Occurrence of Hybrid Escherichia coli Strains Carrying Shiga Toxin and Heat-Stable Toxin in Livestock of Bangladesh

Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) are important causes of diarrhea in humans and animals worldwide. Although ruminant animals are the main source of STEC, diarrhea due to this pathotype is very low in Bangladesh where ETEC remains the predominant group associated with childhood diarrhea. In the present study, E. coli strains (n = 35) isolated from Bangladesh livestock (goats, sheep, and cattle) and poultry (chicken and ducks) were analyzed for the presence of major virulence factors, such as Shiga toxins (STX-1 and STX-2), heat-labile toxin, and heat-stable toxins (STa and STb). Multiplex polymerase chain reaction results revealed 23 (66%) E. coli strains to be virulent possessing either sta (n = 5), stx (stx1, n = 8; stx2, n = 2), or both (n = 8) genes in varying combinations. Thirty-four percent (8/23) of strains from livestock were hybrid type that carried both stx (either stx1 or stx2) and ETEC-specific enterotoxin gene sta. Serotyping results revealed that the ETEC strains belonged to five serotypes, namely O36:H5, O174:H−, O152:H8, O109:H51, and O8:H21, while the STEC-producing strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16 (n = 2), OR:H2 (n = 1), O110:H16 (n = 1), and O152:H8 (n = 1). The STEC–ETEC hybrid strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16, OR:H2, and O152:H8. Forty percent (2/5) of the ETEC and 20% (2/10) of the STEC strains were multidrug resistant with the highest drug resistance (50%) being found in the hybrid strains. Molecular fingerprinting determined by pulsed-field gel electrophoresis and cluster analyses by dendrogram revealed that, genetically, STEC–ETEC hybrid strains were highly heterogeneous. Multidrug-resistant E. coli STEC–ETEC hybrid strains in domesticated animals pose a public health threat for humans in Bangladesh.

Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) are important causes of diarrhea in humans and animals worldwide. Although ruminant animals are the main source of STEC, diarrhea due to this pathotype is very low in Bangladesh where ETEC remains the predominant group associated with childhood diarrhea. In the present study, E. coli strains (n = 35) isolated from Bangladesh livestock (goats, sheep, and cattle) and poultry (chicken and ducks) were analyzed for the presence of major virulence factors, such as Shiga toxins (STX-1 and STX-2), heat-labile toxin, and heat-stable toxins (STa and STb). Multiplex polymerase chain reaction results revealed 23 (66%) E. coli strains to be virulent possessing either sta (n = 5), stx (stx1, n = 8; stx2, n = 2), or both (n = 8) genes in varying combinations. Thirty-four percent (8/23) of strains from livestock were hybrid type that carried both stx (either stx1 or stx2) and ETEC-specific enterotoxin gene sta. Serotyping results revealed that the ETEC strains belonged to five serotypes, namely O36:H5, O174:H−, O152:H8, O109:H51, and O8:H21, while the STEC-producing strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16 (n = 2), OR:H2 (n = 1), O110:H16 (n = 1), and O152:H8 (n = 1). The STEC-ETEC hybrid strains belonged to serotypes O76:H19 (n = 3), O43:H2 (n = 2), O87:H16, OR:H2, and O152:H8. Forty percent (2/5) of the ETEC and 20% (2/10) of the STEC strains were multidrug resistant with the highest drug resistance (50%) being found in the hybrid strains. Molecular fingerprinting determined by pulsed-field gel electrophoresis and cluster analyses by dendrogram revealed that, genetically, STEC-ETEC hybrid strains were highly heterogeneous. Multidrug-resistant E. coli STEC-ETEC hybrid strains in domesticated animals pose a public health threat for humans in Bangladesh. Hybrid  inTrODUcTiOn Escherichia coli is commonly regarded as a non-pathogenic beneficial inhabitant of the gastrointestinal tract but several pathogenic strains acquired specific virulence factors that are accountable for a variety of intestinal and extra intestinal diseases, including diarrhea, acute inflammation, hemorrhagic colitis, urinary tract infections, septicemia, and neonatal meningitis (1)(2)(3)(4). Based on virulence traits, major diarrheagenic E. coli pathogroups include shigatoxigenic E. coli, enterotoxigenic E. coli (ETEC), enteropathogenic E. coli, enteroinvasive E. coli, and enteroaggregative E. coli (EAEC) (3). Shiga toxinproducing E. coli (STEC) is a pathogen of significant public health concern and infection by this strain can result in a spectrum of outcomes ranging from asymptomatic carriage to severe diarrhea, as well as bloody diarrhea and hemolyticuremic syndrome (HUS) (5). The disease causing ability of STEC is associated with the production of phage-encoded Shiga-like toxins (Stx), which are classified into two major families, Stx1 and Stx2 (encoded by stx1 and stx2 genes) on the basis of toxin neutralization assays and sequence analysis of stx genes. ETEC is the leading cause of diarrhea in travelers from industrialized countries and children living in low-income countries. ETEC bacteria are largely defined by the presence of the plasmid-encoded heat-labile (LT) and/or heat-stable (STa/STb) toxins (3). Ruminants, especially cattle, sheep, and goat are the main source of STEC for humans and play an important role in the epidemiology of human infections (6). Meat contamination can facilitate transmission of pathogenic E. coli occurring through unhygienic slaughtering and poor food handling. Screening and characterization of animal STEC helps to identify the origin of human STEC infections. Despite the fact that over 400 STEC serotypes have been identified, only a subset of these have been correlated to human illness (7). Most outbreaks of hemorrhagic colitis and HUS have been associated with STEC O157 strains (5). There are outbreaks reported that are caused by non-O157 (8); infections caused by some non-O157 serotypes have also been frequently associated with severe illness in humans. In some geographic areas, STEC non-O157 strains are more commonly isolated than STEC O157 strains from persons with diarrhea or HUS (9). Antimicrobial resistance patterns may be an additional epidemiological marker for surveys of non-O157 SETC (10).
In human, ETEC strains are associated with one of the most frequent (sometimes fatal) causes of childhood diarrhea in the developing countries and an important causative agent of traveler's diarrhea (3). ETEC is also a major cause of severe diarrheal disease in suckling and weanling animals (11). ETEC infections are transmitted through the fecal-oral route. Exposure to ETEC usually comes from contaminated food and drinking water (3). A close genetic relationship has been found between ETEC strains belonging to certain serotypes with diarrhea, which suggest that serotype analysis can be coupled with genetic typing to study strain clustering in epidemiologic and pathogenic studies. To assess the potential public health risk posed by ETEC and STEC, the detection of virulence factors, such as heat-stable toxin a (STa), heat-stable toxin b (STb), heat-labile toxin (LT), stx1, and stx2, is recommended (12)(13)(14)(15)(16).
Shigatoxigenic E. coli and other diarrheagenic E. coli are capable of acquiring virulence and related genes via horizontal gene transfer leading to the development of pathogroups different from the pre-existing ones (17). Such divergent pathogroups are often reported using different terminology such as "hybrid" (18), "blended virulence profiles, " and "virulence combination" (19). Several studies have reported coexisting STEC-and ETECassociated virulence genes in E. coli strains of human, animal, and environmental origins (17,(20)(21)(22)(23)(24)(25). The notorious sprout-borne outbreak strain O104:H4 in Germany possessed EAEC-and STEC-associated virulence genes (18) pointing to the possibility of extremely pathogenic strains emerging over a short time period (26).
In Bangladesh, the prevalence of STEC in hospitalized patients with diarrhea is very low (0.5%) (27), but it was somewhat higher among community cases with diarrhea (1.9%) (27). In Bangladesh, the predominant group of E. coli associated with childhood diarrhea is ETEC, which accounts for approximately 20% of all diarrheal cases (28). Animals seem to be the potential carrier for pathogenic E. coli but there is little data available concerning animal reservoirs, epidemiology, human pathogenicity, and drug resistance profiles of STEC and ETEC in Bangladesh. In the present study, we report the existence of multidrug-resistant E. coli strains belonging to STEC, ETEC, and a newly emerged STEC-ETEC hybrid in domesticated animals, which pose a potential public health threat for humans in Bangladesh.

MaTerials anD MeThODs
sample collection, isolation, and identification of E. coli Rectal (livestock) and cloacal (bird) samples were collected from a total of 35 different livestock [goat (n = 19), sheep (n = 9), cattle (n = 2)] and poultry [chicken (n = 3) and duck (n = 2)] between June and August 2007 from the district of Mymensingh, Bangladesh. Sterile cotton swabs were used for the collection of rectal and cloacal samples. After collection, the cotton swabs were dipped into Carry-Blair media and stored in a cool box (4°C). Immediately after sampling, all samples were transported to the laboratory for analysis. All samples were streaked onto MacConkey agar (Difco), and the plates were incubated overnight at 37°C. Bright pink lactose-fermenting colonies were selected as presumptive E. coli. These colonies were again grown on eosin methylene blue agar to examine for the production green colonies with a metallic sheen. One presumptive colony per sample was identified by biochemical tests, as described before (29).
Polymerase chain reaction (Pcr) amplification of Virulence genes DNA was extracted from isolated colonies suggestive of E. coli, as described previously (30). The samples were then subjected to molecular analysis of ETEC (sta, stb, elt) and STEC (stx1, stx2) virulence genes using multiplex PCR. The PCR assay conditions are described in Table 1. Each amplification was conducted in a  , and presterilized ultrapure water (Milli-Q) to 20 µl. This mixture was processed in a thermocycler at 94°C for 4 min (denaturation), followed by 30 cycles at 94°C for 1 min (denaturation), annealing temperature (specific for each primer) for 1 min (binding), and at 72°C for 1.5 min (extension). Complete extension of the Taq DNA polymerase was performed at 72°C for 7 min. An aliquot of this reaction containing only water without DNA was used as a negative control. Two multiplex PCR tests were carried out; one for the primer set LT, STa, and STb and the other for the primer set Stx1 and Stx2.

serotyping
The rabbit antisera against O1 to O187strains were prepared in rabbits (SERUNAM) according to procedures described by Ewing (31). The E. coli O1 to O172 strains were obtained from the Laboratory of Gastrointestinal Pathogens, Department of Gastrointestinal, Emerging and Zoonotic Infections, Centre for Infections, Health Protection Agency, London, UK, and the E. coli O173 to O186 strains (32) were obtained from the Statens Serum Institute, Copenhagen. The strains were serotyped by agglutination assay (33)

Pulsed-Field gel electrophoresis (PFge)
Whole agarose-embedded genomic DNA from the E. coli isolates was prepared. PFGE was carried out using a contour-clamped homogeneous electrical field (CHEF-DRII) apparatus (Bio-Rad), according to procedures described previously (36). Genomic DNA of the test strains and Salmonella enterica serovar Braenderup was digested using XbaI, with fragments employed as molecular size markers. Restriction fragments were separated in 1% pulsedfield-certified agarose in 0.5× TBE (Tris-borate-EDTA) buffer. Post-electrophoresis gel treatment included both gel-staining and de-staining. The DNA was visualized using a UV transilluminator, and images were digitized via a one-dimensional gel documentation system (Bio-Rad).

PFge analysis
The fingerprint pattern in the gel was analyzed using a computer software package, Bionumeric (Applied Maths, Belgium, version 3.1). After background subtraction and gel normalization, the fingerprint patterns were typed according to banding similarity and dissimilarity, using the Dice similarity coefficient and unweighted-pair group method employing average linkage (UPGMA) clustering, as recommended by the manufacturer. The results were graphically represented as dendrograms.

Phenotypic characteristics of E. coli
All the E. coli strains (n = 35) included in this study produced bright pink colonies on MacConkey and green colonies with metallic sheen on EMB agar plates. All strains gave biochemical reactions typical of E. coli.

Virulence gene Profile
Among the 35 E. coli strains isolated from livestock and poultry in the present study, 23 (66%) (goats: 17, sheep: 4, chicken: 1, and ducks: 1) carried virulence genes, such as, sta and stx, either stx1 or stx2, but did not carry stb or elt genes, as confirmed by PCR   Table 2). None of the strains tested presented elt or stb.  1). None of the tested strains belonged to the EHEC serogroup, as they did not agglutinate with the antisera specific for O157 ( Table 2).

antibiotic assay
Antibiotic susceptibility assay was performed for the 23 toxigenic strains only, and the results showed 87% of the strains to be resistant to erythromycin, 43% to azithromycin, 22% to streptomycin, 22% to trimethoprim/sulfamethoxazole, 17% to cephalothin, 9% to ciprofloxacin, 9% to nalidixic acid, and 4% to tetracycline ( Table 2). All E. coli strains were sensitive to gentamicin, ampicillin, ceftriaxone, cefixime, and mecillinam. Results also revealed 40% (2/5) of the ETEC and 20% (2/10) of the STEC strains to be multidrug resistant (MDR) showing resistance to erythromycin, trimethoprim/sulfamethoxazole, azithromycin, cephalothin, ciprofloxacin, and nalidixic acid. The highest MDR was found in the hybrid strains, accounting for 50% (4/8) ( Table 2), although the strains varied in their patterns of response to the different drugs tested. Overall drug response results revealed 11 different resistance patterns among the three pathogroups of E. coli (ETEC, STEC, and hybrids of STEC-ETEC). Resistance patterns I (E R , S R ), II (E R , AZM R ), III (E R , SXT R , AZM R , KF R ), and IV (E R , CIP R , SXT R , NA R , AZM R ) were found in ETEC; resistance patterns V (E R ), VI (E R , AZM R , S R , KF R ), and VII (E R , SXT R , AZM R , S R ) in STEC; and   Table 3).

PFge and cluster analysis
The XbaI-digested genomic DNAs of the E. coli strains carrying virulence genes from animal and poultry were subjected to PFGE to determine genetic relatedness and clonal origin. The number of fragments generated by restriction digestion with XbaI varied between 14 and 19, and the molecular size of the fragments ranged from 30 to 600 kb. Diverse PFGE patterns obtained from the 23 toxigenic E. coli strains (Figure 2) belonged to multiple small sub-clusters (Figure 2) when dendrograms were prepared with PFGE images by the BioNeumeric software (Applied Maths) using dice similarity coefficient and UPGMA. Although the E. coli strains exhibited high genetic heterogeneity in the PFGE patterns, four hybrid strains belonging to serotype O76:H19 formed a small tight cluster, suggesting that genetically, they are closely related.

DiscUssiOn
Occurrence of hybrid of sTec and eTec strains Enterotoxigenic E. coli and Shiga toxin-producing E. coli (STEC) are notorious pathogens associated with severe diarrhea and HUS in humans. Although E. coli constitutes an important component of normal gut bacterial flora, many studies have shown the non-toxigenic strains to harbor a number of different virulence factors and to be diarrheagenic (17,18,37). Here, we present data showing the prevalence of E. coli strains carrying an ETEC-specific gene, sta, and STEC-specific gene, stx, which included either stx1 or stx2 in the gut of livestock and poultry in Bangladesh. This study also presents data showing for the first time that a significant proportion (8/23) of the toxigenic E. coli strains from livestock were MDR and hybrid type carrying both stx (either stx1 or stx2) and ETEC-specific enterotoxin gene, sta.
The presence of such a high percentage of MDR hybrid type E. coli strains in livestock poses a potential health threat to people of Bangladesh.

Virulence gene Profiling of E. coli strains
Results obtained in this study confirmed that a very high proportion of the E. coli strains (66%) deriving from different livestock are toxigenic, carrying either sta, stx, or both in varying combinations. What is most important to note is that a significant proportion of E. coli in livestock was confirmed to be ETEC, including 22% carrying the sta gene only, 44% STEC (harboring only stx gene; either stx1 or stx2), and 35% hybrid comprising both est and stx. ETEC is an important cause of diarrhea in humans and animals in the low-income countries and is the main cause of diarrhea in travelers to low-income countries (1). One study carried out in Spain reported a high incidence of ETEC (30%) in piglets with diarrhea (38). A study carried out in Bangladesh revealed 61% of the E. coli strains isolated from the aquatic environment carried the est gene (39). Zoonotic and environmental transmission may be the reason ETEC is the predominant group of E. coli associated with childhood diarrhea in Bangladesh (28). The occurrence of ETEC in farm animals has always been of great public health importance as animals excreting pathogenic bacteria can contaminate water bodies in and around the area they are reared and in many ways can be related to human diseases. This may be one reason why ETEC is largely a pathogen of low-income countries where access to safe drinking water is often low. STEC strains can cause bloody diarrhea known as haemorrhagic colitis, non-bloody diarrhea, and HUS (1). The data presented in this study provide evidence that livestock is an important reservoir for STEC carrying either stx1 or stx2. A study carried out in Bangladesh reported 37.9% of buffalo, 20.1% of cows, and 10.0% of goats were positive for STEC (40). Our data appear to concur with 44% (n = 10) of livestock E. coli isolated mostly from goats (80%, 8/10 or 35%, 8/23) revealed STEC, either stx1 or stx2. Our results also appear to agree with those from a study carried out in Vietnam that showed STEC to account for 27% of E. coli isolated from buffalos, 23% from cattle, and 38.5% from goats (41). Although water plays an important role in the transmission of pathogenic bacteria, a study carried out in Bangladesh reported the presence of stx only in 1.25% of the E. coli strains isolated from natural aquatic environments (39). STEC-associated diarrhea is also very low compared to that of other enteric pathogens, including Vibrio cholerae and ETEC in Bangladesh (27). This low prevalence of STEC as the etiology of diarrhea could be due to STEC-specific antibodies in the human population, as this pathotype is present in a high proportion of E. coli occurring in domesticated livestock, such as goats and sheep, although it was not found in poultry, and only rarely in surface water samples (39). The low prevalence in natural surface water could be related to the incompetence of the STEC strains to survive beyond their livestock reservoirs (cattle, sheep, and goats) (6). Despite the low prevalence of STEC infections in Bangladesh (27), the virulence potential of these livestock strains to emerge as a novel STEC pathogen cannot be ruled out.
In the present study, 34% of the toxigenic livestock E. coli strains were hybrid as those carried toxin genes present in two different pathotypes; stx and est. Notably, majority of these hybrid strains (7/8) carried both sta and stx 1, while one strain carried both sta and stx2 genes. This finding is not unique to our study as a number of past studies have shown hybrid strains carrying virulence marker genes of two different E. coli pathotypes (17), nonetheless, in a significantly lower frequency (2%) than that we observed in Bangladesh. Clinically linked STEC-ETEC hybrid E. coli strains from Brazil, Denmark, France, Germany, Great Britain, Mexico, and the United States were shown to harbor virulence markers for two or three different pathotypes (17). In a study in Germany, 0.6% of the human STEC isolates possessed stx2g and estIa (37). Serotype O100:H of a STEC-ETEC hybrid strain carrying stx2 and estIa genes was isolated from contaminated drinking water during an outbreak in Finland (22). A later study reported a STEC strain carrying stx1a and estIa from cattle in Burkina Faso (42). Although no data exist on the virulence potential of STEC-ETEC hybrid strains isolated from livestock in Bangladesh, the widespread distribution and clinical relevance worldwide might indicate their virulence potential.

antibiotic resistance Profile
Resistance to antimicrobial agents can be a useful epidemiological marker for ETEC, STEC, and STEC-ETEC hybrid strains. In this study, the highest antimicrobial resistance was found against erythromycin 87% (20/23) followed by azithromycin 43% (10/23). In the present study, a significant proportion of ETEC and STEC strains were MDR with the highest MDR [50% (4/8)] being observed in STEC-ETEC hybrid strains. Since 1950s, antibiotics have been used in animal feed to ensure healthy livestock, prevent infections, and excel growth. Such indiscriminate use of antibiotics resulted in the widespread antibiotic resistance in bacteria (21). Erythromycin resistance during the 1990s compelled clinicians to switch to azithromycin as the drug of choice in the treatment of cholera (45) and watery (cholera-like) diarrhea caused by ETEC (46,47).

PFge analysis
As shown earlier by Vu-Khac et al. (48), a high degree of genetic polymorphisms was observed from the PFGE profiles of the STEC non-O157 and ETEC strains in the present study. Similarly, a high degree of heterogeneity was also observed with the STEC-ETEC hybrid strains, as the pulsotypes were different for strains belonging to the same serotypes, suggesting that they were unlikely to be of a single ancestral origin. A few strains showing identical PFGE patterns, however, suggest close clonal relatedness.

cOnclUsiOn
This study is the first to show livestock as the reservoir for multidrug resistant hybrid type E. coli strains carrying both stx and st found in Shiga toxin-producing E. coli (STEC) and ETEC, respectively, in Bangladesh. Serotyping results revealed that none of the E. coli STEC-ETEC hybrid strains were O157:H7 but belonged to serotypes O76:H19, O43:H2, O87:H16, OR:H2, and O152:H8 and were heterogeneous genetically as confirmed by PFGE of XbaI-digested genomic DNA and cluster analyses by dendrogram. Although there may be some sorts of immunity toward Shiga toxin-producing E. coli existing among the healthy human population of Bangladesh (49), as they live in close proximity to the domesticated animals, multidrug resistant STEC-ETEC hybrid E. coli strains occurring in domesticated animals deserve careful attention as they can pose even greater public health threat for the people of Bangladesh.
aUThOr cOnTriBUTiOns AK, MO, HW, and MA contributed to the design of the study, manuscript revision, and final approval of version to be published. F-TJ designed, implemented, and performed the study in the laboratory and wrote the manuscript. SC, CG, AC, SM, SA, BH, and AN contributed to revising the manuscript critically for