1778
Bacillus subtilis R89, Lab. isolate
S. typhimurium ATCC 13311
S. paratyphi R46, Lab. isolate
S. enteritidis, Lab. isolate
P. aeruginosa ATCC 15442
Shigella species A23, Lab. isolate
S. sonnei ATCC 25931
S. dysenteriae A20, Lab isolate
Inhibition by culture supernatant of Lactobacillus spp.

AA07
AA13
AA25
AA36
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+, Presence of inhibition activity; -, Absence of inhibition activity.
24 h. Culture supernatants were separated from the cells by

centrifugation at 10000 rpm for 10 min. The supernatants were
treated with catalase (5 mg/ml) (Sigma) and adjusted to pH 6.7 with
1 M NaOH to eliminate antagonism resulting by hydrogen peroxide
and lactic acid, respectively. Finally, the supernatants were filtered
through a 0.22-m filter (Millipore).
for antimicrobial activity.
Influence of lyophilization and pH on antimicrobial activity
Lactobacillus cell-free supernatants were evaluated by the agar well

diffusion method as described by Todorov and Dicks (2005). The
indicator strains used in this study were listed in Table 1. The
indicator strains were grown in Brain Heart Infusion (BHI) medium
or in Luria-Bertani (LB) medium (1% tryptone, 1% yeast extract,
0.5% NaCl) (Sambrook et al., 2001). The plates were incubated for
24 h at 37C. Antimicrobial activity was then detected by observing
the formation of inhibition zones.
To study the resistance of antimicrobial activity to lyophilization,

culture supernatant were lyophilized. The culture supernatant fluid
of overnight culture of L. salivarius AA13 was separated and filter
sterilized.
Fractions of 10 ml were lyophilized and then shortly resuspended
in the same volume of MRS broth. The antimicrobial activity of the
resuspended lyophilized powder was assayed using well diffusion
agar.
To investigate the effect of pH on the antimicrobial activity,
culture supernatants were adjusted to different pH values such as
3, 5, 7, 9, and 11, and incubated at 37C for 12, 24, and 48 h; and
100C for 30 min. The antimicrobial activities of the supernatants
were assayed.
Effect of enzymes and temperature on antimicrobial activity
Stability of bacteriocin during storage
Samples of culture supernatant were treated with proteinase K (1

mg/ml), papain (1 mg/ml), trypsin (1 mg/ml), pepsin (1 mg/ml),
catalase (1,000 U/ml), lysozyme (1 mg/ml), a-amylase (0.1 M), and
lipase (10 g/ml) (Sigma). The mixtures were incubated at 37C for 2
h. Then, the enzymes were heat-treated at 70C for 20 min. The
remaining antimicrobial activity was detected.
Thermo-resistance of the bacteriocin was studied after the
culture supernatants were heated at 37, 50, and 90C for 30 min
and 120C for 20 min. The supernatants were cooled and examined
The supernatant fluids of L. salivarius AA13 were stored at different

temperature values: 37, 4 and -20C for 160 days. Samples were
taken for determination of antimicrobial activity every 20 days.
Screening of lactic acid bacteria with antagonism
Kinetics of bacteriocin production

MRS broth was inoculated with a 1% concentration of overnight
culture of L. salivarius AA13 and incubated at 37C. Samples were
6592
withdrawn every 2 h for 28 h to determine the antimicrobial activity

of the bacteriocin. Also, the growth of bacteria was detected by
measuring the optical density at 600 nm (OD600). For the activity,
culture supernatants were filter sterilized and serially diluted in MRS
broth.
Twenty-five microliters of each dilution was poured into the wells
of agar plates poured with Listeria monocytogenes. The
antimicrobial activity was expressed in arbitrary units (AU), defined
as the contrary of the highest dilution in milliliters that allowed no
growth of the indicator strain.
All analyses were carried out according to one-way analysis of
variance (ANOVA) and assessed by post hoc comparison of means
using lowest significant differences (LSD) using Statistical Package
for Social Sciences (SPSS) 11.0 software. They were considered
significant at P < 0.05 level. The experiments were performed in
triplicate.

Mode of action
To investigate whether the antimicrobial compound has a
bactericidal or bacteriolytic mode of action, L. monocytogenes was
grown in 250 mlconical flasks containing 50 ml of BHI broth and
incubated at 37C.
A sample of 20% (v/v) of the concentrated supernatant was
added approximately to a mid-log phase culture. The optical
densities (OD600) of the bacterial growth at 600 nm were determined
every hour for 10 h.
Investigation of stress resistance of L. salivarius AA13
Standards for using Lactobacillus strains as probiotics include
functional characteristics such as the ability of bacteria to resist
environmental conditions found in the gastrointestinal tract such as
bile salts and low gastric pH. Resistance to bile salts was examined
by the ability of bacteria to grow on MRS agar containing 2% (w/v)
bile salt (Merck). The cultures were checked after 48 h of anaerobic
incubation at 37C.
Resistance to acidic pH (pH 3.0) was studied by centrifuging the
overnight cultures of the bacteria at 10,000 rpm for 10 min at 4C
and resuspending the pellets in the same volume of 0.9% (w/v)
NaCl, pH 3.0. The suspensions were then incubated at 37C for 3 h
under anaerobic conditions and then centrifuged. Finally, the pellets
were plated onto MRS agar and incubated anaerobically at 37 C
for 48 h.
Antibiotic susceptibility test of L. salivarius AA13

Significant criterion for probiotic bacteria is that they had not carried
any transferable antibiotic resistance genes. Also, patients taking
probiotics are often treated for other diseases. Therefore, it is
important to verify the effect of antibiotics on the growth of probiotic
strains. L. salivarius AA13 was evaluated for susceptibility to
commercially available antibiotics such as chloramphenicol (30 g),
gentamicin (10 g), polymixin (300 UI), tobramycin (10 g),
ampicillin (10 g), amikacin (30 g), fusidic acid (10 g), cefoxitin
(30 g), carbenicillin (100 g), amoxycillin/clavulanic acid (20/10
g), netilmicin (30 g), penicillin (6 g), trimethoprim/
sulfamethoxazole (1.25/23.75 g), ampicillin/sulbactam (10/10 g),
rifampin (5 g), colistin (10 g) and nalidixic acid (30 g) (BIO-RAD
LABORATORIES, SGH).
Assessment was based on the overlay disc diffusion agar as
described previously by Aymerich et al. (2000). Bacterial culture
was suspended in MRS medium at about 106 CFU/ml. One milliliter
of the suspensions was seeded onto MRS agar plates by the
spreading technique. The plates were air dried for 15 min, after
then antibiotic disks were placed on the plates and incubated at
37C for 48 h. Inhibition zones around the disks were determined
and compared to those defined by Charteris et al. (1998) for
lactobacilli.
Isolation, identification, and screening of lactic acid

bacteria for antimicrobial production
Forty one bacterial isolates were obtained from oral
cavity of desert foxes on MRS agar anaerobically. These
bacterial isolates were screened for their antimicrobial
activity against indicator strains (Table 1). Only four
isolates exhibited antimicrobial activity against pathogens
AA07, AA13, AA25, and AA36. Isolate AA13
demonstrated a wide spectrum of inhibition; so, it was
selected for further investigations. The bacterial strain
AA13 was identified morphologically and biochemically
as L. salivarius and confirmed by API CH50. Moreover,
the strain AA13 was further characterized by 16S rRNA
analysis. The 16S rRNA encoding gene of the isolate
AA13 was PCR-amplified and sequenced. The resulting
nucleotide sequences were compared to available
sequences in the databases. A phylogenetic tree showing
the results of 16S rRNA analysis is as shown in Figure 1.
The results indicated the greatest similarity to members
of the Lactobacillus group, which matches the
conclusions of the morphological and biochemical
analysis. As demonstrated, the 16S rRNA sequences of
the isolate AA13 is most closely related to L. salivarius
(Similarly, ~98%).
L. salivarius AA13 was able to inhibit the growth of
many pathogens tested such as Streptococcus faecalis,
Streptococcus
pyogenes,
Streptococcus
mutans,
Streptococcus
pneumoniae,
Escherichia
coli,
Enterococcus
faecalis,
L.
monocytogenes,
Staphylococcus aureus, Staphylococcus epidermidis,
Bacillus cereus, Salmonella typhimurium, Salmonella
paratyphi,
Salmonella
enteritidis,
Pseudomonas
aeruginosa, Shigella sonnei, and Shigella dysenteriae.
Previous results reported that L. salivarius CRL1384
isolated from the crop of a broiler chick produced
inhibitory substance against L. monocytogenes,
Enterococcus hirae, S. enteritidis and S. typhimurium
(Audisio and Apella, 2006). Recent study reported that L.
salivarius SMXD51 isolated from the chicken cecum
exhibited antagonism against Campylobacter jejuni and
Campylobacter coli (Messaoudi et al., 2011). A
bacteriocin-like substance produced by vaginal L.
salivarius CRL 1328 showed antimicrobial activity against
E. faecalis, Enterococcus faecium, and Neisseria
6593
Table 2. Effect of enzyme and temperature treatments on the activity of antimicrobial

produced by L. salivarius AA13.
Treatment
Proteinase K
Papain
Trypsin
Pepsin
Catalase
Lysozyme
-amylase
Lipase
37C for 30 min
50C for 30 min
90C for 30 min
120C for 20 min
Inhibition activity against L. monocytogenes

+
+
+
+
+
+
+
+
+, Presence of inhibition activity after treatment; -, Loss of inhibition activity after treatment.
Table 3. Effect of pH on the activity of antimicrobial produced by L. salivarius AA13.
pH
3
5
7
9
11
12 h at 37C
+
+
+
+
-
Inhibition activity against L. monocytogenes

24 h at 37C
48 h at 37C
30 min at 100C
+
+
+
+
+
+
+
+
+
+
+
+
-
+, Presence of inhibition activity after treatment; -, Loss of inhibition activity after treatment.
gonorrhoeae (Ocana et al., 1999). L. salivarius 1077

(NRRL B-50053) isolated from poultry intestinal materials
demonstrated anti-Campylobacter jejuni activity (Svetoch
et al., 2011).
(Busarcevic et al., 2008). Bacteriocin L-1077 produced by

L. salivarius 1077 was sensitive to proteolytic enzymes
and resistance to lysozyme and lipase (Svetoch et al.,
2011).
Effect of enzymes on bacteriocin activity
Heat, pH, lyophilization, and storage effects on

bacteriocin activity
Chemical properties of the antimicrobial substance

produced by L. salivarius AA13 were different from lactic
acid and hydrogen peroxide, because the supernatant
was adjusted to pH 6.0 and treated with catalase.
Antimicrobial activity of the supernatant was lost after
treatment with proteinase K, papain, trypsin, and pepsin,
but the substance was resistant to catalase, lysozyme, aamylase, and lipase (Table 2). These results indicated
that the antimicrobial substance was revealed to be
proteinceous nature. Lipase did not significantly change
its inhibitory effect, indicative that no lipidic regions were
related to the active site. Antimicrobial substance
produced by L. salivarius BGHO1 completely lost its
activity after treatment with different proteolytic enzymes
The antimicrobial substance retained activity after heating

at 37, 50, and 90C for 30 min and 120C for 20 min
(Table 2). Antimicrobial compound produced by L.
salivarius CRL1384 was heat resistant (121C for 15 min)
(Audisio and Apella, 2006). The supernatant of L.
salivarius BGHO1 culture retained its antimicrobial
activity after heating at 100C for 15 min and after
autoclaving at 121C for 20 min (Busarcevic et al., 2008).
Bacteriocin L-1077 produced by L. salivarius 1077 was
resistance to 100C (Svetoch et al., 2011).
The antagonistic activity was stable at pH values
ranging from 3.0 to 9, but became inactive at alkaline
conditions (pH 11) (Table 3). Previous study showed that
6594
Lactobacillus acidophilus NBRC 13951

Lactobacillus salivarius ZJ602
893

745

707
Lactobacillus salivarius FK10-10
706
1000
493
Lactobacillus salivarius DSPV 333T

922
Lactobacillus salivarius AA13

958
Lactobacillus salivarius HO 66
728
Lactobacillus salivarius P2
549
Lactobacillus cacaonum LMG 24285

Lactobacillus plantarum LCN 28
1000
Lactobacillus paraplantarum DSM 10667

Enterococcus avium E6844
0.01
Lactococcus lactis subsp. Lactis NCDO 604
Figure 1. Phylogenetic tree of L. salivarius strains and related bacteria based on 16S
rDNA gene sequence. Boot values were based on 1000 replicates. Scale shows
percentage of substitutions per nucleotide position.
antimicrobial activity produced by L. salivarius CRL 1328

was detected at pH levels from 2 to 8 (Ocana et al.,
1999).
Moreover, lyophilization did not change the activity of
substance produced by L. salivarius AA13. Previous
result reported that the bacteriocin of L. salivarius CRL
1328 was resistant to lyophilization (Ocana et al., 1999).
The antimicrobial substance of Lactobacillus acidophilus
AA13 could be stored at -20C for at least 160 day and at
4C for at least 120 days without loss of its activity
(Figure 2). However, a little loss of activity was detected
during storage after 80 days at 37C.
According to all these characteristics, this substance
was considered to be a bacteriocin or a bacteriocin like
substance (Piard and Desmazeaud, 1992; Tagg et al.,
1976). These results agree with those of other authors
who reported on bacteriocins synthesized by strains of L.
salivarius, but none of the bacteria was isolated from oral
cavity of desert foxes. Also, there is information of a
bacteriocin produced by L. salivarius isolated from pigs,
but it does not present anti-Listeria effects (Robredo and
Torres, 2000). Therefore, the antimicrobial compound
produced by L. salivarius AA13 met the criteria for being
considered as a bacteriocin (Tagg et al., 1976).
Bacteriocin production
The antimicrobial activity of L. salivarius AA13 was
determined after 2 h of incubation. The highest
concentration was obtained between 8 and 10 h of
incubation, with titers of 1,400 AU/ml of supernatant. The
production kinetic is as shown in Figure 3. These results
indicated that the antimicrobial compound was produced
continuously during growth phase. However, the
maximum level of inhibition was detected at the
beginning of mid-log phase and remained constant at
stationary phase. Previous study reported that the
activity of antimicrobial substance produced by L.
salivarius CRL1384 was detected after 3 h of incubation,
while between 9 and 12 h, the highest concentration
(1,280 AU/ml) was detected (Audisio and Apella, 2006).
Moreover, a bacteriocin produced by L. salivarius CRL
1328 demonstrated the highest level of antimicrobial
production during the late exponential phase (Ocana et
al., 1999).
Mode of action
Mode of action of the antimicrobial substance produced
6595
14
12
10
8
6
4
100)
Antimicrobial activity (AU/ml x 100)
16
2
0
0
20
40
60
80 100 120 140 160
Time (day)
Figure 2. Effect of time at different temperature values on the activity of
antimicrobial compound produced by L. salivarius AA13 during storage. Culture
supernatants were stored at 37C (), 4C () and -20C (). The inhibitory
activity was determined by well diffusion assay using L. monocytogenes as
indicator strain.
by L. salivarius AA13 on L. monocytogenes cells was

investigated. The optical density of L. monocytogenes
cells was decreased from 0.49 to 0.39 after 1 h of
addition of antimicrobial substance AA13 at 30C. Then,
after another hour, the optical density dropped to be
constant at 0.3. However, the optical density of L.
monocytogenes cells without addition of the antimicrobial
compound reached maximum value (OD600=2) after 7 h
of incubation (Figure 4). As no changes were observed in
the optical density of the L. monocytogenes, cellsuspension was detected during the period tested, a lytic
action was rejected. Therefore, the antimicrobial
substance AA13 has bactericidal effect against this
Gram-positive pathogen. Previous results stated that L.
salivarius CRL1384 produced a bacteriocin that had a
bactericidal effect on both L. monocytogenes and S.

enteritidis cells after 24 h of contact (Audisio and Apella,
2006). A bacteriocin produced by L. salivarius CRL 1328
showed a bactericidal mode of action (Ocana et al.,
1999).
As described earlier, the presence of bacteriocin
production among Lactobacillus species in competitive,
complex microbial communities as in oral cavity
suggested that bacteriocins might have a regulatory role
for population dynamics within bacterial ecosystems.
Moreover, natural substances with antimicrobial activity
against Gram-negative and Gram-positive pathogens
bacteria can provide a cost-effective alternative to the
use of antibiotics, especially given current concerns by
the scientific community and by the public over the
6596
16
2.5
1.5
8
1
6
4
(OD
10
Optical density (OD600nm )
2
12
100)
Antimicrobial activity (AU/ml x 100)
14
0.5
2
0
0
0
12
16
Time (h)
20
24
28
Figure 3. Kinetics of bacteriocin production during growth. Growth of L. salivarius

AA13 in MRS broth was determined by optical density () and bacteriocin
concentration is expressed as arbitrary units per milliliter (). The inhibitory activity
was determined by well diffusion assay using L. monocytogenes as indicator
strain.
emergence
of
antibiotic-resistant
microorganisms
(Patterson and Burkholder, 2003; Reid and Friendship,
2002).
determined only in the case of cephalothin, streptomycin,

vancomycin, and colistin (Messaoudi et al., 2011).
However, antibiotic resistance has been demonstrated in
some lactobacilli strains (Karimi Torshizi et al., 2010).
Antibiotic susceptibility of L. salivarius AA13

L. salivarius AA13 as a potential probiotic strain
Utilization of antibiotics is an issue of current concern,
because of the emergence of antibiotic resistance in
human and zoonotic pathogens. L. salivarius AA13 was
examined against 17 antibiotics of different classes. The
strain was sensitive to majority of the antibiotics, with
resistance observed only in the case of penicillin (Table
4). Previous results reported that L. salivarius SMXD51
and L. salivarius NRRL B-30514 were investigated
against 15 antibiotics of eight classes. These strains were
sensitive to the majority of the antibiotics, with resistance
L. salivarius AA13 strain was sensitive to antibiotics,

resistant to bile salt (2%) and low gastric pH (3), and had
antagonistic activity against some food borne pathogens.
Therefore, these results indicated that L. salivarius AA13
could be a potential probiotic strain. A potential probiotic
L. salivarius AA13 could play a beneficial role by
inhibiting the growth of some cariogenic bacteria.
Previous results reported that 36% of lactobacilli isolated
from the tongue have ability to inhibit the growth of S.
6597
2.2
1.6
1.8
1.4
1.2
1
0.8
(OD
Optical density (OD 600nm)
0.6
0.4
0.2
0
0
10
Time (h)
Figure 4. Mode of action of antimicrobial compound produced by L. salivarius AA13. L.
monocytogenes was grown in LB without bacteriocin () and with bacteriocin () at 37C.
The time of adding culture supernatant is indicated by arrow.
Table 4. Antibiotic susceptibilities of L. salivarius AA13.
Antibiotic tested
Chloramphenicol
Gentamicin
Polymixin
Tobramycin
Ampicillin
Amikacin
Fusidic acid
Cefoxitin
Carbenicillin
Amoxycillin/Clavulanic acid
Netilmicin
Penicillin
Trimethoprim/Sulfamethoxazole
Ampicillin/Sulbactam
Rifampin
Colistin
Nalidixic acid
*S, sensitive; R, resistant.
Antibiotic code
C
GM
PB
TM
AM
AN
FA
FOX
CB
AMC
NET
P
SXT
SAM
RA
CS
NA
Disk load
30 g
10 g
300 UI
10 g
10 g
30 g
10 g
30 g
100 g
20/10 g
30 g
6 g
1.25/23.75 g
10/10 g
5 g
10 g
30 g
Mode of action
Protein synthesis inhibitor
Disruption of inner and outer cell membrane
Cell wall synthesis inhibitor
Cell wall synthesis inhibitor (-lactam)
Folic acid and DNA synthesis inhibitor
DNA-dependent RNA synthesis inhibitor
Disruption of outer cell membrane
DNA synthesis inhibitor
Response
S*
S
S
S
S
S
S
S
S
S
S
R*
S
S
S
S
S
6598
mutans (Ahumada et al., 2001). Sookhee et al. (2001)

isolated Lactobacillus paracasei and Lactobacillus
rhamnosus from the oral cavities capable of having an
antimicrobial activity against Streptococcus sanguis, S.
mutans,
Streptococcus
salivarius,
S.
aureus,
Porphyromonas gingivalis, Actinomyces viscosus, and
Candida. Moreover, previous study stated that 69% of
tested lactobacilli inhibited S. mutans, 88% Actinobacillus
actinomycetemcomitans, 82% Porphyromonas gingivalis,
and 65% Prevotella intermedia (Koll-Klais et al., 2005).
Probiotic bacteria such as L. salivarius, Lactobacillus
plantarum, Lactobacillus paracasei and L. rhamnosus
produced the greatest antimicrobial activity. Another
study reported that in an in vitro co-culture system, L.
salivarius inhibited Porphyromonas gingivalis, Prevotella
nigrescens, and Prevotella intemedia within 24 h
(Ishikawa et al., 2003). A strain of L. salivarius was
exhibited to have a mechanical action against S. mutans
and periodontopathic bacteria owing to its ability to attach
to the salivary pellicle on the tooth surface (Sakabe et al.,
2004). Obviously, lactobacilli play a significant role within
the oral ecosystem whether oral health or carious
diseases. Probiotic bacteria may contribute to the
colonization resistance of the host and its protection
against gastrointestinal pathogens (Reid et al., 2001).
Bacteriocins may facilitate the establishment of probiotic
strains in the competitive environment of the gut
(Klaenhammer and Kullen, 1999); however, the
importance of bacteriocin production in vivo remains
under discussion (Ouwehand et al., 1999). Also, L.
salivarius CRL 1328 could be used for the design of a
probiotic to prevent urogenital infections (Ocana et al.,
1999).
Conclusions
L. salivarius AA13, a potential probiotic strain of oral
cavity of desert foxes origin, synthesizes a bacteriocin
with a broad inhibitory spectrum. Bacteriocin production
was maintained at different environmental condition.
Bacteriocin produced by L. salivarius AA13 could be
employed as an antibiotic alternative and a food
preservative.
ACKNOWLEDGEMENTS
We are thankful to Professor Dr. Simon C. Andrews,
Reading University, England, for his assistant. Also, we
are thankful to HH prince Bander Bin Saud, the secretary
general of Saudi Wildlife Authority (SWA), for his
engorgements and support during the period of the study.
Also, we are grateful for the Director of the National
Wildlife Research Center (NWRC), Mr. Ahmad Bouq for
his support in the study. Our deep gratitude goes to Mr.
Moayyad Sher Shah for his viable help in capturing the
animals and his guidance to get the samples and release

the animals.
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UPCOMING CONFERENCES
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African Journal of

Donovan Anthony McGrowder

Dr. Ntobeko A. B. Ntusi

Instructions for Author

Materials and methods should be complete enough

Results should be presented with clarity and precision.

1987a,b; Tijani, 1993,1995), (Kumasi et al., 2001)

Number 36 20 September, 2012

Inhibitory effect of some plant extracts on clinical isolates of Staphylococcus

Role of CSE1034 in bacterial lipids and polysaccharides involved in biofilm

Analytical specificity and sensitivity determination of 16SrRNA gene based

Effect of Bacillus cereus Br on bacterial community and gossypol content

Identification and characterization of a fungal strain with lignin and cellulose

Survival of microorganisms in high pressure treated minced meat during

Partial characterization of a bacteriocin produced by Lactobacillus alivarius

Full Length Research Paper

Conventional and molecular characterization of

*Corresponding author. E-mail: farzadaala@yahoo.com. Tel:

Researchers use two general methods for the

dermatophytes. Sequencing of the Internal Transcribed

(PBS) and finally stored at 70C.

DNA PCR products were purified according to the QIAquick PCR

RESULTS AND DISCUSSION

Morphological characteristics of colonies T. rubrum

All isolates of T. rubrum maintained on Sabourauds dextrose agar

This study used both conventional and molecular

Afr. J. Microbiol. Res.

numbers 1138 and 1059 are white and cottony or fluffy

Figure 2 showed that ITS1 of all isolates T. rubrum had

other species of Trichopthyon is also higher than 90% as

more than 86% among isolates of T. rubrum and isolates

agreement with Li et al. (2008), who revealed that

Afr. J. Microbiol. Res.

Figure 2. PCR amplification of isolates of T. rubrum on 1% agarose gel

> T. rubrum (1138)

> T. rubrum (1059)

> T. rubrum (1208)

> T. rubrum (1008)

Afr. J. Microbiol. Res.

> T. rubrum (2970)

Figure 4. Comparison of nucleotide sequence between T. rubrum ITS1

Afr. J. Microbiol. Res.

T. raubitschekii strain NOMH 789

raubitschekii strain NOMH 789

T.raubitschekii strain NOMH 789

strain ATCC 28188

raubitschekii strain NOMH 789

raubitschekii strain NOMH 789

raubitschekii strain NOMH 789

raubitschekii strain NOMH 789

Afr. J. Microbiol. Res.

raubitschekii strain NOMH 789

raubitschekii strain NOMH 789

raubitschekii strain NOMH 789

T.raubitschekii strain NOMH 789

raubitschekii strain NOMH789

raubitschekii strain NOMH789

raubitschekii strain NOMH789

raubitschekii strain NOMH789

T. raubitschekii strain NOMH789

reference sequence in GenBank (BLAST search) ranged

Borman AM, Linton CJ, Miles SJ, Johnson EM (2008). Molecular

Afr. J. Microbiol. Res.