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Indones Biomed J. 2017; in press
RESEARCH ARTICLE
Corresponding author. E-mail: kambang_sar@yahoo.com
Received date: Jun 11, 2017; Revised date: Sep 14, 2017; Accepted date: Sep 27, 2017
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were resistant to Penicillin and 5.3% were resistant to
ACKGROUND: Diptheria cases are treated with erythromycin. Seven isolates (12%) showed resistance
both anti-diphtheria serum (ADS) and antibiotics. to more than one antibiotics, such as tetracycline and
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Penicillin and erythromycin are the primary choices clindamycin, vancomycin and clindamycin, penicillin and
for any Corynebacterium diphtheriae (C. diphtheriae) tetracycline, also penicillin and vancomycin. Moreover, 4
recultured by using Blood Agar (BA) and Cystine Tellurite KEYWORDS: Corynebacterium diphtheriae, resistance,
Blood Agar (CTBA). Afterward, these isolates were antimicrobial
identified by using API Coryne. The antibiotic susceptibility
pattern was determined by using Kirby Bauer Method based Indones Biomed J. 2017; in press
on CLSI M45-A2.
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The Indonesian Biomedical Journal, 2017 (in press) Print ISSN: 2085-3297, Online ISSN: 2355-9179
in communities. Penicillin and erythromycin are Twenty-four isolates are from Banten, 14 isolates are from
recommended antibiotics to treat diphtheria by the World West Kalimantan, 1 isolate is from West Java, 10 isolates
Health Organization (WHO). However, it has been reported are from Jakarta, 1 isolate is from Central Kalimantan and
that there are C. diphtheriae which resistant to penicillin, 7 isolates are from East Java. All isolates were stored in
oxacillin, erythromycin, rifampicin, tetracycline and Trypticase Soy Broth (TSB) with 20% glycerol, at -80°C.
clindamycin.(4-6). Another problem that has been identified Laboratory examination was conducted at the Bacteriology
in the treatment of diphtheria is the increase of multidrug Laboratory, Center for Biomedical and Basic Technology of
resistance C. diphtheriae. Therefore, it is necessary to Health, Ministry of Health Indonesia in January-December
monitor the antimicrobial susceptibility of C. diphtheriae 2015.
continuously.
In 2008, 14.8% isolates of C. diphtheriae in Brazil Re-culture and Antimicrobial Susceptibility Testing of
showed resistance to penicillin G, even erythromycin and C. diphtheriae
azithromycin were still effective, but the susceptibility to All isolates were recultured on Blood Agar (BA) and
erythromycin had decreased.(6) Meanwhile, in Canada Cystine Tellurite Blood Agar (CTBA) medium. The isolates
around 2011, multidrug resistant C. diphtheriae were were reidentified by using API Coryne®. The susceptibility
isolated from a skin wound. These isolates were resistant test was performed by disk diffusion method. Mueller
to clindamycin and erythromycin.(7) A study in Indonesia Hinton agar medium containing 5% sheep blood was used
showed that C. diphtheriae is resistant to tetracycline since in this method.(8-13) The antibiotic susceptibility test was
1982. During that time, 86% of 133 C. diphtheriae isolated performed by using disc diffusion method, based on Clinical
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from diphtheria patients were resistant to tetracycline.(8) and Laboratory Standards Institute (CLSI) M45. Medium
The gold standard of antimicrobial susceptibility test that used in antibiotics susceptibility test was Mueller
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for C. diphtheriae is broth microdilution method. However, Hinton agar medium containing 5% sheep blood.(8-13)
this method is quite challenging and has some limitations, Antibiotics used in this antibiotic susceptibility testing
such as time-consuming, a high risk of contamination were erythromycin (15 mg), rifampin (5 mg), linezolid (30
and more complicated rather than disk diffusion method. mg), clindamycin (2 mg), moxifloxacin (5 mg), gentamicin (10
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Disk diffusion is another alternative method to determine mg), Trimethoprim-sulfamethoxazole, (15 mg), vancomycin
the antimicrobial susceptibility of C. diphtheriae. This (30 mg), tetracycline (30 mg) and benzylpenicillin (10 mg).
method is simpler, easier and cheaper. The reason for There was no breakpoint criteria for C. diphtheriae, therefore
choosing Staphylococcus aureus and Streptococcus spp. as in this study, the breakpoint referred to Streptococcus spp.
a susceptibility breakpoints for C. diphtheriae is because for antibiotic other than penicillin, and Staphylococcus spp.
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both of them are gram-positive bacteria. for breakpoints of penicillin(6,7) We used Streptococcus
In Indonesia and other developing countries, antibiotics pneumoniae ATCC 49619 as internal quality control for
are available without a prescription. Consequently, the C. diphtheriae antibacterial susceptibility testing.
emergence of multidrug resistant C. diphtheriae become
another problem in controlling diphtheria. The aim of Statistical Analysis and Ethical Approval
this study is to determine the pattern of resistance and the Data analysis of antimicrobial resistance pattern and profile
effectiveness of some antibiotics against C. diphtheriae. This was performed by using WHONET software version 5.6.
study presents the resistance pattern of mono and multidrug The Ethical approval was obtained from Ethical Committee
resistance C. diphtheriae to antibiotics that frequently used of National Institutes of Health Research and Development,
in diphtheria treatment in Indonesia. Ministry of Health Indonesia, with the clearance number:
LB.02.01/5.2/KE.315/2015.
Methods
Results
Bacterial Isolates
Fifty-seven C. diphtheriae isolates were isolated from Biochemical identification of 57 reculture isolates on CTBA
diphtheria outbreaks investigation in Indonesia during and BA was shown in Table 1. Meanwhile, the antimicrobial
2010-2015. All isolates were obtained from nasopharynx susceptibility pattern of 57 C. diphtheriae isolates by using
and throat swab of diphtheria patients in several provinces. disc diffusion method was shown in Table 2.
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Antimicrobial Susceptibility of C. diphtheriae from Indonesian’s Outbreak (Sariadji K, et al.)
Indones Biomed J. 2017; in press
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GEN 10 µg 57 1.8 3.5 94.7
Erythromycin.
VAN 30 µg 57 8.8 5.3 ES 86
TCY 30 µg 57 84.2 7 8.8
Table 2 shows that all 57 isolates are sensitive to
CLI 2 µg 57 5.3 5.3 89.5
moxifloxacin and linezolid. There are 84.2% C. diphtheriae
LNZ 30 µg 57 0 0 100
isolates that resistant to tetracycline, 22.8% resistant
RIF 5 µg 57 1.8 0 98.2
trimethoprim-ssulfamethoxazole, 10.5% resistant to
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SXT 1,25/23,75 57 22.8 0 77.2
penicillin, 8.8% resistant to vancomycin, 5.3% resistant to
ERY 15 µg 57 5.3 3.5 91.2 erythromycin and 5.3% resistant to clindamycin.
%R: Resistance Percentage, % I : Intermediate percentage, %S: The profile of antibiotic susceptibility result of
Susceptible percentage, PEN: Penicillin G, MFX: Moxifloxacin,
C. diphtheriae is shown on Table 3, there are some isolates
GEN: Gentamicin, VAN: Vancomycin, TCY: Tetracycline,
CLI: Clindamycin, LNZ: Linezolid, RIF: Rifampicin, SXT: that resistance to two or more antibiotics, commonly called
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The Indonesian Biomedical Journal, 2017 (in press) Print ISSN: 2085-3297, Online ISSN: 2355-9179
was resistant to clindamycin, erythromycin, tetracycline and Some studies reveal that some C. diphtheriae
trimethoprim-sulfamethoxazole.(7,17-20) isolates are resistant to some drugs, such as penicillin
In our study, there were 6 isolates (10.5%) that G, oxacillin and other antibiotics which are used for the
resistance to two antibiotics, such as tetracycline and treatment of diphtheria, for instance rifampin, tetracycline
clindamycin, vancomycin and clindamycin, vancomycin and clindamycin. The presence of b-lactams resistance
and tetracycline, also penicillin and vancomycin. Then against C. diphtheriae should be concerned, especially
4 isolates (7%) resistance to 3-5 antibiotics. All isolates in the administration of penicillin for patients who have
susceptible to moxifloxacin and linezolid and 15 out of endocarditis because of C. diphtheriae infections. It could
57 C. diphtheriae isolates (26.3%) are susceptible to all increase the risk of treatment failure.(6) A study in Brazilian
antimicrobials tested. reported that a patient with endocarditis bacterialis caused
A study conducted in Cipto Mangunkusumo Hospital, by multidrugs resistance C. diphtheriae infection was
Jakarta in 1982 reported that 33 out of 188 samples are showing a treatment failure. Based on the study, clinician
identified as C. diphtheriae subtype mitis.(8) All isolates must reconsider the use of resistant to b-lactams
were tested by disc diffusion and E-test, simultaneously. It antibiotic if the clinical condition of patient with
showed that the agreement value between disc diffusion and systemic infection does not improve after being treated
E-test method were 94.9%.(6) with b-lactams antibiotics.(6)
Tetracycline resistance C. diphtheriae is uncommon According to CLSI document M45-A2 guideline,
in some countries, but some study in Europe countries the microdilution broth method is recommended in
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revealed C. diphtheriae that resistant to tetracycline, C. diphtheriae susceptibility testing. We already conducted
after treatment of intravenous tetracycline. Tetracycline ES our research with this method by using an automated
resistance C. diphtheriae was also reported in 1982 in reading incubation system (ARIS) (Thermo Scientific,
Cipto Mangunkusumo Hospital, Jakarta. In that study, Massachusetts, USA) to determine dentification and
133 samples (86%) showed resistance to tetracycline.(10) antimicrobial susceptibility testing of C. diphtehriae,
Moreover, the study reported all the isolates susceptible to nevertheless there are several limitations of the sensititre
penicillin and erythromycin. It study have similarity with ARIS. First, it only detects genus level of corynebacterium
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our research, especially in tetracycline resistance. On the species. Second, the breakpoints of determining of the
contrary, the research of susceptibility test in Brazil reported antibiotic concentration have small range values. Therefore,
that 12.8% out of 47 isolates of C. diphtheriae was resistant when examining Streptococcus pneumoniae ATCC 49619
to tetracycline, by using minimum inhibitory concentration as a validity test, the results are out of the range. Third,
(MIC) method. the sensititre plate with lysed horse blood medium for
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Antimicrobial Susceptibility of C. diphtheriae from Indonesian’s Outbreak (Sariadji K, et al.)
Indones Biomed J. 2017; in press
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