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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~ Conclusion
 ~  References
 ~  Article Figures
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  Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 2  |  Page : 210-218
 

Characterisation of virulence genes associated with pathogenicity in Klebsiella pneumoniae


Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Chennai, Tamil Nadu, India

Date of Submission10-Jun-2019
Date of Decision29-Jun-2019
Date of Acceptance14-Sep-2019
Date of Web Publication19-Nov-2019

Correspondence Address:
Ms. P A Remya
Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai - 600 116, Tamil Nadu
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijmm.IJMM_19_157

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 ~ Abstract 


Purpose: This study was undertaken to characterise the virulence factors in clinical strains of Klebsiella pneumoniae and analyse their association with various infections caused and also to determine the association between virulence factors and antimicrobial resistance profile. Materials and Methods: A total number of 370 clinically significant, non-duplicate isolates of K. pneumoniae isolated from both hospitalised patients and patients attending clinics were included in this study. Polymerase chain reaction (PCR) was carried out for the detection of various virulence genes such as mucoviscosity-associated gene A (magA), gene associated with allantoin metabolism (allS), Klebsiella ferric iron uptake(Kfu), capsule-associated gene A (K2A), regulator of mucoid phenotype A (rmpA), enterobactin (entB), yersiniabactin (YbtS), aerobactin, Fimbrial adhesin (FimH) and uridine-diphosphate galacturonate 4-epimerase (uge). Antimicrobial susceptibility testing and PCR-based detection of beta-lactamase-encoding genes such as extended-spectrum beta-lactamases, AmpCs and carbapenemases were performed. Univariate analysis was done to find the association between virulence genes and mortality. Results: The siderophore, entB, was present in most (90.5%) of the isolates. Of the 370 isolates, 345 carried multiple virulence genes; 15 harboured single virulence genes and 10 did not harbour any of the studied virulence genes. The most common combination of occurrence was entB and FimH. A mortality rate of 12.75% (38/298) was observed among hospitalised patients. None of the virulence genes had any significant association with mortality. Conclusion: Pathogenic K. pneumoniae can harbour single to multiple virulence genes. Invasive infection with even a single virulence gene-harbouring K. pneumoniae can lead to poor outcomes. Both multidrug-resistant (MDR) and non-MDR K. pneumoniae can harbour a variety of virulence genes. None of the virulence genes have a significant association with mortality.


Keywords: Antimicrobial resistance, beta-lactamases, Klebsiella pneumoniae, virulence factors


How to cite this article:
Remya P A, Shanthi M, Sekar U. Characterisation of virulence genes associated with pathogenicity in Klebsiella pneumoniae. Indian J Med Microbiol 2019;37:210-8

How to cite this URL:
Remya P A, Shanthi M, Sekar U. Characterisation of virulence genes associated with pathogenicity in Klebsiella pneumoniae. Indian J Med Microbiol [serial online] 2019 [cited 2020 Sep 30];37:210-8. Available from: http://www.ijmm.org/text.asp?2019/37/2/210/271167





 ~ Introduction Top


In humans, Klebsiella pneumoniae (K. pneumoniae) colonise skin, oropharynx or gastrointestinal tract. It causes various infections including pneumonia, bacteraemia, suppurative infections, urinary tract infections, cholangitis and rarely osteomyelitis or meningitis, especially in the immunocompromised or in those suffering from underlying disease conditions such as diabetes mellitus.[1],[2] Variations in the clinical spectrum of infections can be partly attributed to the presence or expression of the virulence factors in addition to the antimicrobial susceptibility profile.

The virulence factors that have been well characterised to date in K. pneumoniae consist of capsule, lipopolysaccharides (LPS), siderophores and fimbriae. These are important in adherence, colonisation, invasion and development of infection. The more recently identified virulence factors include outer membrane proteins, porins, efflux pumps, iron transport systems and genes involved in allantoin metabolism.[3],[4]

Capsules are made up of strain-specific capsular polysaccharides termed K antigens, and they protect bacteria from phagocytosis. Among the 77 capsular serotypes, K1 and K2 are associated with severe infections in humans.[3] Mucoviscosity-associated gene A (MagA) is restricted to the capsular serotype K1, whereas capsule-associated gene A (K2A) is restricted to serotype K2.[5] Both serotypes are frequently associated with liver abscess.[6],[7] However, data on the infections caused by K1 or K2 serotypes at non-hepatic sites are relatively limited.[8] Regulator of mucoid phenotype A (RmpA) gene is a plasmid or chromosome-mediated regulator of capsular polysaccharide synthesis.[9],[10] It enhances capsule production in hypervirulent K. pneumoniae (hvKP).[3] MagA and K2A are considered important in pathogenesis of hvKP infections.

LPS protect bacteria from complement-mediated lysis.[11] Its production is regulated by the Uridine diphosphate galacturonate 4-epimerase (uge) gene. In the absence of thisgene, K. pneumoniae are less capable of causing urinary tract infections, pneumonia and sepsis.[3],[12] FimH-1 is the gene encoding for fimbriae and mediates bacterial adhesion.[13] Type 1 fimbriae are expressed in 90% of K. pneumoniae and mediateadherence to many types of epithelial cells, especially the bladder epithelium.[3]

Siderophores are small molecules which can steal iron from the host iron-chelating proteins. Siderophores expressed in K. pneumoniae include enterobactin (entB),  Yersinia More Detailsbactin (YbtS) and aerobactin. The production of multiple siderophores helps the organism to overcome neutralisation by the host. The most common siderophore is the entB.[3] Compared to entB, YbtS is significantly overexpressed in the respiratory tract strains than in blood, urine or stool.[14] Aerobactin which is more often encountered in hvKP enhances their virulence.[3]

Klebsiella ferric iron uptake (Kfu) gene is a regulator of iron transport system that is involved in the acquisition of iron. It is associated with capsule formation, hypermucoviscosity, purulent tissue infection and is found in invasive strains.[3],[4] The gene associated with allantoin metabolism (allS) is used by bacteria to obtain carbon and nitrogen from the environment.[15] This gene was detected in primary liver abscess strains.[3]

All the above virulence genes singly or in combination contribute in various degrees to the initiation, invasion, spread, severity and outcome of infection with K. pneumoniae.

The purpose of this study was to characterise the virulence factors in clinical strains of K. pneumoniae and analyse their association with the infections caused, to study the relationship between virulence factors and antimicrobial resistance and to determine the association of virulence factors with mortality among hospitalised patients.


 ~ Materials and Methods Top


Bacterial isolates

The study was approved by the Institutional Ethics Committee. The isolates were collected over a period of 1 year from September 2014 to August 2015. A total of 370 clinically significant, consecutive, non-duplicate isolates of K. pneumoniae were included in this study. The isolates were identified up to species level by automated system (VITEK2 GN-card; BioMerieux, Brussels, Belgium) and standard biochemical tests.[16] They were obtained from urine (170), exudative specimens (132), respiratory secretions such as bronchial wash, endotracheal secretion, bronchoalveolar lavage and pleural fluid (38) and blood (30). Care was taken to ascertain that the study isolates were clinically significant by correlating with gram stain, significant colony count in culture and clinical history.

Phenotypic screen test for hypervirulent Klebsiella pneumoniae

String test was performed for all isolates as a screen test for hvKP. Hyperviscosity was tested by evaluating the formation of a mucoviscous string of >5 mm, when an inoculation loop was used to stretch a colony grown on an agar plate.[17]

Antimicrobial susceptibility testing

Antibiotic susceptibility testing was done by Kirby–Bauer disc diffusion method for cefotaxime (30 μg), ceftazidime (30 μg), amikacin (30 μg), ciprofloxacin (5 μg), piperacillin/tazobactam (100 μg/10 μg) and imipenem (10 μg) (HiMedia laboratories, Mumbai, Maharashtra, India) as per the Clinical and Laboratory Standards Institute guidelines.[18]

Detection of virulence-associated genes

Bacterial DNA was extracted by boiling method.[19] Multiplex polymerase chain reaction (PCR) was performed to detect magA, allS, Kfu, K2A, rmpA, entB and YbtS as described in previous studies.[20] Multiplex PCR was carried out with a final volume of 25 μl. Each reaction contained 10 pmol of each primer (Sigma-Aldrich, India), 10 mM of deoxynucleotide triphosphates (dNTP) mixture (Takara, India) and 5U Taq polymerase (Takara, India) in 2.5 μl of ×10 Taq polymerase buffer (Mg2 + plus). One microlitre of template DNA was added to 24 μl of the master mix. The PCR conditions were as follows: initial activation at 95°C for 15 min, followed by 30 cycles at 94°C for 30 s; annealing at 60°C for 90 s, followed by extension at 72°C for 60 s and a final extension at 72°C for 10 min. The amplicons were separated at 100 V for 2 h in a 2% (w/v) agarose gel containing ethidium bromide. Previously characterised strains were used as positive controls. Negative control was PCR mixture with water instead of template DNA.

Individual PCR was performed for aerobactin, FimH and uge as described in earlier studies.[4],[21]

Polymerase chain reaction for beta-lactamase-encoding genes

Multiplex PCR was performed to detect beta-lactamase-encoding genes such as extended-spectrum beta-lactamases (ESBLs) (TEM, SHV and CTX-M), plasmid-mediated AmpC beta-lactamases and carbapenemases (KPC, IMP, VIM, NDM and OXA-48) as described in previous studies.[19],[22],[23]

Statistical analysis

The data were analysed with the SPSS software 16.0 version (IBM Corp., New York, USA).

Univariate analysis was done to find the significance of association between virulence factors and mortality. Proportions were compared using Chi-square test. Differences were considered statistically significant if P < 0.05.

DNA sequencing

PCR-positive amplicons were purified and sequenced by BigDye 3.1 cycle sequencing kit using the Sanger AB13730 XL DNA analysing instrument (AgriGenome, India). The nucleotide sequences analysed were compared with the sequence available at the National Center for Biotechnology Information website (www.ncbi.nIm.nih.gov). The DNA sequences were submitted to GenBank and the following accession numbers were obtained: magA (MF188924), allS (MF188923), Kfu (MF188920), K2A (MF188922), rmpA (MF188921), entB (MF188916), YbtS (MF188918), aerobactin ( MF188915), FimH (MF188917) and uge (MF188919).


 ~ Results Top


Ninety per cent of the isolates carried the entB gene, followed by FimH (89.1%), uge (48.6%), YbtS (44.3%), Kfu (27.8%), aerobactin(5.4%), rmpA (5.1%), K2A (2%), allS (1%)and magA (0.2%) [Figure 1], [Figure 2], [Figure 3]. Ten isolates did not harbour any of the genes looked for.
Figure 1: Lane 1 and 2 – Positive control and test strain of magA. Lane 5 and 6 – Positive control and test strain of K2A. Lane 8 and 9 – Positive control and test strain of rmpA. Lane 3, 7 and 10 – Negative controls. Lane 4 – 100 bp ladder

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Figure 2: Lane 1 and 2 – Positive control and test strain of allS. Lane 13 – 100 bp ladder. Lane 4 and 5 – Positive control and test strain of Kfu. Lane 3, 6, 9, 12 and 16 – Negative controls. Lane 10 and 11 – Positive control and test strain of YbtS. Lane 14 and 15 – Positive control and test strain of aerobactin

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Figure 3: Lane 1 – 100 bp ladder. Lane 2 and 3 – Positive control and test strain of FimH. Lane 4 and 7 – Negative controls. Lane 5 and 6 – Positive control and test strain of uge

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Distribution of the studied virulence genes in various clinical specimens is depicted in [Table 1].
Table 1: Distribution of the virulence genes in various clinical specimens

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In total, 345 isolates of K. pneumoniae carried multiple virulence genes in them. Some genes occurred singly in 15 isolates which included FimH (1.6%), entB (1.3%), uge (0.5%) and YbtS (0.5%).

In 329 isolates, two or more genes coexisted. A combination of entB and FimH was the most common prototype observed. The pattern of occurrence of multiple virulence genes is depicted in [Table 2]. On the contrary, certain combinations of genes (16) were encountered only in single isolates [Table 3].
Table 2: Virulence genes combinations and their frequency of occurrence

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Table 3: Virulence genes combinations in single isolates

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The string test was positive in 21 isolates. All the K1 (1) and K2 (9) serotypes were string test positive, whereas only 11 of the rmpA carriers exhibited string test positivity.

Antimicrobial susceptibility pattern

The susceptibility of the study isolates to different antimicrobial agents is as follows: imipenem (87.83%), amikacin (82.16%), piperacillin/tazobactam (80.27%), ciprofloxacin (62.97%), ceftazidime and cefotaxime (41.09%).

Beta-lactamases and virulence factors

Among 370 isolates, 58.91% produced ESBLs, 9% AmpCs and 13.78% were carbapenemase producers.

[Table 4] depicts the distribution of the virulence genes among the different beta-lactamase-producing K. pneumoniae. EntB followed by FimH, uge, YbtS and Kfu was predominantly encountered in those isolates which produced CTX-M. One isolate which was an AmpC producer harboured the YbtS gene. Thirty-three isolates which coproduced ESBL and AmpC harboured entB (75%), FimH (84%) and uge ( 39%). Those isolates which were ESBL and carbapenemase producers also had similar distribution of the virulence genes in them. Notably those which were third-generation cephalosporins susceptible and harbouring the TEM and SHV alone had a high proportion of all virulence genes distributed among them. Of interest was the fact that two of the hypervirulent K2A positive K. pneumoniae carried NDM-1, OXA-48 and CTX-M.
Table 4: Beta-lactamases and virulence genes

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A mortality rate of 12.75% (38/298) was observed among the patients admitted in the hospital. The virulence genes profiles, beta-lactamases-encoding genes detected and underlying disease conditions of these patients are given in [Table 5]. Univariate analysis of mortality-associated virulence genes did not reveal a significant association between mortality and any of the virulence genes [Table 6].
Table 5: Mortality-associated virulence genes and beta-lactamases

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Table 6: Univariate analysis: Virulence factors and mortality

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 ~ Discussion Top


K. pneumoniae causes a wide range of infections both in the community and health-care setting leading to increased morbidity and mortality.[24] Pathogenicity of K. pneumoniae is due to the presence of various virulence factors such as capsule, endotoxins, siderophores, iron-scavenging systems and adhesins. These factors help this bacterium to evade immune system and cause various infections.[4],[25]

Presence of multiple virulence genes was observed in 93.24% (345/370) of the study isolates.

Majority of K. pneumoniae carried one or more siderophores. Among the 370 isolates, 90.5% (335/370) expressed entB gene. Expression of this gene was high in exudative (95.4%), followed by respiratory (92.1%), urine (88.2%) and blood isolates (80%). Several previous studies have also reported entB as the most common siderophore detected in K. pneumoniae.[4],[13],[26],[27]

YbtS gene was present in 44.3% (164/370) of the study isolates. This is in agreement with the earlier report that YbtS is the second common siderophore in K. pneumoniae after entB.[3] In the present study this gene was observed in sepsis causing K. pneumoniae (63.3%) and in 60% of respiratory tract isolates. Historically, YbtS has been more prevalent among respiratory isolates contributing to the risk of infections in the respiratory tract.[14] In China and Algeria, the presence of YbtS gene was 83.7% and 46.3%, respectively.[28]

Among the siderophores, aerobactin exhibits lowest affinity toward iron.[3] Only 5.4% (20/370) of K. pneumoniae harboured aerobactin gene in this study.

Most K. pneumoniae express fimbrial adhesins.[29] In the present study, FimH gene encoding for type 1 fimbriae was present in 89% (330/370) of the total isolates. It was detected highest in urinary isolates (91.1%) supporting the fact that type 1 fimbriae contribute to the pathogenesis of urinary tract infections.[3] It was also detected in 87.8% of exudative, 89.4% of respiratory and 83.3% of blood isolates. A previous study has reported the presence of type 1 fimbriae from blood and wound isolates.[30] El Fertas-Aissani et al. and Aljanaby and Alhasani observed the presence of FimH gene in all of urinary isolates.[4],[13]

Previous animal studies have cited that K. pneumoniae carrying uge gene were more virulent.[12] Nearly one-half (48.6%) harboured uge gene in this study. Its distribution in various clinical specimens was blood (50%), urine (48.8%), exudates (48.4%) and respiratory secretions (47.3%). Aljanaby and Alhasani observed the presence of uge gene in 84.6% of urinary tract isolates and in all the blood and respiratory isolates.[4] The occurrence of uge gene in K. pneumoniae has varied widely (41.6% to 86%) in different studies.[25],[30],[31]

Kfu gene codes for an iron uptake system and is associated with capsule formation and invasiveness.[3],[30] Presently 27.8% (103/370) of the study isolates carried this gene. It was detected in blood isolates, followed by exudates, respiratory secretion and urine. Similar observations have also been made from Iraq.[4] This gene has been reported from bacteremic isolates of K. pneumoniae previously.[32],[33]

Capsular types K1 and K2 are considered to be the most virulent serotypes in humans.[3] In this study, K2A gene (specific to K2 serotype) was detected in 2% (9/370) and magA (specific to K1 serotype) gene was detected in 0.2% (1/370) of the isolates. In Taiwan, K2A gene was more prevalent (7/18) compared to magA (4/18).[8] Abdul-Razzaq et al. also observed K2 serotype as being more common than K1 serotype (8/43).[34]

The absence of rmpA gene in phenotypically hypermucoviscous strains has been attributed to the presence of other genes. Discrepancies between phenotypic and genotypic markers of hvKP have been cited by other authors.[35]

K1 serotype was detected in one patient with perianal abscess in the present study. Liu et al. observed that K. pneumoniae is one of the major causative agents for perianal abscess in diabetic patients, but they did not investigate the serotypes associated with perianal abscess.[36] The K2 serotype was isolated from nine patients; one had sepsis due to cholangitis and other one had sepsis due to pneumonia. Malignancy, chronic kidney disease and chronic liver disease were the other risk factors identified in other patients. Although K1/K2 serotypes are frequently associated with liver abscess, they have been isolated from extrahepatic sites also.[8]

In this study, K1 and K2 serotypes coexisted with other virulence factors such as siderophores, fimbriae and LPS. It has been suggested that the increased virulence associated with K1 and K2 serotype may be due to the co-carriage of multiple virulence genes, compared to other strains.[3]

In the present study, very few (5.1%) carried the rmpA gene. It coexisted with magA 5% (1/20), K2A 10% (2/20) and non-magA/K2A 85% (17/20). Elsewhere, a substantial number of study isolates carried the rmpA gene (62.5%).[4] Al-Jailawi et al. have also observed the presence of rmpA gene in K1, K2 and non-K1/K2 serotypes in 21.7%, 45.5% and 16.7% of K. pneumoniae, respectively.[9] In Taiwan, all rmpA-positive isolates coharboured magA and K2A.[37]

A correlation between the presence of rmpA gene and aerobactin has been described wherein 96% of rmpA gene-positive isolates coproduced aerobactin.[38] In our study, 75% (15/20) of rmpA gene were also aerobactin producers. Tan et al. reported that 48.8% of bacteremic isolates harboured the aerobactin gene.[32] However aerobactin carrying isolates in this study were obtained from all specimen types.

The allS gene was presented in only three exudative isolates and one urine isolate. This gene has been encountered in abscess-derived K. pneumoniae in China (41.7%).[39] The concurrent presence of allS and K1 is in agreement with previous observations.[19],[39]

The distribution of virulence genes among the various beta-lactamase-producing isolates was analysed. FimH has previously been associated with KPC-positive K. pneumoniae.[40] However, we found that the lone KPC producer harboured multiple virulence genes, namely YbtS, entB, Kfu and uge in addition to FimH. Studies using clinical samples have proposed that KPC-producing K. pneumoniae lack magA, K2A, rmpA and aerobactin, and this is in alignment with the present study.[41] It has been reported that ESBL-producing K. pneumoniae are able to produce more fimbrial adhesins.[42] In this study, 195 ESBL producers harboured the FimH, and this was the second most prevalent virulence gene among them.

The genes rmpA, rmpA2, aerobactin and allS are more frequently found in hvKP isolates than in classical K. pneumoniae.[43] From India, Shankar et al. have detected Kfu, allS, entB and YbtS in hvKP. All the K. pneumoniae isolates coded for genes responsible for allantoin metabolism in their study. By whole-genome analysis, K1 capsular types were identified in five isolates, whereas there were no K2 capsular serotypes. The international clone ST23 and two novel sequence types were reported from their study.[33] Presently, two of the K2A-positive hvKP harboured both blaNDM and blaOXA-48. Significantly, the non-multidrug-resistant K. pneumoniae carried multiple virulence genes in them.

Ten isolates 2.7% (10/370) did not harbour any of the studied virulence genes. It can be reasonably assumed that these isolates could have harboured the genes that were not included in the PCR. Among them, beta-lactamase genes detected were as follows: ESBLs (2), ESBLs +AmpC (3), ESBLs + carbapenemase (1) and SHV-1 alone (3). One had no beta-lactamase-encoding gene. This can be explained by the fact that SHV-1 has been identified only in up to 80%–90% of K. pneumoniae.[44]

A proportion of the study isolates harboured single virulence genes only (15/370): entB, FimH, uge and YbtS. FimH alone was detected in five urinary isolates and one blood isolate. EntB gene was also observed singly in urinary tract (2) and blood isolates (3). Two uge genes were detected from patients with urinary tract and wound infections. YbtS gene was isolated from one patient with sepsis and the other with wound infection. Fatal outcomes was observed in three patients who harboured single virulence genes namely; FimH, entB and YbtS.

The mortality rate was 12.75% (38/298) among the hospitalised patients. The isolates from these patients carried 1 to 7 virulence genes. By univariate analysis, none of the virulence genes had significant association with mortality [Table 6]. Reports of increased mortality with magA- and K2A- harbouring K. pneumoniae have been cited before.[45] Similarly, YbtS has been associated with high mortality.[46] Aerobactinhas been determined to be an important virulence characteristic for mortality in bacteraemic isolates, both in human and animal studies.[39] However, clear association between the virulence factors and 30-day mortality was not evident in a study from Singapore.[47]


 ~ Conclusion Top


Pathogenic K. pneumoniae harbour multiple types of virulence genes. Invasive infection with even a single virulence gene harbouring K. pneumoniae can lead to poor outcomes. EntB followed by FimH are the most prevalent genes that contribute to pathogenesis of various types of infections. Detection of virulence genes by PCR will help to characterise them and help to understand how they function in different host environments. Occurrence of a variety of virulence genes is encountered in ESBL, carbapenemase and AmpC-producing K. pneumoniae. Non-multidrug resistant K. pneumoniae may also possess multiple virulence genes which contributes to their establishment and invasion in host tissues. There is no significant association between mortality and any of the virulence factors.

Acknowledgement

All the authors are grateful for the financial assistance provided by Sri Ramachandra Institute of Higher Education and Research as Founder Chancellor Fellowship.

Financial support and sponsorship

This study was supported by Founder Chancellor, Shri. N.P.V Ramasamy Udayar Research Fellowship, provided by Sri Ramachandra Institute of Higher Education and Research.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

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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