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Journal of Clinical Microbiology, June 2002, p. 2009-2015, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2009-2015.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Antibiotic Resistance, Virulence Gene, and Molecular Profiles of Shiga Toxin-Producing Escherichia coli Isolates from Diverse Sources in Calcutta, India

Asis Khan,¹ S. C. Das,² T. Ramamurthy,¹ A. Sikdar,² J. Khanam,³ S. Yamasaki,⁴ Y. Takeda,⁵ and G. Balakrish Nair^1,6*

National Institute of Cholera and Enteric Diseases, Beliaghata,¹ Eastern Regional Station, Indian Veterinary Research Institute, Belgachia,² Jadavpur University, Jadavpur, Calcutta, India,³ Department of Veterinary Sciences, Graduate School of Agriculture and Biological Sciences, Osaka Prefecture University, Osaka,⁴ Faculty of Human Life Sciences, Jissen Women's University, Tokyo, Japan,⁵ International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Dhaka, Bangladesh⁶

Received 27 December 2001/ Returned for modification 11 February 2002/ Accepted 12 March 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibiotic resistance, virulence gene, and molecular profiles of Shiga toxin-producing Escherichia coli (STEC) non-O157 strains isolated from human stool samples, cow stool samples, and beef samples over a period of 2 years in Calcutta, India, were determined. Resistance to one or more antibiotics was observed in 49.2% of the STEC strains, with some of the strains exhibiting multidrug resistance. The dominant combinations of virulence genes present in the strains studied were stx₁ and stx₂ (44.5% of strains) and stx₁, stx₂, and hlyA (enterohemorrhagic E. coli hemolysin gene) (19% of strains). Only 6.4% of the STEC strains harbored eae. The diversity of STEC strains from various sources was assessed by random amplification of polymorphic DNA (RAPD). STEC strains that gave identical or nearly similar DNA fingerprints in RAPD-PCR and had similar virulence genotypes were further characterized by pulsed-field gel electrophoresis (PFGE). Identical RAPD and PFGE profiles were observed in four sets of strains, with each set comprising two strains. There was no match in the RAPD and PFGE profiles between strains of STEC isolated from cows and those isolated from humans. It appears that the clones present in bovine sources are not transmitted to humans in the Calcutta setting although these strains showed evolutionary relatedness. Maybe for this reason, STEC has still not become a major problem in India.

Top Abstract Introduction Materials and Methods Results Discussion References

 
During the past decade, Shiga toxin-producing Escherichia coli (STEC) has evolved from a clinical novelty to a global public health concern. STEC infections have been reported from over 30 countries and can cause a spectrum of human illness ranging from symptom-free carriage to severe bloody diarrhea and even life-threatening sequelae such as hemolytic-uremic syndrome (HUS) (4, 31). STEC is a serologically diverse group of food-borne, zoonotic pathogens, of which the serotype O157:H7 has been epidemiologically significant worldwide because of its notoriety of being associated with life-threatening disease (18). However, in some geographic areas, non-O157 strains are more commonly isolated from persons with diarrhea or HUS than are O157 STEC strains (40).

The ability of STEC to cause serious disease in humans is related to the production of one or more Shiga-like toxins (Stx1, Stx2, or their variants), which inhibits protein synthesis of host cells, thus leading to cell death (26, 27). Additional virulence factors—including the presence of a pathogenicity island designated the locus of enterocyte effacement (LEE) and especially eae, one of the constituent genes of LEE, which is responsible for attaching and effacing lesions (17, 28)—are also shown to be necessary for STEC infection. The large plasmid of STEC O157 carries determinants characteristic for STEC that presumably harbor additional virulence factors: hlyA (the enterohemorrhagic E. coli hemolysin gene), which acts as a pore-forming cytolysin on eukaryotic cells (38); the bifunctional catalase peroxidase (KatP) (6); the etp gene cluster (36); and the secreted serine protease (EspP) (5, 35).

Cattle and sheep are the primary reservoirs of STEC, while other animals such as deer, horses, dogs, and birds are recognized as a major risk factor (1). However, newer vehicles of STEC infection continue to be identified, and the massive waterborne outbreak in Missouri points to environmental contamination and intensive farming as major risk factors (41). Rural drinking water and recreational water are increasingly being recognized as important vehicles (7).

Epidemiological investigation of STEC is complicated by its ubiquitous nature and lack of heterogeneity between clonal strains. The discriminatory capacity of many methods used to subtype strains of E. coli causing enterohemorrhagic disease is restricted by clonal strains that are responsible for the majority of cases of infection. The phenotypic and genotypic methods are particularly useful in this context, maximizing the likelihood of detecting the often minimal amount of variation between strains (2, 11, 13, 33, 35). These typing methods are of great use to identify the sources and routes of transmission of organisms and make intervention strategies. In this study, one of our main objectives was to determine the relatedness of STEC strains isolated from different sources and thereby obtain clues on whether transmission occurs between human and bovine sources in our setting. This represents the first systematic study of strains of STEC in India, where these organisms are still not a potential cause of human diarrhea and do not portray the same threat as they do in other Western countries (19).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains. Sixty-three STEC strains were isolated while surveillance of sporadic cases of diarrhea in humans, cow stool samples, and beef samples from a city abattoir was being conducted from January 1999 to December 2000. The virulence properties and serotypes of 30 of these strains have been reported recently (19). The remaining 33 strains were isolated using similar procedures detailed previously (19) and characterized in this study.

Serotyping. For serotyping, the slide agglutination test was performed using commercially available O157 (polyvalent) antisera (Denka Seiken Co., Tokyo, Japan) for E. coli O157 antigen determination.

PCR detection of virulence factors. PCR for detecting both chromosome (stx₁, stx₂, and eae)- and plasmid (hlyA, katP, and etpD)-encoded virulence genes was performed as described earlier (19) in a total volume of 20 µl containing a 2.5 mM concentration of each deoxynucleoside triphosphate, a 30 µM concentration of each primer, 2 µl of 10x PCR buffer, and 1 U of recombinant Taq DNA polymerase (both from Takara, Shuzo, Otsu, Japan) using a thermal cycler. The primer sequences and PCR conditions are given in Table 1.

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TABLE 1. PCR primers and conditions used in this study

Antibiotic resistance. The STEC strains were examined for resistance to ampicillin (10 µg), chloramphenicol (30 µg), co-trimoxazole (25 µg), ciprofloxacin (5 µg), gentamicin (10 µg), neomycin (30 µg), nalidixic acid (30 µg), norfloxacin (10 µg), streptomycin (10 µg), tetracycline (30 µg), cephalothin (30 µg), amikacin (30 µg), ceftazidime (10 µg), kanamycin (30 µg), and ceftriaxone (30 µg) using commercial disks (Hi Media, Bombay, India) by the disk diffusion test. E. coli ATCC 25922, which is sensitive to all the drugs, was used as the control strain. The characterization of strains as being susceptible, having reduced susceptibility, or being resistant was as recommended by the National Committee for Clinical Laboratory Standards (22).

RAPD of STEC strains. Genomic analysis of STEC strains was done by RAPD-PCR analysis using a single primer, 1247 (5'-AAGAGCCCGT-3') (35). Genomic DNA (10 to 20 ng) was used as a template. PCR for RAPD-PCR analysis was done using the Gene Amp PCR system 9600 (Perkin-Elmer Applied Biosystems, Weiterstadt, Germany) in a volume of 50 µl containing a 200 µM concentration of each deoxynucleoside triphosphate, 30 pmol of primer, 5 µl of 10-fold-concentrated polymerase synthesis buffer, 3 mM MgCl₂, and 2.0 U of Taq DNA polymerase (Takara). After a hot start at 80°C for 5 min, the DNA was subjected to 35 cycles of denaturing at 94°C for 1 min, annealing at 40°C for 1 min, and extension at 72°C for 2 min. A final extension step was done for 10 min at 72°C. The PCR products were separated on 1.5% (wt/vol) agarose gels in 1x TAE (0.04 M Tris acetate, 0.001 M EDTA [pH 8.0]) buffer at 100 V for 5 h. After being stained with ethidium bromide, the gels were digitized for computer-aided analysis (Gel Doc system; Bio-Rad, Hercules, Calif.).

Interpretation of RAPD-PCR results. Using Gel Doc 2000 (Bio-Rad), gel images are stored in a PC that runs on the Windows system. All the images were retrieved and aligned using Adobe software and analyzed in the Diversity Database fingerprinting software (Bio-Rad). Comparison of differences in the patterns of RAPD-PCR bands were made to ascertain the phylogenetic relationship between isolates. An unrooted phylogenetic tree was made with the neighbor-joining method that is available in the software. The minimum and maximum distance values between nodes on the sorted branches were removed to present the tree in a compressed form.

Pulsed-field gel electrophoresis (PFGE). A 1-day PFGE protocol described by Zhao et al. (44) was adapted in our study. Briefly, bacterial strains were grown overnight on Luria agar (LA) plates at 37°C. Bacterial colonies were suspended in cell suspension buffer (100 mM Tris-HCl, 100 mM EDTA [pH 8.0]) and adjusted to an optical density of 1.3 to 1.4 at 610 nm. The cell suspension (200 µl) was mixed with 10 µl of proteinase K (20 mg/ml) and an equal volume of melted 1% SeaKem agarose containing 1% sodium dodecyl sulfate. The mixture was carefully dispensed into a sample mold (Bio-Rad). After solidification, the plugs were transferred to a 2.0-ml Eppendorf tube containing 1.5 ml of cell lysis buffer (50 mM Tris-HCl, 50 mM EDTA [pH 8.0], 1% sarcosyl) and 0.5 mg of proteinase K per ml. Cells were lysed for 2 h in a water bath with continuous agitation. After lysis, the plugs were washed twice with sterile distilled water and four times with TE buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8.0]) for 15 min per wash at 54°C with vigorous shaking. The agarose-embedded DNA was equilibrated in restriction enzyme buffer for 1 h at 37°C. For complete digestion of the DNA, 50 U of XbaI (5'-TCTAGA-3'; Takara) was used. The resulting fragments were resolved by the contour-clamped homogeneous electric field method on a CHEF Mapper system (Bio-Rad) with 1% PFGE-grade agarose in 0.5x TBE (44.5 mM Tris-HCl, 44.5 mM boric acid, 1.0 mM EDTA [pH 8.0]) at 6 V/cm for 40 h 24 min at 14°C using the autoalgorithm mode. Gels were stained with ethidium bromide and digitized for computer-aided analysis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Of the 63 strains isolated during the study period, 40 (63.5%) were from cow stool samples, 19 (30.2%) were from human stool samples, and the remaining 4 (6.3%) strains were isolated from beef samples obtained from a city abattoir.

Antibiotic sensitivity profile. The STEC strains were tested for resistance against 15 antimicrobial agents. Resistance was observed most commonly to ampicillin (25.4%), tetracycline (23.8%), and streptomycin (14.3%) and less frequently to cephalothin (11.1%), co-trimoxazole (9.5%), nalidixic acid (6.4%), and neomycin (3.2%) (Fig. 1). More than one-third of the strains (35%) showed reduced susceptibilities to different antimicrobial agents but were not completely resistant to any of the antibiotics, and 14.3% were sensitive to all the antibiotics used in this study. Multidrug resistance was seen in 14 strains, and there was no common resistance pattern among the strains.

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FIG. 1. Antibiotic resistance patterns of STEC strains. Abbreviations: A, ampicillin; Ch, cephalothin; G, gentamicin, Ak, amikacin; Cf, ciprofloxacin, Ca, ceftazidime; K, kanamycin; T, tetracycline; C, chloramphenicol; Ci, ceftriaxone; Co, co-trimoxazole; Na, nalidixic acid; Nx, norfloxacin; S, streptomycin; N, neomycin.

Virulence gene profile. In STEC strains, stx₁, stx₂, and eae are chromosomally encoded, while hlyA, etpD, and katP are plasmid-encoded virulence marker genes (2, 35). We used a PCR-based method for detection of these virulence genes. Of the 63 strains, 36.5% carried stx₁ and 19% carried stx₂, while 44.5% of strains gave positive amplicons for both stx₁ and stx₂. Nineteen percent of the STEC strains possessed stx₁, stx₂, and hlyA genes; 4.8% of the strains carried stx₂ and hlyA genes; and one strain (P-179-10) isolated from a cow stool sample carried the stx₁ and hlyA genes (Table 2). The eae gene was found in 14.3% of strains and was exclusively associated with stx₁-positive strains in this study. Of the 63 strains of STEC, the etpD gene was found in 3 strains isolated from cows, and in all 3 of these strains it was associated with stx₁. Two strains which carried stx₁ and eae genes were positive for etpD and katP. Two strains, AK38 and P-33-2-26, which were isolated from cow stool samples, carried all the potential virulence genes searched for in this study (stx₁, eae, hlyA, etpD, and katP). Only one of the STEC strains (AK47) isolated from a human stool sample possessed more than three virulence marker genes (stx₁, eae, hlyA, and katP). A strain (AK17) isolated from a beef sample gave positive amplicons for stx₁, stx₂, hlyA, and katP. Overall, the most-prevalent toxin genotype was that of STEC strains possessing both stx₁ and stx₂ (23.8%), and this was followed by that of strains carrying stx₁, stx₂, and hlyA (19%) (Table 2).

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TABLE 2. Virulence gene profiles of STEC strains

RAPD profile of STEC strains. All 63 STEC strains were characterized by RAPD-PCR to analyze the genetic diversity among the strains. All the strains were typeable with primer 1247, with amplified fragment sizes ranging from 0.2 to 6.0 kb. The RAPD profile of strains SD8, SD10, and SD12, isolated from different cow stool samples during different time periods, were identical (Fig. 2, panel I, lanes 4, 5, and 6). Three sets of bovine isolates (SD2 and SD5 [Fig. 2, panel I, lanes 2 and 3], SD4 and SD7 [Fig. 2, panel II, lanes 2 and 3], and AK48 and AK54 [Fig. 2, panel IV, lanes 2 and 3]) which had the same virulence genotype had the same RAPD profile, while SD1 and SD3 gave the same profile in RAPD-PCR (Fig. 2, panel II, lanes 4 and 5), despite having a different virulence genotype (Fig 2). Two human strains (SDH2 and SDH3) were closely identical in RAPD profile, with only a one-band difference. Interestingly, two strains (AK33 and AK36) that were isolated from human and cow stool samples showed an identical RAPD profile (Fig. 2, panel III, lanes 2 and 3). The remaining strains showed RAPD patterns that were different from each other.

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FIG. 2. RAPD-PCR results for STEC strains that exhibited identical DNA banding patterns. In panel I, lanes 2 and 3 contain strains SD2 and SD5; lanes 4 to 6 contain strains SD8, SD10, and SD12, respectively. In panel II, lanes 2 and 3 contain SD4 and SD7; lanes 4 and 5 contain SD1 and SD3, respectively. In panel III, lanes 2 and 3 contain AK33 and AK36, respectively. In panel IV, lanes 2 and 3 contain AK48 and AK54, respectively. Lane 1 in each panel contains a 1-kb DNA ladder marker.

The phylogenetic analysis of STEC strains reveals seven major clusters (clusters A through G in Fig. 3). Even though the RAPD banding patterns were not identical, the phylogenetic analysis combined many human and cow isolates of STEC. Except for cluster B, the rest comprised human and cow STEC strains. Even though there is no complete homology, three out of four STEC strains isolated from beef samples are combined with strains of human origin at cluster C through E, indicating some degree of relatedness. To confirm the RAPD-PCR results, we performed PFGE with representative strains that exhibited close relatedness in the assay.

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FIG. 3. Phylogenetic analysis of STEC strains. The unrooted neighbor-joining method was used to summarize the similarity of RAPD-PCR profiles of STEC strains in a dendrogram. Seven major clusters (A through G) are shown near the nodes of each cluster.

PFGE profile of STEC strains. We analyzed the XbaI pattern by PFGE to determine the clonal relatedness among STEC strains that gave identical or nearly similar DNA fingerprints in RAPD-PCR and had similar virulence genotypes (Fig 4). PFGE of 25 strains produced 12 to 23 fragments ranging in size from approximately <48.5 to 450 kb. SD2 and SD5, SD4 and SD7, and AK48 and AK54 (strain pairs which gave the same RAPD profiles) also showed similar PFGE results (Fig. 4, panel II, lanes 3 and 6; panel II, lanes 5 and 7; and panel IV, lanes 2 and 3). SD8, SD10, and SD12 had identical RAPD profiles, but SD10 showed a one-band difference compared to SD8 and SD12 in PFGE (Fig. 4, panel I, lanes 2, 3, and 4). However, strains AK33 and AK36, which were isolated from human stool and cow stool samples, respectively, and possess the same RAPD profile, showed different PFGE profiles (Fig. 4, panel III, lanes 2 and 3). Likewise, SD1 and SD3 also showed identical RAPD profiles but showed different PFGE profiles (Fig. 4, panel II, lanes 2 and 4).

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FIG. 4. PFGE profiles of XbaI digest of STEC strains. PFGE results for STEC strains that exhibited identical profiles by RAPD-PCR (panels I to IV) and differences upon virulence gene analysis (panels V and VI) are shown. Lanes 1 in all panels and lane 7 in panel V contain ladder molecular size markers.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In less than 20 years, strains of E. coli that produce Shiga toxin have emerged as a cause of serious human gastrointestinal disease and HUS. The most common serotype of STEC implicated worldwide is O157:H7. In addition to E. coli O157:H7, a large number of other STEC strains have been isolated from human patients (11). The significance of non-O157 STEC strains in human disease is less understood than that of the STEC O157:H7 strains and is probably underestimated. Among the clinical STEC strains that have been isolated, a subset of enterohemorrhagic E. coli strains has been found which carry a common set of virulence genes that encode the factors for attachment to host cells, elaboration of effective molecules, and production of two different types of Shiga toxins. The sets of virulence genes are found in the LEE, lamboid phages, and a large virulence-associated plasmid (9, 10, 21, 23, 24, 25, 36, 38).

In the present study, we characterized the virulence gene profile of the STEC strains isolated in Calcutta, India. STEC strains with different combinations of virulence genes were observed. The dominant combinations of virulence factors present in the strains studied were stx₁ and stx₂ (44.5% of strains) and stx₁, stx₂, and hlyA (enterohemorrhagic E. coli hemolysin gene) (19% of strains). This pattern of distribution of virulence genes was similar to the virulence gene profiles of STEC strains isolated in New South Wales, Australia, where 47. 5% of the STEC harbored stx₁, stx₂, and hlyA (8). Such a high presence of stx₁ and stx₂ genes in STEC strains is, however, in contrast to the report from Germany, where STEC strains usually possessed stx₁ and only rarely contained the stx₂ gene (35, 40). In France, the prominent toxin genotype was that of STEC strains carrying stx₂ (32).

Intimin is the only intestinal adherence factor identified to date for STEC, and the eae gene, which encodes intimin, is widely distributed in STEC strains (17). Previous studies suggest that eae may be the single most important accessory factor correlated with severe disease (3, 15). In this study, only 6.4% of the STEC strains harbored eae. Interestingly, a case of HUS reported recently was associated with a strain of STEC O5:H^-, which lacked the eae gene (39). eae-lacking STEC strains belonging to serogroups O91, O104, or O113 have also been isolated from patients with HUS and hemorrhagic colitis (20, 30). The absence of eae in STEC strains indicates that there are additional adherence factors yet to be discovered for STEC. Unlike E. coli O157:H7, in which the production of Stx2 toxin is more common than Stx1, only 19% of STEC possessed the stx₂ gene in this study. No eae and etpD gene sequences were observed among the STEC strains carrying the stx₂ gene sequence. From these results, it appears that STEC strains containing the stx₁ gene are more frequently encountered than those carrying stx₂. This also contrasts with the situation observed in O157:H7, in which the possession of stx₂ is associated with a higher likelihood of causing HUS (35). Another noteworthy feature was that two strains (AK38 and P-33-2-26) isolated from two different cow stool samples at different time periods possessed all the putative virulence genes searched for in this study. Only one isolate (AK47) from a human diarrhea stool sample carried four virulence genes (stx₁, eae, katP, and hlyA), while 52.4% of STEC strains did not carry any virulence marker genes except the toxin gene(s). Thus, it appears that the STEC in this part of the world lacks some factors that are essential for it to become a frequent cause of diarrhea.

E. coli O157:H7 is reportedly susceptible to many antibiotics (34, 42). However, non-O157 strains isolated from humans and animals have developed antibiotic resistance and many are resistant to multiple antimicrobials commonly used in human and veterinary medicine (12, 14, 37). Approximately 49.2% of the present strains showed antibiotic resistance to one or more of the antimicrobial agents used in this study. Some of the strains exhibited multidrug resistance.

We used different subtyping methods to principally understand the relatedness between the STEC strains isolated from different sources since we were unable to serotype these strains. Both RAPD and PFGE showed that the STEC strains were genetically heterogeneous except for some sets of strains (such as SD2 and SD5, SD4 and SD7, and AK48 and AK54) which gave identical RAPD and PFGE profiles. One of the aims of our study was to determine whether the non-O157 STEC strains isolated from human patients were related to those in cow or beef isolates. Except for two strains (AK33 and AK36) that showed an identical RAPD profile but different PFGE profile, there was no match in the RAPD and PFGE profiles between strains of STEC isolated from cows and humans. Phylogenic analysis, however, revealed the clustering of strains from human and cow origins, indicating an evolutionary relationship. A high level of heterogeneity was also found among STEC strains isolated in France, where non-O157 STEC strains isolated from HUS patients were related, but not identical, to those isolated from cattle and food (32). Our study indicates that clones present in bovine sources are not transmitted to humans in the Calcutta setting. This may be related to food habits in this part of the world, where beef consumption is relatively low due to religious connotations. It should be mentioned that the non-O157 STEC strains isolated in this study form a small population of organisms that have been randomly isolated, and it would be quite easy to miss the clonal strains. Human STEC has still not become a major problem in India.

AK33 and AK36 had the same RAPD profile but were isolated from human and cow stool samples, respectively. SD1 and SD3 had different virulence gene profiles but yielded identical RAPD profiles. However, these two sets of strains exhibited different restriction enzyme profiles upon PFGE. The incongruity in the results between RAPD and PFGE typing reflects the difference in the molecular bases of RAPD and PFGE, since PFGE explores the whole length of the chromosome whereas RAPD explores only randomly selected parts of it (33). As a consequence, the clustering of isolates can vary considerably for RAPD and PFGE. However, in general, agreement between the results of the two methods was good for strain differentiation, as most strains emerged as a single lineage by both typing methods.

The standardization of the RAPD-PCR and PFGE methods and computer-based submission of the genomic profiles enable discriminative and rapid comparison of STEC strains among laboratories. In the United States, a national network (PulseNet) of public health laboratories that performs DNA fingerprinting has been developed for this purpose (16). However, at the European level, the genomic profiles of STEC isolates can, at present, be compared only on an intralaboratory basis. In this part of the world, standardization of the RAPD-PCR and PFGE method would be needed for efficient international epidemiologic research of the vast number of non-O157 STEC strains. The fact that STEC has not become a major problem in India also gives an opportunity to investigate the natural history of this organism should it emerge as an enteric pathogen in the near future.

 
This work was supported in part by the Council of Scientific and Industrial Research [projects 27 (0103)/EMR-II and 37 (1019)/99/EMRII] and by the Japan International Cooperation Agency (JICA/NICED project 054-1061-E-O).

 
* Corresponding author. Mailing address: Laboratory Sciences Division, International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), Mohakhali, Dhaka-1212, Bangladesh. Phone: 880-2-9886464. Fax: 880-2-8823116. E-mail: gbnair{at}icddrb.org.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, June 2002, p. 2009-2015, Vol. 40, No. 6
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.6.2009-2015.2002
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