Accepted Manuscript

Isolation, identification and characterization of Klebsiella

pneumoniae from infected farmed Indian Major Carp Labeo
rohita (Hamilton 1822) in West Bengal, India

A. Das, S. Acharya, B.K. Behera, P. Paria, S. Bhowmick, P.K.

Parida, B.K. Das

PII: S0044-8486(17)31092-X
DOI: doi: 10.1016/j.aquaculture.2017.08.037
Reference: AQUA 632801
To appear in: aquaculture
Received date: 1 June 2017
Revised date: 26 August 2017
Accepted date: 31 August 2017

Please cite this article as: A. Das, S. Acharya, B.K. Behera, P. Paria, S. Bhowmick, P.K.
Parida, B.K. Das , Isolation, identification and characterization of Klebsiella pneumoniae
from infected farmed Indian Major Carp Labeo rohita (Hamilton 1822) in West Bengal,
India, aquaculture (2017), doi: 10.1016/j.aquaculture.2017.08.037

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 ACCEPTED MANUSCRIPT

Isolation, identification and characterization of Klebsiella pneumoniae from Infected farmed

Indian Major Carp Labeo rohita (Hamilton 1822) in West Bengal, India.

A. Das1, S.Acharya2, B. K. Behera*1, P. Paria1, S. Bhowmick1, P. K. Parida1 and B. K. Das1

1. ICAR-Central Inland Fisheries Research Institute, Barrackpore, Kolkata-700120, West Bengal,

India.

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2. Department of Zoology, Vidyasagar University, Medinipur-721102, West Bengal, India.

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*E mail: beherabk18@yahoo.co.in

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Abstract
The present study was conducted to identify and characterize the etiological agent causing mortality

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in Indian Major Carp, Rohu (Labeo rohita) in West Bengal, India. Diseased fish samples having
haemorrhages near the tail and intraperitonial region was collected from culture ponds of Burdwan,
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West Bengal for the isolation of the pathogenic bacteria. Primarily the bacterium was characterized
using biochemical and antibiogram studies. The 16S rRNA gene sequence of the isolated bacteria
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revealed that the isolate was having 100% homology with Klebsiella pneumoniae (NCBI Accession
Number- KY003130). Intraperitoneal injection with 1.05 X 106 CFU per fish causes 50% mortality.
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The challenged fish showed hemorrhages in the intraperitonial region. The histopathology of the
challenged fish liver showed vacuolation, necrosis and disruption of hepatocytes. However, focal
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necrosis and vacuolation was observed in kidney tissue. This study highlights the first time
involvement of Klebsiella pneumoniae in the disease outbreak of cultured Labeo rohita.
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Understanding the pathology and pathogenesis studies of this emerging pathogen in cultured carps
would help in control of this disease in aquaculture.
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Key words: Klebsiella pneumoniae, 16S rRNA gene, Histopathology, LD50, Biochemical test.
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Introduction
Labeo rohita (Rohu) is one of the most cultivable carps in India with high consumer
preference due to its rapid growth and high quality flesh. In modern aquaculture systems, ponds are
being treated with inorganic chemicals and fertilizers. Farmers are over stocking in the aquaculture
ponds and providing artificial feeds for faster growth of the farmed fish. However, all these
innovative practices are leading to stress, resulting in various infections in fish species (Mastan,
2013). Like other carps, Rohu is often severely affected by various types of pathogenic bacteria,

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which cause huge mortality in farms and hatcheries (Das et al., 2014; Ramkumar et al., 2013;

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Tiwari and Pandey, 2014).

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Among the known pathogenic bacteria Klebsiella sp. is found to be infectious to various
organisms including fish. Klebsiella genus comprises of non-motile, Gram-negative, oxidase-

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negative, rod-shaped bacteria that are found in different environments and part of the natural
microflora of soil effluents and natural water bodies (Oliveira et al., 2014; Janda and Abbott, 2005).
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The Klebsiella sp. belongs to the Enterobacteriaceae family and subdivided into a range of species
including Klebsiella pneumoniae, K. granulomatosis, K. mobilis, K. ornithinolytica, K. oxytoca, K.
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planticola, K. singaporensis, K. terrigena, K. trevisanii and K.variicola (Euzéby, 1997; Drancourt

et al., 2001). Among these species Klebsiella pneumoniae is mainly responsible for wide range of
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infections including pneumonia, urinary tract infections, wound infections, bacteremia in humans
(Holden et al., 2016; Magill et al., 2014), severe enteritis and septicemia in dogs (Roberts et al.,
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2000), pleuritis and suppurative pneumonia in Zalophus californianus (Jang et al., 2010),
hemorrhage, red spottiness along the body of Cyprinus carpio (Oliveira et al., 2014; Al-Imarah.,
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2008), skin discoloration with ulcer and exopthalmia in Nemipterus japonicus (Diana and
Manjulatha., 2012), O. niloticus (Takyi et al., 2012), skin haemorrhages in Amphiprion nigripes
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(Gopi et al., 2016). Klebsiella pneumoniae posses many virulence genes including uge (encoding
uridine diphosphate galacturonate4-epimerase), wabG (involved in the biosynthesis of the outer
core lipopolysaccharide), urea (related to the urease operon), magA (mucoviscosity-associated gene
A), mrkD (type 3 fimbriae adhesion), allS (activator of the allantoin regulon), kfuBC (iron-uptake
system), rpmA (regulator of mucoid phenotype) and fimH (fimbrial gene encoding type 1 fimbrial
adhesion), which play prominent roles in bacterial pathogenesis (Gao et al., 2014; Brisse et al.,
2009; Lascols et al., 2013; Fang et al., 2004).
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Till date, very scanty works have been reported on the infection of Klebsiella pneumoniae in
fish. In the present study, K. pneumoniae was first time isolated from diseased L. rohita in India. The
experimental challenge was conducted through intraperitoneal injection of the pure culture of the
bacterium isolated from the diseased fish to confirm the pathogenicity. The aim of the study was to
investigate the causative agent behind the bacterial disease outbreak in cultured L. rohita based on
morphological, biochemical characteristics and 16S rRNA gene sequence analysis along with the
pathogenecity of the etiological agent.

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2. Material and methods

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2.1 Sampling
Infected L. rohita samples were collected from cultured pond of Burdwan (N 23º18′285′′; E
88º11′038′′) West Bengal, India, for the isolation of pathogenic bacteria. Hemorrhages near the

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pectoral fin were observed in the moribund fishes. Clove oil (Merck, Germany) (50 μl/l) was used
for anesthetizing the fish. The fish was cleaned with alcohol and then dissected. Samples of blood,
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liver, and kidney were taken aseptically and plated directly on Tryptic Soya Agar (TSA) (Hi-
media). The plates were incubated for 24 h at 37oC for the bacterial colonies to appear. Dominant
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colonies from Tryptic Soya Agar were selected and streaked again on Tryptic Soya Agar plate and
also on different selective media plates to obtain pure culture. The pure culture of K. pneuminae
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(BB12) was grown in Tryptic Soya Broth (TSB) and maintained as the glycerol stock at -20 ºC.
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2.2. Biochemical characterization of bacterial isolate

The preliminary characterization of the isolated strain was carried out by Gram-staining,
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followed by biochemical tests such as: Malonate utilization, Esculin hydrolysis, Arabinose, Xylose,
Adonitol, Rhamnose, Cellobiose, Melibiose, Saccharose, Raffinose, Trehalose, Glucose and
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Lactose Oxidase, Ornithine utilization, Phenylalanine deamination, H2S production, ONPG, Lysine
utilization, Urease, Nitrate reduction, Voges Proskauer’s, Citrate utilization, Methyl red and Indole
were performed (KB-003, HiMedia). Additional 41 biochemical tests were carried out using an
automated bacterial identification system (VITEK 2 compact, BioMerieux, France).

2.3. DNA isolation and 16S rRNA gene amplification

Total bacterial genomic DNA was extracted by Sarkosyl method (Sambrook and Russel,
2001). The extracted DNA was run in ethidium bromide-stained 1% agarose gel electrophoresis.
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PCR amplification was performed using 16S rRNA gene specific primers with the thermal cycler
Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA). The primers chosen for
amplification of 16S rRNA gene and its annealing temperature have been shown in Table 4. PCR
product was visualized on 2% agarose gel containing ethidium bromide.
2.4. Bacterial identification (DNA sequencing and phylogenetic analysis)
The PCR amplified sample was sequenced in both directions using an ABI 3730xl capillary
sequencer (Applied Biosystems, Foster City, CA) to check the validity of the sequence data (Behera

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et al., 2014).The forward and reverse sequences of the isolated strain BB12 were aligned using the

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software DNA Bazer v0.7.0 version. 16S rRNA gene sequence of BB12 was around 1407 bp which

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was then compared with other sequences available in GenBank using the NCBI–BLAST program
facility (http://www.ncbi.nlm.nih.gov\BLAST). The 16S rRNA gene sequence of BB12 was aligned

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with 16S rRNA gene sequences of the most identified Klebsiella sp. retrieved from NCBI GenBank
database using MEGA 6 software (Tamura et al., 2013). 16S rRNA gene sequence of Rickettsia
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conorii was included as an outgroup. Maximum-likelihood algorithms were applied for
Phylogenetic analysis to ensure coherency of the clusters formed. Bootstrapping was performed
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(10,000 replications) using the MEGA 6 program.

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2.5. Antibiotic susceptibility test

According to the criteria of the Clinical and Laboratory Standards Institute (Oxoid,
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Hampshire, UK) antibiotic susceptibility of the bacterium was determined by the disc diffusion
method on Muller–Hinton agar with 25 antibiotic discs. The following antimicrobial agents (disc
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content indicated in parentheses) were tested : Ampicillin (25g), Tetracycline (10g),

Nitrofurantoin 200g), Erythromycin (10g), Dicloxacillin (1g), Streptomycin (254 g),
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Doxycycline (10 g), Ofloxacin (2 g), Amoxicillin (30g), Ceftazidime (30 g), Cefixime (5g),
Rifampicin (5g), Nalidixic acid (30g), Piperacillin (10g), Chloramphenicol (30g),
Polymyxin B (300g), Colistin (10g), Imipenem (10 g), Trimethoprim (5 g), Ciprofloxacin
(5g), Netilmicin sulphate (30g), Tobramycin (10 g), Cefepime (30g), Gentamicin (10g),
Fosfomycin (200g).
2.6. Acclimatization of fish for Experimental Challenge
Live, healthy and disease free Labeo rohita of average weight of about 20-23 g were
collected from the local hatchery and transported to the laboratory in aerated bags. Fishes were
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acclimatized in well aerated 200 L FRP tanks with provision of supplementary feed at the rate of
2% of their body weight and were being fed twice a day. The fishes were acclimatized for two
weeks in the FRP tanks.

2.7. LD 50 determination

The Experimental challenge study was conducted to find out the lethal dose - 50% end point (LD50)

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in L. rohita. The bacteria were sub-cultured in 10 ml of Brain Heart Infusion (BHI) broth and were

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incubated at 37 ºC for 24h. After 24h incubation, 1 ml of culture was centrifuged at 5000 rpm for 5
min. After discarding the supernatant, bacterial pellet was washed thrice with normal saline (NS). 1

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ml of cell suspension was suitably diluted up to 10−8 in NS and spread plate method was used to
enumurate the number of cells/ml of suspension. The plates were incubated at 37 ºC for 24 h. The

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test was conducted with batches of 10 animals per dose by intraperitonial injection in L. rohita with
24 hrs of bacterial suspension. The fishes were injected with 0.2 ml of bacterial suspension
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intraperitoneally with final concentration of 1.8 X 103, 1.8 X 104, 1.8 X 105, 1.8 X 106, 1.8 X 107,
1.8 X 108, 1.8 X 109, 1.8 X 1010 CFU ml−1, respectively. The control fishes were injected with 0.2
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ml of NS. The challenge study was carried out in triplicates. A total number of 10 fishes were kept
in each tank (1×3 tanks taken as control and 8×3 tanks were taken for experimental challenge
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purpose). Fish mortality was recorded in every 24 h interval for 10 days. LD50 was calculated using
Reed and Muench (1938) method. In order to satisfy Koch's postulate the bacteria was reisolated
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and identified from the kidney, blood and liver of the moribund fishes.
2.8. Histopathology
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After observing and recording the external signs at the end of exposure time, tissues were collected
from the infected fish for histological analysis. Liver and kidney were dissected out from the
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infected fishes and were cut into 1-2 mm size.Then the tissues were fixed in 10% neutral buffered
formalin solution for 24 h. Tissues were dehydrated with a series of different concentrations of
alcohol and cleared in xylene and embedded into paraffin following the impregnation technique
(Leica EG 1140H, Germany). Paraffin blocks of liver and kidney were cut at a 5 μm thickness and
stretched on decontaminated glass slides. After deparaffinization, sections were stained with
haematoxylin and eosin (Luna, 1968) and were observed under light microscope.
3. Results
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3.1. Biochemical characterization and Molecular identification of the bacterium

The biochemical tests revealed that BB12 isolate was Gram negative, rods. The other tests revealed
that it was positive for urease, malonate, arabinose, cellobiose, melibiose, glucose, oxidase, ONPG,
lysine, ornithine, nitrate, VP, esculin hydrolase, adonitol, rhamnose, saccharose, raffinose,
trehalose, lactose, sorbitol, sucrose and mannitol; whereas it was negative for Ornithine, H2S,
Phenylalanine deamination, Citrate, Methyl red and Indole (Table 1). Additional biochemical test
showed that the bacteria was positive for L pyrrolydonyl–arylamidase, Beta-galactosidase, Gamma-

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glutamyl transferase, Fermentation/glucose, Beta-glucosidase, D-Maltose, D-Mannitol, D-Mannose,

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Beta-xylosidase, Palatinose, Tyrosine arylamidase, D-Tagatose, D-Trehalose, Alpha-galactosidase,

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Phosphatase, Lysine decarboxylase, Glu–Gly–Arg–arylamidase (Table 2).

The PCR amplified 16S rRNA gene from the isolated bacterium BB12 was sequenced

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(GenBank Accession Number: KY003130) and analyzed by using NCBI- BLAST program. The
BLAST result showed that the 16S rRNA gene sequence of BB12 is having 100% homology with
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K. pneumoniae. The biochemical and molecular result confirmed that the isolated bacterial
pathogen BB12 is K. pneumoniae.
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3.2. Phylogenetic analysis

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The phylogenetic cladogram was constructed using maximum likelihood algorithm. It revealed the
relationship of the isolate BB12 with other Klebsiella species The phylogenetic cladogram revealed
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that 16S rRNA gene sequence of BB12 was evolutionary very close to Klebsiella pneumoniae
(KR856389) with highest bootstrap value (Fig. 1).
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3.3. Antibiogram study

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Antibiogram study of Klebsiella pneumoniae (BB12) revealed that it was resistant to

Dicloxacillin (D/C), Ampicillin (AMP), Amoxicillin (AMX), Rifampicin (RIF5), Piperacillin
(PIT100/10), Ceftazidime (CAZ30), Fosfomycin (FO200), Trimethoprim(TR5), Cefepime
(CPM30), Cefixime (CFM5), Imipenem (IPM10) whereas sensitive to Doxycycline, (DO10),
Streptomycin (S25), Ofloxacin (OF2), Tetracycline (TE), Erythromycin (E10), Nalidixic acid
(NA30), Chloramphenicol (C30), Polymyxin B (PB300), Colistin (CL10), Ciprofloxacin (CIP5),
Netilmicin sulphate (NET30), Tobramycin (TOB10), Nitrofurantoin (NIT), Gentamicin (GEN10)
(Table 3).
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3.4. Cumulative mortality and determination of LD50

The cumulative mortality rate of Labeo rohita after infection with K. pneumoniae (BB12)
are shown in Fig. 2. No mortality found in the control fishes injected with NS upto 10 days of post
injection. Most of the fishes challenged by intra-peritoneal injection developed hemorrhagic
subcutaneous ulcers and reddening at injection sites. Bacterial strains were re-isolated from the
kidney, liver, and blood of infected fish and found to be the same as that of the injected strain. The
LD50 value of K. pneumoniae was calculated to be 1.05 X 106 CFU per fish.

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3.6. Histopathology

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Histopathology of the infected kidney tissue revealed Focal necrosis, ultra structural changes in the
glomeruli and renal tubules (Fig. 3A) and vacuolation in kidney (Fig. 3B). The tissue section of

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liver exhibits vacuolation, necrosis and disruption of hepatocytes (Fig. 4A) and formation of
melanomacrophages center (Fig. 4B)
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Discussion
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Diseases are the main barrier towards successful aquaculture practices. The present study was
conducted to identify the causative agents behind the mortality of the farmed L. rohita. In the
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present study, K. pneumoniae was isolated from the infected L. rohita fishes exhibiting
haemorrhages in the intraperitonial region. Usually K. pneumoniae is present everywhere in nature
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and have the capacity to infect wide range of organism including Homo sapiens, Zalophus
californianus, Nemipterus japonicus, O. niloticus, Cyprinus carpio, Amphiprion nigripes (Holden et
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al., 2016; Magill et al., 2014, Roberts et al., 2000, Jang et al., 2010, Takyi et al., 2012, Diana and
Manjulatha, 2012, Oliveira et al., 2014; Al-Imarah, 2008). K. pneumoniae as the causative agent
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responsible for infection in L. rohita have been confirmed as by 16S rRNA sequence analysis along
with the biochemical tests and challenge studies. Biochemical tests of the isolate BB12 was found
similar with the work reported by other workers (Gopi et al., 2016; Takyi et al., 2012; Diana and
Manjulatha, 2012).
The phylogenetic analysis of 15 different Klebsiella species revealed the 16S rRNA gene
sequence of BB12 have 100% homology with K. pneumoniae (KR856389). They were clustered in
a phylogenetic tree with highest bootstrap value. Gopi et al., 2016 had identified infection of K.
pneumoniae in Amphiprion nigripes using 16S rRNA gene sequence analysis.
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The antibiotics resistance is one of the most important problems in case of pathogenic
bacteria like K. pneumoniae. Antibiogram study of BB12 revealed that it was resistant to
Dicloxacillin (D/C), Ampicillin (AMP), Amoxicillin (AMX), Rifampicin (RIF5), Piperacillin
(PIT100/10), Ceftazidime (CAZ30), Fosfomycin (FO200), Trimethoprim(TR5), Cefepime
(CPM30), Cefixime (CFM5), Imipenem (IPM10). Most of the antibiogram susceptibility test results
were found similar with result reported by the earlier workers (Lin et al., 2016). This bacterium is
found to be resistant against most of the third and fourth generation antibiotics used in the present

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study.

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The complete pathological alterations were observed in the kidney of experimentally

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challenged Labeo rohita. Focal necrosis, ultra structural changes in the glomeruli and vacuolation
was observed in the kidney of the diseased fish. Similar types of histological changes in kidney of

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African catfish Clarias gariepinus infected by Edwardsiella tarda was observed by Abraham et al.,
2015. Vacuolation, necrosis and disruption of hepatocytes and formation of melanomacrophages
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center was observed in the liver section of L. rohita challenged with K. pneumoniae. The liver
tissues of infected fishes (Hoplostethus atlanticus, Aphanopus carbo, Phycis blennoides,
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Coryphaenoides rupestris) were studied by Feist et al., 2015 and observed the trabecular
arrangement of hepatocytes and significant variation in the individual hepatocytes in H & E stained
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sections. Hepatic necrosis and irregular cytoplasmic vacculation with converging sinusoids was
observed by Ramkumar et al., 2014 in the experimentally challenged Labeo rohita with
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Providencia vermicola. Gopi et al., 2016 have observed similar alteration in tissue structure of liver
and kidney of Amphiprion nigripes due to infection with K. pneumoniae.
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The present study is the first detailed report about pathogenic K. pneumoniae infection in
cultured L. rohita. In this study, LD50 value for K. pneumoniae in Labeo rohita was found to be
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1.05 × 106 CFU per fish. This LD50 value of K. pneumoniae estimated by in vivo experiments would
serve as a baseline data for future immune response studies. The clinical symptoms of the
experimentally challenged fishes were found same as the collected diseased fishes from cultured
fish pond. Bacterial colonies isolated from liver, kidney and blood of artificially infected moribund
and dead fishes were reconfirmed as K. pneumoniae by biochemical characteristics and 16S rRNA
gene sequence analysis. Since K. pnuemoniae was found to be pathogenic against L. rohita so, L.
rohita should be screened by molecular diagnostic techniques for the identification of this
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bacterium and better management practices should be followed by the fishers. The study would help
in managing the emerging bacterial pathogens in aquaculture system and its control in future.

Conflict of interest

The authors have declared no conflict of interest.

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Acknowledgments

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This work was funded by the National Fisheries Development Board (NFDB) (G/Nat.
Surveillance/2013 dated 16.08.2013), Hyderabad under “National Surveillance Program for Aquatic

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Animal Diseases (NSPAAD)” Project. The authors are thankful to Dr. Joykrushna Jena, Deputy
Director General (Fishery Science), Indian Council of Agricultural Research, New Delhi for his
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support and guidance. The authors would like to express their sincere gratitude to Prof. T. J
Abraham, Dept of Aquatic Animal Health, Faculty of Fishery Sciences, West Bengal University of
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Animal and Fishery Sciences, India for providing the equipment for biochemical analysis. The
authors are also thankful to Mr. Asim Kumar Jana for laboratory assistance.
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Fig.1. Phylogenetic tree analysis of Klebsiella sp. based on 16S rRNA nucleotide sequences.
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Phylogenetic tree was generated using Maximum-likelihood method by the MEGA 6 software.
The numbers next to the branches indicate percentage values for 10,000 bootstrap replicates.
Bootstrap values are shown at the nodes. The isolate BB12 identified in this study are indicated
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by the shaded triangle.

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Fig. 2. Cumulative mortality curves for the determination of LD50 values in Indian Major Carp

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Labeo rohita challenged with Klebsiella pneumoniae (BB12) by intra-peritoneal injection at
different concentrations.
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Fig. 3. A–B. Photomicrograph of Kidney of BB12 challenged Labeo rohita showing [A]: Focal
necrosis, ultrastuctural changes in the glomeruli and renal tubules (*) (H & E staining;
60X); [B]: vacuolation in kidney (X) (H & E staining; 60X)

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A B

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Z

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Fig. 4. A–B. Photomicrograph of Liver of BB12 challenged Labeo rohita showing [A]:
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vacuolation, necrosis and disruption of hepatocytes were evident (Z) (H & E staining;
60X); [B]: Formation of melanomacrophages center (M) (H & E staining; 60X)
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Table 1
Comparative biochemical analysis of BB12 isolated from Labeo rohita fingerlings with published
biochemical data of Klebsiella pneumoniae
Biochemical BB1 ** K. pneumoniae
Test 2
ONPG (β- + +
galactosidase)
Lysine + +

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Ornithine - -
Urease + +

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Phenylalanine - -
deamination

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Nitrate + +
H₂ S - -
Citrate - -

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V.P + +
Methyl red - -
Indole - -
Oxidase + +
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Malonate + +
Esculin + +
hydrolase
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Anabinose + +
Xylose + +
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Adonitol + +
Rhamnose + +
Cellobiose + +
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Mellibiose + +
Saccharose + +
Raffinose + +
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Trehalose + +
Glucose + +
Lactose + +
Sorbitol + +
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Mannitol + +
Sucrose + +
β haemolysin + +
Catalase + +
**
Phenotypic characteristics of Klebsiella pneumoniae observed earlier as described in Diana and
Manjulatha., 2012 , Brisse et al.,2006.
“+”: positive; “−”: negative
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Table 2
Additional biochemical characteristics of Klebsiella pneumoniae (BB12) strain as assessed by using
VITEK 2 compact (Biomerieux, France).

Biochemical Test Result

Ala–Phe–Pro–arylamidase (APPA) -
L pyrrolydonyl–arylamidase (PyrA) +
L-Arabitol (IARL) -
Beta-galactosidase (BGAL) +

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Beta-N-acetyl-glucosaminidase (BNAG) -

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Glutamyl arylamidase pNA (AGLTp) -
Gamma-glutamyl transferase (GGT) +
Fermentation/glucose (OFF) +

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Beta-glucosidase (BGLU) +
D-Maltose (dMAL) +
D-Mannitol (dMAN) +

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D-Mannose (dMNE) +
Beta-xylosidase (BXYL) +
Beta-alanine arylamidase pNA (BAlap) -
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L-Proline arylamidase (ProA) -
Lipase (LIP) -
Palatinose (PLE) +
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Tyrosine arylamidase (TyrA) +

D-Tagatose (dTAG) +
D-Trehalose (dTRE) +
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5-Keto D-gluconate (5KG) -

L-Lactate alkalinisation (ILATk) -
Alpha-Glucosidase (AGLU) -
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Succinate alkalinisation (SUCT) -

Beta-N-acetyl-galactosaminidase (NAGA) -
Alpha-galactosidase (AGAL) +
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Phosphatase (PHOS) +
Glycine arylamidase (GlyA) -
Lysine decarboxylase (LDC) +
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Orinithine decarboxylase (ODEC) -

L-Histidine assimilation (IHISa) -
Coumarate (CMT) -
Beta-glucoronidase (BGUR) -
O/129 resistance (O129R) -
Glu–Gly–Arg–arylamidase (GGAA) +
L-Malate assimilation (IMLTa) -
Ellman (ELLM) -
L-Lactate assimilation (ILATa) -
“+”: positive; “−”: negative
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Table 3

Antimicrobial susceptibility analysis of Klebsiella pneumoniae (BB12) isolates from Labeo rohita
fingerlings.

Antibiotics Results
Ofloxacin ( OF2) S
Streptomycin (S25) S

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Erythromycin (E10) S

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Dicloxacillin (D/C) R
Nitrofurantion (NIT) S

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Ampicilin (AMP) R
Tetracycline (TE) S
Amoxycilin (AMX) R
Doxycycline (DO10) S

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Rifampicin (RIF5) R
Nalidixic acid (NA30) S
Piperacillin (PIT100/10) R
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Chloramphenicol (C30) S
Ceftazidime (CAZ30) R
Polymixin B (PB300) S
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Colistin (CL10) S
Imipenem (IPM10) R
Trimethoprim(TR5) R
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Ciprofloxacin (CIP5) S
Netilmicin sulphate (NET30) S
Tobramycin (TOB10) S
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Cefipime (CPM30) R
Cefixime (CFM5) R
Gentamycin (GEN10) S
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Fosfomycin (FO200) R
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Table 4

Primer details for 16S rRNA gene PCR.

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Primer Annealing PCR product Sequence Reference

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name Temperature size (bp)
(°C)

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UFF2 52°C 1407 5/-GTTGATCATGGCTCAG-3/ Behera et
al., 2017
URF2 5/-GGTTCACTTGTTACGACTT-3/

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Highlights

Labeo rohita (Rohu) is one of the most cultivable carps in India with high consumer preference due
to its rapid growth and high eminence flesh. The modern aquaculture systems ponds are being
treated with inorganic chemicals and fertilizers. Farmers are over stocking and providing artificial
feeds for faster growth in aquaculture ponds. However, all these innovative practices had led to
stress resulting in various infections in fish species. The present study was conducted to identify
and characterize the etiological agent causing mortality in Indian Major Carp, Rohu (Labeo rohita)
in West Bengal, India. Diseased fish samples were collected from Burdwan, West Bengal, India for

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the isolation of pathogenic bacteria. The 16S rRNA gene sequence of the bacteria revealed that the

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isolate was 100% identical with Klebsiella pneumoniae (NCBI Accession number KY003130).

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