An Epidemiological and Environmental Study of Shiga Toxin

Abstract

Shiga toxin–producing Escherichia coli (STEC) are foodborne pathogens of worldwide importance, but a shortage of data exists for STEC isolation from India. Therefore, an epidemiological and environmental study that covers a large geographic area in north India was conducted. Ruminant stool samples (n=650) were collected from 59 dairies. Meat samples (n=450) were collected from local abattoirs and the main slaughterhouse of the region. Additionally, 600 human cases of diarrhea and hemolytic uremic syndrome were screened for STEC. Isolates were characterized for the virulence gene profiles and for the serogroups and were submitted to molecular typing by the multilocus variable-number tandem-repeat analysis (MLVA). Overall, 12.3% of animal stool samples and 6.3% of mutton samples (n=160) were positive for STEC. Additionally, STEC were isolated from 1.7% and 1.6% of watery (n=290) and bloody (n=310) stool specimens, respectively. Animal stool isolates were significantly more prevalent in hilly areas (p<0.05) than in plain areas. Polymerase chain reaction demonstrated the presence of stx1, stx2, hly, espP, saa, toxB, and iha genes in 117 (83.5%), 94 (67.1%), 77 (55%), 33 (23%), 62 (44.2%), 29 (20.7%), and 51 (36%) of the isolates, respectively. Five new serogroups (O55, O33, O173, O165, and O136) are being reported for the first time from India. Four isolates from serogroup O103 were found in mutton and stool specimens of cattle and humans (n=160). One isolate from serogroup O104 was isolated from a mutton sample. MLVA suggested the potential transmission of STEC from contaminated meat and bovine sources. This study confirms the frequent contamination of mutton samples (24%), whereas chicken and pork samples were negative for STEC. This study demonstrates the presence of STEC that carry a large repertoire of virulence genes and the potential transmission of STEC from contaminated mutton and animal stools in north India.

Introduction

Shiga toxin–producing Escherichia coli (STEC) are important foodborne zoonotic pathogens that cause hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although the O157:H7 serotype is the most common serotype associated with the various sporadic outbreak cases of human illness (Tarr et al., 2002; Osek, 2003), infections from various non-O157 STEC are increasingly acknowledged in many countries (Caprioli et al., 1997; Schmidt et al., 1999; WHO, 2011). According to the Centers for Disease Control and Prevention, the following six non-O157 serotypes, O111:H8 or NM; O103:H2, H11, H25; O26:H11 or NM; O45:H2 or NM; O121:H19 or H7; and O145: NM are responsible for 71% of the non-O157 STEC illnesses in the United States (Bosilevac and Koohmaraie, 2011).

STEC are transmitted to humans from inadequately cooked beef, unpasteurized dairy products, contaminated sprouted seeds, juices, fresh produce, etc. (Vally et al., 2012). Additionally, STEC are transmitted from person-to-person and from animal-to-person transmission, particularly in sporadic cases (Mead and Griffin, 1998). Several large waterborne outbreaks have also been reported (Owen, 2000).

The pathogenesis of human illness that is caused by STEC is multifactorial. Shiga toxins (Stx1 and Stx2) are the hallmark virulence factors of STEC (Strockbine et al., 1986). However, epidemiological studies have shown that not all STEC strains that produce Stx are clinically important. Therefore, the accessory virulence genes may also contribute to the human disease (Amézquita-López et al., 2013). Other virulence factors of epidemiological and clinical significance include the eae gene that has encoding for intimin, is located on the locus of enterocyte effacement (LEE), and is responsible for attaching and effacing lesions (Perna et al., 1998). Other plasmid-borne virulence factors exist, such as enterohemorrhagic E. coli enterohemolysin (hly) (Schmidt et al., 1995), extracellular serine protease (espP), bifunctional catalase peroxidase (kat P) (Brunder et al., 1996), and the etp gene that encodes a part of the type II secretion system (Schmidt et al., 1997). Although intimin is the major adhesin, other putative adhesins, including toxB (Tarr et al., 2002), efa 1 (Nicholls et al., 2000), Iha (chromosomal iron- regulated gene A) (Tarr et al., 2000), and saa (autoagglutinating adhesion), (Paton et al., 2001) have been described.

In India, STEC have been demonstrated in animal reservoirs and food chains in various studies (Khan et al., 2002; Wani et al., 2006; Bandyopadhyay et al., 2011, 2012). However, a comprehensive search for this organism has been uncommon, and a shortage of data exists for STEC isolation from human illness. A study conducted in Delhi in 1989 did not find STEC in children with diarrhea (Bhan et al., 1989), whereas in a study from Kolkata, 1.4% and 0.6% of human bloody and watery diarrhea specimens, respectively, were positive for STEC (Khan et al. 2002). No outbreaks of human illness have been reported to date. No data are available from India to support the role of STEC in causing HUS. A large geographic area in north India was chosen for the study. This region in north India is a major milk-producing area, and animal rearing is one of the main sources of income. In this region, animals are reared in close proximity to humans, and hygienic conditions are generally poor. An investigation for STEC as causative agents of human illness, including bloody diarrhea and HUS was also performed. Furthermore, the virulence gene profiles and serogroups were studied, and STEC isolates from different sources were submitted to molecular typing by multilocus variable-number tandem-repeat analysis (MLVA).

Materials and Methods

Study area and sample categories

Chandigarh (latitude: 30° 43' N, longitude: 76° 47' E) is an organized city, but the surrounding areas in the states of Punjab, Haryana, and Himachal Pradesh are a mixture of semi-urban and rural populations, where animal (cows, buffalos, sheep/goats) rearing is commonly practiced. A survey was performed to locate the dairies in an area 300 km across and Chandigarh in the center. The purposive sampling for animal stool collection was performed from November 2007 to October 2009 from 59 diaries that included 40 small (<25 animals), 12 medium (25–100 animals), and 7 large (>100 animals) dairies. In the study region, the total number of diaries was 69 medium diaries and 7 large diaries. The number of small diaries was not counted because every house has a small diary. Meat samples were randomly collected from local abattoir shops in these areas and from the slaughterhouse of Chandigarh. Chutney samples (a grounded mixture of raw onion, coriander, mint, garlic, etc.) were collected from small vendors in the study area. Water samples were collected from water bodies surrounding Chandigarh. The total samples included 650 animal stool samples (250 cows, 300 buffalos, and 100 sheep/goats), 450 meat samples (chicken, n=210; mutton, n=160; and pork, n=80), 45 chutney samples, and 125 water samples. Beef samples were not available because beef is not eaten in this area because of religious beliefs. A total of 150 animal stool samples and 100 meat samples were collected from hilly areas, and 500 animal stool samples and 350 meat samples were collected from plain areas.

Human stool specimens

The study was conducted at the Enteric Laboratory of the Postgraduate Institute of Medical Education and Research in Chandigarh, India. Stool specimens were collected from all patients, who had bloody diarrhea and HUS, and from every fifth case patient with nonbloody diarrhea. Ten clinical isolates of STEC from human cases of diarrhea were obtained from the National Institute of Cholera and Enteric Disease in Kolkata and were included in this study for geographical comparison. Forty-eight stool specimens from animal handlers were collected from the dairies located in the study area.

Culture of human stool specimens

Approximately 5–10 mL of fresh stool was collected in a wide-mouth sterile container. The stool was grossly examined for the presence of blood and mucus and cultured for Salmonella, Shigella, Vibrio cholerae, and Aeromonas using standard conventional techniques. Enteroaggregative, enterotoxigenic, and enteropathogenic E. coli were confirmed (Chandra et al., 2012).

Sample processing for STEC detection

Briefly, 5 g of a human or animal stool (cattle and sheep/goat stool) sample was directly inoculated into 10 mL of an enrichment culture (EC) medium (Difco) for enrichment. Approximately 10 g of finely minced raw meat was mixed with 50 mL of EC broth in a sterile conical flask. Ten milliliters of chutney was added to 50 mL of EC broth. The enrichment broths were incubated overnight at 37°C and screened by polymerase chain reaction (PCR) as described below.

One liter of water collected in sterile bottles was filtered through 0.22-μm-pore-sized nitrocellulose acetate filter membranes (Millipore), and the portions of the membranes were inoculated into the EC broth and processed as above.

Isolation and identification

PCR for the detection of the stx1 and stx2 genes was performed using primers and PCR conditions (Paton and Paton, 2002). The positive cultures for either the stx1 and/or stx2 genes by PCR were processed for the isolation of colonies (Kumar et al., 2012).

A loopful of sample was inoculated into modified tryptone soy broth (Difco) and incubated overnight at 37°C. Immunomagnetic separation was performed using Dynabeads anti-E. coli O157 (Invitrogen, Norway) according to the manufacturer's instructions and was further processed (Amézquita-López et al., 2012). Ten to 15 presumptive E. coli O157 colonies were confirmed by standard biochemicals, serotyping, and rfb O157 PCR using the primer sequences (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/fpd).

Phenotypic characterization

A commercially available enzyme immune-assay (Ridascreen-Biopharm, Germany) was used to detect Shiga toxins per the manufacturer's instructions. STEC isolates were serogrouped on the basis of their O-antigens at the National Salmonella and Escherichia Centre at Central Research Institute in Kasauli, India.

Molecular typing

PCR assays were performed for the following genes: stx1, stx2, eae, hly, etpD, espP, katP, saa, iha, toxB, and efa1 by using primers as shown in Supplementary Table S1 (Appendix online). Amplification conditions were used as described previously (Kumar et al., 2012).

MLVA was performed on 140 isolates (80 animal, 39 mutton, and 21 human strains). The reference strain used was EDL933. Five variable number of tandem repeats loci, including CVN001, CVN004, CVN007, CVN014, and CVN015, as described by Lindstedt et al. (2007), were chosen. After amplification, products were separated on a 6% denaturing polyacrylamide gel and silver-stained using the Promega silver staining kit (Promega, Madison, WI). On the basis of amplicon size, an allele number was assigned to each locus, and an allele string was made for each isolate in the following order: CVN001-CVN004-CVN007-CVN014-CVN015. The absence of amplification was assigned an arbitrary number (e.g., 50). Nei's diversity index was calculated for each locus (Noller et al., 2003). The Simpson's index of diversity was calculated to evaluate the discriminatory power of the typing method (Hunter and Gaston, 1988). The dendrogram was divided into clades (strains>one locus similarity) and MLVA-type complexes (MTCs) that were assigned for related isolates of the same allele for more than three common loci.

Statistical methods

To compare the frequency of the positive isolates in the samples from different sources, the chi-square test was used, and a p value of<0.05 was considered significant.

Results

Prevalence of STEC in various samples

A total of 12.3% animal stool samples and 24.3% mutton samples were positive for STEC, while the chicken, pork, chutney, and water samples were negative. Animal stools had a higher prevalence of STEC in hilly areas (p=0.0199) compared to plain areas. The prevalence of STEC in mutton samples from hilly areas was also higher, but the difference was not statistically significant (p=0.4583). STEC were isolated as the sole pathogen in 11 of 600 (1.8%) human diarrhea specimens (five isolates each from watery and bloody diarrhea and one from a case of HUS). Males were predominately affected (10/11 cases), and bloody diarrhea was more common than watery diarrhea for persons below the age of 2 years old (Table 1). No specimen from the animal handlers was positive for STEC.

Table 1.

Age, Sex, and Type of Disease Distribution of Human Patients Whose Stool Samples Grew Shiga Toxin–Producing Escherichia coli (STEC)

	Diarrhea		Bloody diarrhea and HUS
Age group	Male	Female	Male	Female	Other organisms isolated (number positive, %)
<2 y (n=182)	30	42	74	36	Shigella flexneri (10, 5.4%), Shigella dysenteriae (2, 1.09%), enteroaggregative E. coli (15, 8.2%)
STEC^a isolated	1	0	2	1	STEC (4, 2.2%)
2–5 y (214)	58	26	74	56	Shigella flexneri (12, 5.6%), enterotoxigenic E. coli (15, 7%), enteropathogenic E. coli (8, 3.7%)
STEC^a isolated	0	0	2	0	STEC (2, 0.9%)
>5 y (204)	70	64	40	30	Salmonella (2, 1.04%), Aeromonas (2, 1.0%)
STEC^a isolated	4	0	1	0	STEC (5, 2.4%)
Total (n=600)	158	132	188	122	STEC (11, 1.8%), Shigella flexneri (32, 5.3%), Shigella dysenteriae (2, 0.3%), enteroaggregative E. coli (15, 2.5%) enterotoxigenic E. coli (15, 2.5%), enteropathogenic E. coli (8, 1.3%), Salmonella (2, 0.3%), Aeromonas (2, 0.3%)
STEC^a (n=11)	5	0	5	1

STEC was isolated as a sole pathogen.

HUS, hemolytic uremic syndrome.

Detection of virulence markers

Overall, the stx1 gene was significantly more prevalent when compared with the stx2 gene (p=0.0023). However, the stx2 gene was significantly more prevalent in animal stool isolates than in mutton and human stool isolates (p=0.0003). The eae gene was present in 11 (13.75%) animal stool and 4 (19%) human stool isolates and was absent in all mutton isolates. The hly (p<0.0001) and espP (p=0.0003) genes were significantly more prevalent in animal stool isolates than in mutton and human stool isolates (Table 2).

Table 2.

Prevalence of the Different Virulence (Plasmid and Chromosomal) and Adhesion Genes in Clinical and Environmental Shiga Toxin–Producing Escherichia coli (STEC) Isolates

	Chromosomal genes (%)			Plasmid genes (%)				Adhesion genes (%)
Origin	stx1	stx2	Eae	hly	etpD	espP	Katp	saa	toxbB	efa1	ihA	EIA for Shiga toxins (%)
Animal stool l (n=80)	64 (80)	59 (73)^a	11 (13.7)	58 (72)^a	7 (8)	28 (35)^a	2 (2.5)	33(41)	17 (21)	3 (3.7)	36 (45)	28 (35)
Mutton (n=39)	32 (82)	25 (64)	—	16 (41)	1 (2.5)	1 (2.5)	—	23 (59)	7 (18)	2 (5)	8 (20.5)	11 (28.2)
Human stool (n=21)	21 (100)	10 (47)	4 (19)	3 (14)	1 (4.7)	4(19)	—	6 (28)	5 (23)	2(9.5)	7 (33)	4 (19)
Total (n=140)	117 (83.5)^a	94 (67.1)	15 (10.7)	77 (55)	9 (6.4)	33 (23)	2(1.4)	62 (44.2)	29 (20.7)	7 (5)	51 (36)	42 (30)

p<0.05 was considered to be significant. The chi-square test was used to calculate the difference in frequencies of the virulence genes in STEC from different sources.

EIA, enzyme immunoassay.

Ridascreen enzyme immunoassay (EIA)

The results of the Ridascreen EIA assay are summarized in Table 2. The assay had the following detection rate: 30.7% for stx1, 31.9% for stx2, and 32.2% for both the stx1 and stx2 gene positive isolates.

STEC serogrouping

Forty-one isolates were O-antigen untypeable, and 17 isolates were rough (Supplementary Table S2). Several serogroups, including O69, O103, O168, O85, and O95, were common among animal stool, human stool, and mutton isolates, whereas serogroups O5, O104, O141, O152, O11, and O21 were uniquely common in mutton isolates. The serogroup O64 was present in only human stool isolates. Mutton and animal stool isolates shared serogroups O2, O22, and O43 (Table 3). The enterohemorrhagic Escherichia coli isolates belong to serogroups O85, O43, O103, O22, O173, and O136 from animal stool and belong to O168, O69 from human stool specimens. The serogroup O157 was not detected in any of the samples even after employing the immunomagnetic separation technique.

Table 3.

Distribution of Shiga Toxin–Producing Escherichia coli Serogroups and Multilocus Variable-Number Tandem-Repeat Analysis–Type Complexes (MTCs) from Diverse Sources

Sources	Serogroups	MTCs
Unique to animal stool	O3, O25, O30, O33, O41, O52, O55, O60, O76, O97, O110, O112, O113, O136, O165, O172, O173	3, 4, 6, 8, 9, 10, 11, 12, 13,14, 15, 16, 26
Unique to mutton	O5, O11, O21, O104, O141, O152	20, 21, 22, 23, 24
Unique to humans	O64	1, 2, 5^a
Shared between animal stool and mutton	O2, O22, O43	18, 25
Shared between animal stool and humans	O102	None
Shared between humans and mutton	O28	19
Shared between animal stool, mutton, and humans	O69, O85, O95, O103, O168	7,17^b

Four human strains did not fall into any MTC.

Small MTCs containing two isolates each.

MTC 7 (six human isolates, two meat isolates, and one cattle stool isolate) and MTC 17 (four human isolates, six meat isolates, and one cattle isolate).

MLVA typing

A total of 40 alleles were observed. The loci, including CVN001, CVN004, CVN007, CVN014, and CVN015 had 7, 4, 4, 22, and 3 alleles, respectively. The following Nei's diversity indices were obtained: CVN014 (0.94), CVN015 (0.61), CVN001 (0.60), CVN004 (0.55), and CVN007 (0.52). Overall, the Simpson's index of diversity, which is common to all loci, was 0.99. In the dendrogram analysis, 141 isolates could be grouped into 26 different MTCs of related strains: clades I and II each having 16 and 10 MTCs, respectively. Overall, 104 MLVA genotypes were observed (Fig. 1): 86 represented by 1 strain each, and 15 shared by 2 or more isolates. Cattle stool and mutton isolates formed separate lineages (clade 1 had cattle stool isolates and clade II had mutton isolates predominantly, although few strains were interspersed). Figure 1 and Table 3 show the sourcewise distribution of serogroups and MTCs.

FIG. 1.

Dendrogram based on allelic profiles of multilocus variable-number tandem-repeat analysis (MLVA) for 141 strains, including reference strain EDL933, was constructed using START 1.0.5 (Jolley et al., 2001). Allele numbers were entered into START as character data, and the dendrogram was constructed using unweighted-pair group method with arithmetic mean. Strains with a≥80% similarity level were grouped into MLVA-type complexes. C, cattle stool isolates; S, sheep/goat stool isolates; M, mutton isolates; H, human stool isolates.

Human isolates exhibited a scattered distribution; 16 were present in clade I (MTCs 1, 2, 5, 7, and 4 did not fall into any MTC categories), and 5 were present in clade II (MTCs 17 and 19). The human isolates showed a statistically significant and greater odds ratio for clustering with mutton samples (odds ratio of 8.2, 95% CI: 2.6-25) compared with the clustering with animal (odds ratio of 0.12 at 95% confidence interval [CI]: 0.04–0.37) isolates. Serogroup-specific clustering was observed for strains M4 and M5 (serotype O22) in MTC 21, H3, and H5 (serogroup O69) in MTC 5, and H1 and M36 (serogroup O69) in MTC 7. MTCs 7 and 17 were the most interesting. Both contained mutton and human stool isolates of different serogroups, which were isolated from different geographic areas, collected at different time periods, and exhibited the same MLVA profiles. Seven of 10 Kolkata strains were grouped in clade IB with cattle stool and mutton isolates from north India. No relationship was found between the presence of stx1 or stx2 genes or both and the MLVA profile of the isolates.

Discussion

In India, STECs have been reported in 10.5%–18% of stools from healthy animals and 6%–37% of diarrheic animals (Khan et al., 2002; Wani et al., 2006; Bandyopadhyay et al., 2011). STEC have also been reported in 20.6% of yak milk and its products and 1.8%–50% of raw beef samples (Bandyopadhyay et al., 2011, 2012). However, the sample sizes in these studies were smaller, and the sampling was performed in small geographic areas. The present study covered a large geographic area in north India that is a Hindu-dominated region where mutton, chicken, and pork are the only types of meat consumed. All large dairies, 17% of medium dairies, and 29 small dairies in 15 districts of north India, which is a major animal- rearing area, were covered in this study. Meat, chutney, and water samples were also collected from the same areas.

In India, limited data are available for the isolation of STEC from human illnesses, due to the difficulty of isolation and a lack of surveillance for foodborne pathogens. Reports of STEC isolation have varied from 0% to 2% in children with diarrhea (Bhan et al., 1989; Rajendran et al., 2009). The surveillance study for patients in Kolkata (Khan et al., 2002) reported STEC in 1.4% of bloody stool and 0.6% of watery stool specimens. In the present study, STEC were found in 1.7% of acute watery diarrhea and 1.6% of bloody diarrhea specimens, which is similar to the findings reported in the Netherlands from cases of bloody diarrhea (1.7%, van Duynhoven et al., 2008) and in Canada for stool specimens suspected for viral gastroenteritis (1.4%, Couturier et al., 2011). STEC was also isolated from a fatal case of HUS and is the only report available from India (Kumar et al., 2012). In the present study, the isolation rate for STEC for community-acquired diarrhea was higher than for Salmonella (1.04%). Therefore, a justification is routinely needed for the detection of this organism in a diagnostic microbiology laboratory. Until now, no outbreaks of STEC diarrhea or HUS have been reported in India.

No single isolate belonged to serogroup O157, even though the immunomagnetic separation method was used. This observation signifies either the absence or presence of this serogroup in very low numbers in this region. The 10-year data from the National Salmonella and Escherichia Centre in India has shown the presence of the E. coli O157 serogroup in 0.5%, 0.9%, 1.8%, 8.4%, and 1.6% of 17,093 isolates from human, meat, milk and milk products, seafood, and water samples, respectively. The maximum number of isolates of E. coli O157 was detected in samples received from the coastal belt areas (Sehgal, 2008). In the present study, the serogroup distribution was diverse, and 28 of 33 different O groups were the same as reported in earlier studies (Khan et al., 2002; Wani et al., 2006; Lanjewar et al., 2010; Bandyopadhyay et al., 2011, 2012). Five serogroups (O55, O33, O173, O165, and O136) were new and are being reported for the first time from India. One isolate of the serogroup O103 that was isolated from cattle stool harbored a combination of seven virulence genes (stx1, stx2, eae, hly, saa, toxB, and efa1) and is a matter of concern. Additionally, a single isolate from the serogroup O104 was also found that had recently caused an outbreak in Germany (WHO, 2011).

The stx1 gene (78%) was more prevalent in comparison with the stx2 gene (71%), which is similar to the findings by Wieler et al. (1996). However, this finding is in contrast to a study by Monaghan et al. (2011) from Washington, DC, which found a higher prevalence of the stx2 gene in bovine isolates. Bandyopadhyay et al. (2012) also found that the stx2 gene (83.3%) was more common than the stx1 gene (75%) in stool samples from healthy yaks. However, the stx2 gene was significantly more prevalent in animal stool isolates than in mutton or human stool isolates (p<0.05) and demonstrated the virulence potential of STEC isolates present in fecal reservoirs of animals and the potential public health threat posed by these organisms. The low prevalence of the eae and stx2 genes in human isolates may explain the low severity of illness in this region, which was also demonstrated in earlier studies from the Indian subcontinent (Khan et al., 2002; Islam et al., 2008). Although eae ⁺ STEC isolates are more pathogenic, several HUSs and occasional outbreaks due to LEE strains have been reported (Paton et al., 1999). Among the other adhesins, the saa gene was the most prevalent (44.2%), followed by iha (36%), toxb (20.7%), and efa genes (5%). The saa gene was reported earlier in 43% of diarrheic lamb stool isolates and 32.4% of milk and milk product isolates (Bandyopadhyay et al., 2012) from India. The latter study also reported toxB and efa1 in 5.4% of yak milk and 8.1% of yak cheese, respectively. The present study reports the presence of these virulence genes in human isolates from India for the first time.

Various other toxins, including espP, etpD, hly, and katP, which may contribute to the severity of STEC illness, were also variably present. In the present study, both espP and hly genes were more significantly present in animal stool isolates (35% and 72%) in comparison with the mutton and human stool isolates (p<0.05), which further signified the virulence potential of environmental isolates again. This study also found the katP and etpD genes in seven isolates and two isolates each, respectively, which are rarely observed in non-O157 isolates (Bosilevac and Koohmaraie, 2011; Monaghan et al., 2011).

Although no source detection was performed for human cases of STEC, contaminated mutton could be the preferred mechanism of transmission because human isolates showed a statistically significant and greater odds ratio for clustering with mutton samples in comparison with the clustering with animal isolates and signified a closer genetic relationship of human isolates with mutton. Only two animal stool isolates clustered with human strains, which suggested that some of the human infections may be from cattle, perhaps through contact with the animals or a contaminated environment. MLVA showed distinct lineages for cattle stool and mutton isolates, with only a few isolates of each type intermingling in clade I and clade II and signified that only a few of the cattle stool isolates have a potential for further transmission from the intestines of animals. Hygienic conditions at the dairies were invariably poor because all of the small dairies were located in the backyard of houses and were in close proximity to humans. The presence of large volumes of animal manure in the environment containing virulent STEC is a public health concern because environmental isolates may become pathogenic due to gene exchange, especially if the gene transferred involves an important gene such as stx that is phage encoded. To the best of our knowledge, this study is the first study to molecularly characterize STEC isolates using MLVA on strains from diverse sources across a large region in India.

Footnotes

Acknowledgments

We gratefully acknowledge the Director, National Salmonella and Escherichia Centre, Central Research Institute in Kasauli 173204 (India) for serogrouping the isolates. We would like to acknowledge Dr. G.B. Nair and Dr. T. Ramamurthy from the National Institute of Cholera and Enteric Diseases in Kolkata for providing the isolates with STEC and the control strain. The financial, administrative, and technical support of the Indian Council of Medical Research (ICMR) in New Delhi, India (Grant no. 5/8-1(213)-D/2006 ECD-II) is acknowledged.

Disclosure Statement

No competing financial interests exist.

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Supplementary Material

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An Epidemiological and Environmental Study of Shiga Toxin–Producing Escherichia coli in India

Abstract

Introduction

Materials and Methods

Study area and sample categories

Human stool specimens

Culture of human stool specimens

Sample processing for STEC detection

Isolation and identification

Phenotypic characterization

Molecular typing

Statistical methods

Results

Prevalence of STEC in various samples

Detection of virulence markers

Ridascreen enzyme immunoassay (EIA)

STEC serogrouping

MLVA typing

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Supplementary Material