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  Table of Contents  
ORIGINAL ARTICLE
Year : 2020  |  Volume : 38  |  Issue : 3  |  Page : 390-396
 

Detection of various beta-Lactamases in Escherichia coli and Klebsiella sp.: A study from Tertiary Care Centre of North India


1 Department of Microbiology, Government Medical College and Hospital, Chandigarh, India
2 Department of Microbiology, Government Medical College and Hospital; Department of Parasitology, Post Graduate Institute of Medical Education and Research, Chandigarh, India
3 Department of Microbiology, Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Date of Submission13-Jun-2020
Date of Decision16-Jul-2020
Date of Acceptance17-Sep-2020
Date of Web Publication4-Nov-2020

Correspondence Address:
Dr. Meenakshi Singh
Department of Microbiology, Government Medical College and Hospital, Chandigarh
India
Dr. Varsha Gupta
Department of Microbiology, Government Medical College and Hospital, Chandigarh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_20_253

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 ~ Abstract 


Objective: The emergence of carbapenem-resistant Escherichia coli and Klebsiella species is a global threat. We aimed to compare two phenotypic methods and evaluate the genotypic method for the detection of beta-lactamases produced by E. coli and Klebsiella spp. Materials and Methods: One hundred and twenty-six E. coli and Klebsiella isolates were examined for phenotypic production of beta-lactamases by using disc diffusion, combined disc test (CDT) and modified carbapenem inactivation method (mCIM). All strains were also studied for the presence of various genes by polymerase chain reaction. Results: Out of 126 isolates, 96% of the isolates were extended-spectrum β-lactamase (ESBL) producers based on the presence of various ESBL genes. CDT method showed higher number of total (89%) carbapenemases in comparison to mCIM (81%). Among carbapenemases none of the isolates were Klebsiella pneumoniae carbapenemase producer by CDT, while 69% isolates were metallo-beta-lactamase (MBL) producers. Another method, mCIM/ethylene diamine tetraacetic acid mCIM showed 100% agreement for MBL detection. As regards, AmpC and class D carbapenemases; 0.04% and 16% positivity was detected, respectively, based on CDT method. Molecular analysis revealed 91% of the isolates harbouring carbapenemase genes. blaNDMwas the most common gene detected followed byblaOXA-48. Nine of the blaNDM-positive isolates also possessed blaOXA-48gene. Conclusion: Our finding shows high percentages of ESBL and carbapenemases in E. coli and Klebsiella spp. Among phenotypic methods, CDT seems to be a better choice as prevalence of carbapenemases shows lots of variation in our country. For Class B enzymes, both CDT and mCIM/eCIM can be used in the routine laboratories.


Keywords: Carbapenemase, combined disc test, extended-spectrum β-lactamases, Escherichia coli, Klebsiella, modified carbapenem inactivation method


How to cite this article:
Gupta V, Singh M, Datta P, Goel A, Singh S, Prasad K, Chander J. Detection of various beta-Lactamases in Escherichia coli and Klebsiella sp.: A study from Tertiary Care Centre of North India. Indian J Med Microbiol 2020;38:390-6

How to cite this URL:
Gupta V, Singh M, Datta P, Goel A, Singh S, Prasad K, Chander J. Detection of various beta-Lactamases in Escherichia coli and Klebsiella sp.: A study from Tertiary Care Centre of North India. Indian J Med Microbiol [serial online] 2020 [cited 2020 Nov 24];38:390-6. Available from: https://www.ijmm.org/text.asp?2020/38/3/390/299820





 ~ Introduction Top


The emergence and spread of carbapenem-resistant Escherichia coli and Klebsiella species (CREK) is a global health concern particularly in developing countries.[1] Carbapenems have been considered the most effective antibiotics for the treatment of serious human infections caused by multidrug-resistant Gram-negative bacteria (GNB).[2] Their utility has been significantly compromised by the rapidly increasing prevalence of Enterobacteriales, with carbapenem resistance conferred by various enzymes. Not only the CREK strains can be transmitted among patients in a hospital setting, the resistance genes can also be transferred to other susceptible bacteria within the same infected host.[3] Nowadays, infections due to Enterobacteriales possessing different types of enzymes are of concern for many reasons particularly increased length of stay, limited therapeutic options causing higher mortality rates and increased hospital costs.[4] These organisms are responsible for hospital associated infections as well as can be found in community infections such as urinary tract infections, septicaemia, pneumonia, peritonitis, meningitis, etc.[5]

The main mechanisms of resistance to carbapenems in GNB is either due to acquisition of genes coding for enzymes leading to breakdown of carbapenems or overexpression of a beta-lactamase with weak carbapenemase activity coupled with modifications in permeability of porins and/or overexpression of efflux pumps.[6] The carbapenemases are classified according to Ambler in three classes as Class A including the serine carbapenemases (Klebsiella pneumoniae carbapenemase [KPC]; non-metallocarbapenemase/imipenem hydrolysing beta-lactamase; and serratia marcescens enzyme); class B including the metallo-β-lactamases (MBL) (verona integron-encoded MBL [VIM]; active on imipenem [IMP]; and New Delhi MBL [NDM]); and class D β-lactamases (OXA-48, OXA-181, etc.). The clinical role of a fourth class (Ambler class C) is unknown but possesses some extended activity towards carbapenems.[7] These enzymes as such have weak activity towards carbapenems, if any activity at all. If an AmpC enzyme is found in a strain with other resistance mechanisms like extended-spectrum β-lactamase (ESBLs), outer membrane permeability and/or efflux pump overproduction, the resistance towards carbapenems may be enhanced.[8]

There is a variation in distribution of different carbapenemases produced by GNB according to geographical areas.[9],[10],[11],[12] In the United States, KPC carbapenemases are more prevalent, while in India, NDM-1 has been reported to be the most extensively observed carbapenemase among Enterobacteriales.[13],[14] The differences in occurrence of these enzymes causing antibiotic resistance alerts clinical microbiologists to identify these organisms in regard to limited treatment options and epidemiological concerns as well. In resource-limited settings also, early detection of specific carbapenem resistance mechanisms can be performed parallel to routine antimicrobial susceptibility testing by applying simple phenotypic methods. The data thus obtained from testing can help in timely institution of appropriate treatment based on antibiotic protocols formulated and reduce the spread of multi drug resistant bacteria with prevention and containment strategies. Based on detection, specific therapy can be decided like considering combination therapy in KPC/MBL carrying isolates in the form of carbapenems with colistin/tigecycline/aminoglycosides.[2] Further newer antibiotics such as ceftazidime-avibactam, meropenem-vobarbactam, cefidericol and aztreonam-avibactam which act on ESBL/KPC/Class C and OXA 48-producing strains, KPC-producing strains and the other two on all kinds of carbapenemases, respectively, can be tried depending on the availability.[15]

Various phenotypic tests have been adopted in the routine clinical microbiology laboratories for the detection of CREK along with molecular methods to identify and characterise the genetic determinants conferring carbapenem resistance.[16] Among them, one of the phenotypic methods is combined disc test (CDT) suggested by the European Committee for antibiotic susceptibility testing (EUCAST) which uses meropenem and meropenem containing various inhibitors and temocillin.[17] This method is commercially available and has been well-validated in various studies.[18] Clinical Laboratory Standards Institute (CLSI) also advocates various tests for ESBL and carbapenemase detection.[19]

In this setting, the aim of this study was to compare two phenotypic methods: CDT and modified carbapenem inactivation method (mCIM)/ethylene diamine tetraacetic acid mCIM (eCIM) and evaluate molecular method for the identification of types of ESBLs and carbapenemases produced by E. coli and Klebsiella spp.


 ~ Materials and Methods Top


Study design and settings

This prospective study was carried out in the Department of Microbiology, Government Medical College Hospital, Chandigarh, during a period of January to July 2019 on one hundred and twenty-six E. coli and Klebsiella species isolates from various clinical samples including urine, body fluids, pus, throat swab and high vaginal swabs received from symptomatic patients. The antibiotic susceptibility testing was done by Kirby–Bauer disk diffusion test according to CLSI guidelines using the Mueller–Hinton agar.[19] The bacterial isolates were identified to species level according to standard microbiological procedures. The following drugs (Hi-media, Mumbai) were tested: ceftriaxone (30 μg), cefotaxime (30 μg), gentamicin (10 μg), amikacin (10 μg), nalidixic acid (30 μg), ciprofloxacin (5 μg), norfloxacin (10 μg), nitrofurantoin (30 μg) and co-trimoxazole (25 μg). For second-line agents, susceptibility was done for cefoperazone-sulbactam (75/30 μg), piperacillin-tazobactam (100/10 μg), imipenem (10 μg) and meropenem (10 μg).[19] Those strains which showed reduced susceptibility (≤19 mm) based on disc diffusion test to meropenem/imipenem were confirmed for carbapenem resistance by microbroth dilution as per CLSI (MIC ≥4 μg/ml).[17]

Phenotypic detection

Extended-spectrum β-lactamases by disc diffusion test

Briefly, discs containing ceftazidime (30 μg), ceftazidime + clavulanic acid (30/10 μg), cefotaxime (30 μg) and cefotaxime + clavulanic acid (30/10 μg) (HiMedia), were placed independently, 30 mm apart (centre to centre) on a lawn culture of 0.5 McFarland on a Mueller–Hinton agar plate and incubated for 18–24 h at 35°C. An increase in the size of the zone of inhibition of ≥5 mm by addition of clavulanic acid was considered positive for ESBL.[19]

Carbapenemases by combined disc test

For confirmation of carbapenemase-production, CDT was performed using meropenem disc alone and in combination with various carbapenemase inhibitors by commercially available kit (Liofilchem® s.r.l.). All the isolates were assessed for the different classes of carabpenemases by evaluating synergistic effect of meropenem with different inhibitors [Table 1].
Table 1: Interpretative table: Combined disc test based on synergy of meropenem with carbapenemase inhibitors

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Modified carbapenem inactivation method along with ethylene diamine tetraacetic acid carbapenem inactivation method[19]

The modified CIM method as described in the CLSI M100 Performance Standards for Antimicrobial Susceptibility Testing assesses growth of a susceptible control strain (E. coli ATCC 25922) around a carbapenem disk previously incubated with a suspension of a suspected carbapenemase-producing test strain. If the test strain produces a carbapenemase, drug in the disk will be inactivated, thus allowing growth of the control strain up to the edge of the disk in the Mueller–Hinton Agar plate. In contrast, a zone of inhibition indicates that the antibiotic in the disk remains active and the test strain lacks carbapenemase activity. Further eCIM is used together with mCIM to differentiate MBLs from serine carbapenemases in Enterobacteriales. The mCIM was considered positive when the inhibition zone diameter measured between 6 and 15 mm and negative for carbapenemase when zone size is ≥19 mm. The eCIM results were interpreted only if the mCIM results indicated the presence of carbapenemase. A ≥5-mm increase in the zone diameter for eCIM as compared for mCIM was taken as the strain producing MBL.

Genotypic characterisation of extended-spectrum β-lactamases and carbapenemases

Genotypic identification of various genes was done at Microbiology Department of Sanjay Gandhi Postgraduate Institute of Medical Sciences (SGPGI), Lucknow, Uttar Pradesh, India, as per protocol.

DNA extraction

Briefly, DNA was extracted from bacterial strains using the boiling lysis method. Briefly, 1–2 colonies from overnight grown culture was suspended in 100 μl ultrapure water and boiled at 90°C for 20 min, following which centrifugation was done at 15000 rpm for 5 min, the supernatant was removed and stored at −20°C for further use.

Nucleic acid amplification test

Uniplex polymerase chain reaction (PCR) was performed for all genes encoding ESBLs (blaCTX-M, blaSHV, blaTEM) and carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP and blaOXA-48 type). Amplification was carried out in C1000 Touch™ Thermal Cycler (Bio-Rad, USA) in 25 μl reaction volume containing 2 μl of genomic DNA, 1 X DreamTaq PCR master mix (Thermo Fisher, USA), and forward and reverse primers (0.8 μM) specific for each gene were used. The resulting PCR products were analysed by 1.5% agarose gel electrophoresis and images were documented by a Bio-Rad Gel Doc imaging system. The positive controls used in the study were strains collected at SGPGI, Lucknow. These strains were PCR positive for respective genes. Sanger sequencing followed by nucleotide BLAST analysis on the National Center for Biotechnology Information (NCBI) confirmed their 100% identity with previously reported ones in the NCBI. Positive controls also served as PCR reaction control. E. coli strain ATCC 25922 was used as a template for negative control.[20],[21]


 ~ Results Top


Out of a total of 126 carbapenem resistant isolates with (MIC ≥4 μg/ml), E. coli were (n = 76), K. pneumoniae were (n = 44) and Klebsiella oxytoca (n = 6). Among them; 67 (53%) isolates were phenotypically positive for ESBLs (40 E. coli, 24 K. pneumoniae and 3 K. oxytoca) [Table 2]. On molecular analysis, 121 (96%) of the total isolates were ESBL producers based on the presence of various ESBL genes. The most prevalent β-lactamase was blaCTX-M(81%), followed by blaSHV(54%) and blaTEM(52%) [Table 3]. [Figure 1] and [Figure 2] show PCR results of blaCTX-M and blaTEM genes, respectively. Thirty-seven of the isolates carried all the three ESBL genes, while 24 isolates carried only combination of two ESBL genes.
Table 2: Phenotypic detection of beta lactamases

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Table 3: Numbers of carbapenemase producing isolates

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Figure 1: Polymerase chain reaction products of blaCTX-Mgene

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Figure 2: Polymerase chain reaction products of blaSHVgene

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The results of the phenotypic CDT showing total of (112/126) KPC, MBL, AmpC and Class D carbapenemases are depicted in [Table 2] and [Figure 3]. All the phenotypically positive ESBL producers were also showing MBL production. The second phenotypic method adopted for identifying CREK based on CLSI was mCIM showing (102/126) positive isolates [Table 2] and [Figure 4]. The overall concordance of eCIM for MBL detection was 100% in comparison with CDT of EUCAST [Table 2]. Further, mCIM followed by eCIM showed 100% specificity for MBL's detection as shown in CDT of EUCAST.
Figure 3: Synergy of meropenem with carbapenem inhibitors for confirmation of carbapenem resistant Escherichia coli and Klebsiella species. EDTA: Ethylene diamine tetraacetic acid

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Figure 4: Modified carbapenem inactivation method: Four different strains showing zone ≥22 mm

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The results of the PCR showed; ninety (71%) of the isolates harboured blaNDM gene while three of the phenotypically negative MBL strains carried blaNDM-1 gene. The isolated gene of Class D was found in 44/126 (35%) of the isolates. Nine of the blaNDM positive isolates also possessed blaOXA-48 gene. None of the isolate was positive for blaKPC, blaVIM and blaIMP[Table 3]. Furthermore amongst these, many of the strains harboured multiple genes. Twenty-two of the isolates also harboured different combinations of any of the three ESBL types (CTX-M, SHV and TEM) and one carbapenemase type of gene (blaNDM).


 ~ Discussion Top


The emergence of CREK strains is an important growing concern in developing countries due to significant potential impacts on antibiotic usage and patient outcomes. The carbapenemase production is associated with clinical issues due to increased mortality with very limited therapeutic options. The rapid identification of CREK employing cost-effective methods is of relevance to clinicians as well; apart from epidemiological importance due to their rapid dissemination in almost all geographic regions. There is a new concept of individualised therapy in accordance with the source and severity of infection and the susceptibility profile of the bacteria, among other factors.[22] Currently, ceftazidime-avibactam, ceftolozane-tazobactam, meropenem-vaborbactam, have been added as newly licensed antibiotics.[15]

There is a great variation among ESBLs occurring worldwide and is rapidly changing over time.[23] In the present study, based on CLSI phenotypic detection test, 53% of E. coli and Klebsiella were found to be ESBL producers. However on genotypic analysis, 96% of the isolates were carrying ESBL genes. This is in agreement with other studies where the prevalence ranges from 4.7% to 90%.[24] The difference in the prevalence based on phenotypic versus molecular tests has been highlighted in many studies. A study by Gautam et al. showed 40.8% of PCR-positive ESBLs as undetectable by phenotypic methods.[25] Similarly, study by Yazdi et al. showed 13.8% genotypic positive ESBLs unrecognised by phenotypic method.[26] This variation could be due to existence of multiple enzymes which can mask the inhibition of clavulanic acid in the medium.[27] It is also possible that some β-lactamases fail to reach a level to be detectable by disk diffusion tests and it may be based on the quality of Muller–Hinton agar used. However, there is also possibility that the gene is present but not expressed due to the absence of effective promoter or presence of repressor genes which may lead to varied levels of enzyme expression and the poor specificity of some antibiotic combinations.[28] Although the molecular methods can accurately detect ESBL but facilities for them are not available in most of the laboratories, especially in developing countries. Thus, phenotypic methods with variations are being recommended for routine use to detect ESBL production in Gram-negative bacilli.[29]

In our study, three main genes of ESBLs i.e., blaCTX-M, blaTEM and blaSHV were studied. The most common β-lactamase was blaCTX-M(81%), followed by blaSHV(54%) and blaTEM(52%). Among ESBLs, blaCTX-M enzymes are most prevalent across the world as compared to other enzymes.[30] However, the prevalence of individual ESBL genes differ among other study centres. Shahid et al.[31] reported more frequency of blaCTX-M(28.8%) over blaSHV(13.7%), while Ahmed et al.[32] showed increased number of blaCTX-M(71.4% in E. coli and 68.4% in Klebsiella over blaTEM(55.1% E. coli and 58% Klebsiella). Until the year 2000, blaTEM was the most prevalent ESBL gene in the Indian bacterial population but was replaced by blaCTX-M in the following decade.[33] The carriage of multiple ESBL genes by a single isolate has been reported in the literature earlier and the same has been reflected in our study.[25],[26] Thirty seven (30.6%) of the isolates carried all the three ESBL genes. In addition, 24 (19.8%) isolates carried only combination of two ESBL genes. A study done by Yazdi et al., showed 36.6% isolates carrying all the above three ESBL genes together, while 62.3% isolates included blaSHV and blaTEM, 55.9% blaTEM and blaCTX-M and 49.5% blaSHV and blaCTX-M genes together.[26] This highlights the emerging complexity of antibacterial resistance repertoire and warrants further studies.

In India, the prevalence of carbapenem resistance in Enterobacteriales varies widely from 12% to 97% across the country.[9],[10],[12],[34] The study for carbapenemases based on CLSI phenotypic evaluation showed overall positivity of 81% (102/126) by mCIM/eCIM. The other phenotypic methods as per CLSI have their advantages and disadvantages. The Modified Hodge Test has been replaced because of subjective interpretation, false positives with some Enterobacter spp. possessing AmpC enzymes and porin alterations and false negatives with NDM-1 carbapenemases. The advantages of CarbaNP include speed in providing results, simplicity of execution, objectiveness in interpretation and increased sensitivity and specificity while disadvantages of CarbaNP test are requirement of special reagents, and inconsistent detection of Oxa-type carbapenemases.[18],[35] Further, sample processing may also become expensive and time consuming if a large number of isolates are tested with this method. Hence, mCIM is used in the study based on thein vitro inactivation of meropenem by hydrolysis and eCIM which is used together with mCIM to differentiate MBLs from serine carbapenemases in Enterobacteriales. Eighty-seven out of 126 (69%) strains showed positive MBL results by both phenotypic methods (CDT and mCIM/eCIM) and molecular analysis revealed blaNDM gene in all of these isolates by PCR in addition to three more positives 90/126 (71%). New Delhi MBLs are more prevalent in the Indian subcontinent and Eastern Europe whereas KPC serine carbapenemases are mostly found in the Americas, the Mediterranean countries and China.[13],[14] In the present study also, 71% of the isolates harboured blaNDM gene and no other MBL gene which is in accordance with other studies.[31],[36],[37]

The second phenotypic detection CDT method is better for identifying CREK as compared to mCIM method due to simultaneous detection of various classes of carbapenemases.[18] The CDT method is based on inhibitors and has the advantage of showing excellent performance in discrimination of KPC, MBL and OXA-48 carbapenemases.[16] In this study also, the results of the CDT method showed higher number of total (89%) carbapenemases in comparison to mCIM. Among these none of the isolates were positive for KPC both by phenotypic and genotypic methods which are reflected in other studies also.[37],[38] Class C beta-lactamases, AmpC, are chromosomal enzymes that are produced by many species of GNB. but in E. coli and Klebsiella species, they are present on mobile elements.[39] In the present study, 5 (0.04%) of the isolates were found to be positive for AmpC production by phenotypic method using cloxacillin as inhibitor. A study done by Gupta et al. showed four strains out of fifty as positive for AmpC by using boronic acid and cloxacillin.[40] Various studies have showed co-production of AmpC with ESBLs in 4.7%–88% of the isolates.[1],[41],[42] The AmpC-enzyme is usually produced at low levels in E. coli and Klebsiella and thus rarely contributes to β-lactam resistance. However, few plasmidic AmpC enzymes may contribute to carbapenem resistance in porin deficient Enterobacteriales.[43]

Class D carbapenemases, Oxa-type conferring resistance to carbapenems are most commonly present in Acinetobacter spp. and occasionally in Enterobacteriales and Pseudomonas spp. Oxacillinases hydrolyse carbapenems weakly and are poorly inhibited by clavulanate. These enzymes have been previously reported in Mediterranean and European countries but now they are being progressively disseminated to other geographical areas including India.[44] Currently there is no available inhibitor for class D carbapenemases so an antibiotic temocillin which is highly resistant to Oxa-48 strains has been incorporated in CDT to demonstrate Oxa-48 like enzymes.[45] In the present study, only 16% of the isolates were presumed to be class D carbapenemases by CDT but this percentage was raised upto35% for Oxa-48 isolates on molecular characterization. Furthermore, combination of blaNDM-1 and blaOXA-48 gene was seen in nineteen isolates of CREK as has been reported earlier. The emergence of blaOXA carbapenemases in the Enterobacteriales, particularly Klebsiella spp., is of major significance because these bacteria are true pathogens that are able to infect immunocompetent individuals.

Hence, the phenotypic test based on CLSI and EUCAST both are missing some strains carrying blaNDM gene though number is very less, i.e., only three for MBLs and also blaOXA48 genes.[36],[37] The phenotypic testing has its limitation over isoelectric focussing and PCR which are considered as the gold standard methods.[40] Thus, attempts are going on to improve phenotypic testing with better sensitivity and specificity as doing molecular techniques on routine basis is not feasible in low budget diagnostic laboratories. Moreover, there is a limit of using molecular techniques in identifying different carbapenemases as they may lack sequence similarity to genes already described.[39] Other drawbacks of using molecular techniques for end users are lack of trained professionals, labour intensive and time consuming.[40]


 ~ Conclusion Top


Keeping in mind that carbapenems are the last drug of choice for multidrug-resistant strains, it has become important to keep a track on the surveillance of carbapenem resistant Enterobacteriales. It is necessary to determine the various classes of enzymes as treatment can vary for different classes of enzymes like KPC and MBL-producing K. pneumoniae can be treated by combination of carbapenems with colistin/tigecycline/aminoglycosides and for Oxa-48, carbapenems may not be a reliable treatment option.[46] Further, monitoring of various carbapenemases prevalent in the regions is of concern to see the change in trend from one type to another like NDM to OXA-48 group shift in enzymes as is being suggested in some studies.[38]

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
 ~ References Top

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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2004 - Indian Journal of Medical Microbiology
Published by Wolters Kluwer - Medknow

Online since April 2001, new site since 1st August '04