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Iranian Journal of Veterinary Research logoLink to Iranian Journal of Veterinary Research
. 2015 Winter;16(1):31–35.

Molecular characterization and antibiotic susceptibility pattern of caprine Shiga toxin producing-Escherichia coli (STEC) isolates from India

A Mahanti 1, I Samanta 2,*, S Bandyopadhyay 3, S N Joardar 2
PMCID: PMC4789236  PMID: 27175147

Abstract

The present study was conducted to detect the occurrence, serotype, genotype, phylogenetic relationship and antimicrobial resistance pattern of STEC from healthy goats of West Bengal, India. From the 125 faecal samples collected from healthy goats, 245 isolates were identified as Escherichia coli. The E. coli harbouring any gene for Shiga toxins (stx1/stx2) was detected in 36 (14.7%) of the 245 E. coli isolates. These STEC strains belonged to 22 different serogroups (O2, O5, O20, O21, O22, O25, O41, O44, O45, O60, O71, O76, O84, O85, O87, O91, O103, O112, O113, O120, O156, and O158) and three were untypeable. The stx1 and stx2 was detected in 26 (72.2%) and 21 (58.3%) of Shiga toxin producing-E. coli (STEC) isolates, respectively. Further, E. coli harbouring eaeA only (Enteropathogenic E. coli) and ehxA was detected in 22 (61.1%) and 28 (77.7%) isolates, respectively. Whereas the saa was present in 8 (22.2%) E. coli isolates. The subtyping of the 26 E. coli strains possessing stx1 showed that 73.% (19/26) of these isolates were positive for stx1C subtype. Of the 21 isolates with the stx2 gene, 42.8% (9/21) were positive for stx2C, and 38.1% (8/21) were positive for stx2d gene. The phylogenetic analysis of STEC strains after RAPD reveals eight major clusters. However, no serogroup specific cluster was observed. Resistance was observed most frequently to erythromycin (80.5%), amikacin (52.7%), cephalothin (50%), kanamycin (41.6%), neomycin (36.1%) and gentamycin (36.1%) and less frequently to norfloxacin (2.7%), enrofloxacin (2.7%), and ciprofloxacin (2.7%). Multidrug resistance was observed in eleven STEC isolates.

Key Words: Goat, EPEC, STEC, West Bengal

Introduction

Shiga toxin producing-Escherichia coli (STEC) are food borne pathogens associated with a spectrum of human illness ranging from bloody diarrhoea to life-threatening infection such as hemolytic uraemic syndrome (HUS), thrombotic thrombocytopenic purpurea (TTP) and hemorrhagic colitis (HC). The bacteria produce the life-threatening infection with the help of Shiga toxins, encoded by stx1 and stx2 genes and the products of the locus of eneterocyte effacement (LEE) pathogenicity island such as eaeA gene. The eaeA gene encodes for the “intimin” protein which is involved in the intimate adhesion of bacteria to the enterocytes and production of attaching and effacing (AE) lesion (Paton and Paton, 1998a ▶). Some LEE negative strains can also possess saa gene encoding a novel outer membrane protein which acts as auto agglutinating adhesin (Paton et al., 2001 ▶). The majority of STEC strains also have hemolytic phenotype in washed sheep blood agar due to production of enterohemolysin, encoded by ehxA gene (Beutin et al., 1989 ▶).

The domestic and wild ruminants act as the major reservoir of STEC which can secrete the bacteria through the faeces (Wani et al., 2004 ▶). Due to absence of clinical manifestation the reservoir animals are included in the food production. The food products from the reservoir animals are at risk of contamination with STEC either with their faeces or intestinal contents during slaughter (Asakura et al., 2014 ▶). Thus, STEC can enter the human food chain through contaminated food products and is responsible for several food borne outbreaks as reported earlier (CDC, 2008 ▶; Ethelberg et al., 2009 ▶). So, the present study was conducted to detect the occurrence, serotype, genotype, phylogenetic relationship and antimicrobial resistance pattern of STEC from healthy goats of West Bengal, India. Multiplex PCR was used for primary screening of E. coli isolates for Shiga toxin genes, eaeA and ehxA. PCR based subtyping of the Shiga toxin genes, serogrouping and RAPD based phylogenetic relationship were determined among the isolates.

Materials and Methods

Sampling

A total of 125 faecal samples were collected from healthy goats in different locations of West Bengal, India. The samples were transported to the laboratory in transport medium in sterile vials on ice for further processing. None of the samples were held for more than 24 h at 4°C.

Isolation of Escherichia coli

The collected samples were inoculated into the nutrient broth and were incubated at 37°C for 24 h (HiMedia, India). The growth in the nutrient broth was transferred to MacConkey’s agar (HiMedia, India) and again incubated at 37°C overnight. The next day 2-3 rose pink colonies were randomly picked and transferred to EMB agar (HiMedia, India) followed by an overnight incubation at 37°C. The colonies were observed for metallic sheen and single colony was streaked into nutrient agar slant for further biochemical confirmation. All the pure cultures obtained from nutrient agar slant were subjected to Gram’s staining and standard biochemical tests as described earlier (Quinn et al., 1994 ▶). The strains were preserved in glycerol broth at -70°C for subsequent characterization.

Multiplex-PCR for detection of stx 1 , stx 2 , eaeA , ehxA , saa gene sequences

For PCR based detection of STEC genes, all the E. coli isolates were subjected to DNA extraction as per previously described method of Samanta et al. (2014) ▶. All the E. coli isolates were subjected to multiplex-PCR (m-PCR) for the detection of Shiga toxin gene(s) (stx1, stx2), intimin (eaeA), enterohaemolysin (ehxA) and STEC autoagglutinating adhesin (saa) as per the previously described primers and protocol (Paton and Paton, 1998b ▶, 2002; Paton et al., 2001 ▶). The m-PCR was performed in a gradient thermocycler (Eppendorf ProS, Germany). All the reagents and oligo nucleotide primers were procured from Genetix Biotechnology Asia Private Limited, India. The amplified DNA products were visualized by Gel Documentation System (UVP, UK) after gel electrophoresis in 2% agarose containing ethidium bromide (0.5 µg ml-1) (SRL, India) as per the standard protocol (Sambrook and Russel, 2001 ▶).

Subtyping of stx 1 and stx 2 gene

All the stx1 positive E. coli isolates were subjected to PCR amplification for the presence of stx1c gene by using the previously described method of Zhang et al. (2002) ▶. Further, all the stx2 positive E. coli isolates were subjected to m-PCR amplification for the presence of stx2c, stx2d, as described (Wang et al., 2002 ▶).

Serogrouping

The E. coli isolates harbouring characteristic virulence genotype were O-serogrouped from National Salmonella and Escherichia Centre, Central Research Institute, Kausali, Himachal Pradesh, India.

Molecular characterization of STEC isolates and phylogenetic analysis

All the STEC isolates were subjected to RAPD-PCR as described (Mahanti et al., 2013 ▶). The amplified DNA products were visualized by Gel Documentation System (UVP, UK) after gel electrophoresis in 1% agarose containing ethidium bromide (0.5 µg ml-1) (SRL, India) as per the standard protocol (Sambrook and Russel, 2001 ▶). An unrooted phylogenetic tree was created by using neighbour joining method in the Doc-itLs image analysis software (UVP, UK).

Antibiotic sensitivity test of STEC isolates

All the STEC isolates were tested for their sensitivity and resistance to different antibiotics by the disc diffusion method (CLSI, 2008 ▶). The antibiotics used were chloramphenicol (25 µg), co-trimoxazole (25 µg), ciprofloxacin (5 µg), gentamicin (10 µg), neomycin (30 µg), norfloxacin (10 µg), streptomycin (30 µg), oxytetracycline (30 µg), cephalothin (30 µg), amikacin (30 µg), ceftazidime (30 µg), kanamycin (30 µg), ceftriaxone (30 µg), levofloxacin (5 µg), amoxicillin + clavulanic acid (30 µg), cefaclor (30 µg), cefuroxime (30 µg), azithromycin (30 µg), piperacillin + tazobactam (100 + 10 µg), cefepime + tazobactam (30 + 10 µg), ampicillin + cloxacillin (10 µg), enrofloxacin (10 µg), amoxicillin (25 µg), erythromycin (10 µg), doxycycline hydrochloride (30 µg) and pefloxacin (5 µg).

Results

Prevalence of Shiga toxin producing- E. coli

From the 125 faecal samples collected from healthy goats of different places in West Bengal, India, 245 isolates were identified as E. coli after morphological and biochemical confirmation. The E. coli harbouring any gene for Shiga toxins (stx1/stx2) was detected in 36 (14.7%) of the 245 E. coli isolates. These STEC strains belonged to 22 different serogroups (O2, O5, O20, O21, O22, O25, O41, O44, O45, O60, O71, O76, O84, O85, O87, O91, O103, O112, O113, O120, O156, and O158) and three were untypeable (Table 1).

Table 1.

Serogroup and genotype of the STEC and EPEC isolates from goats in West Bengal, India

Serial No. Serogroup Genetype
G1 O5 stx 2C, eae, ehxA
G2 O45 stx 1, eae, ehxA
G3 O84 eae, ehxA
G4 O76 stx 1, stx2, ehxA, saa
G5 O91 stx 1C, stx2d, ehxA
G6 O76 eae, ehxA
G7 O25 stx 1C, stx2d, eae, ehxA
G8 O112 stx 1C, stx2d, saa
G9 O91 stx 2C, eae, ehxA
G10 O6O stx 1C, eae, ehxA
G11 O120 stx 1C, stx2d, ehxA, saa
G12 O5 stx 1C, eae, ehxA
G13 O44 stx 1C, eae, ehxA
G14 O113 stx 1, eae, ehxA
G15 O21 stx 2d
G16 O22 stx 1, eae, ehxA
G17 OUT stx 1C, stx2, eae, ehxA
G18 O156 stx 1, stx2d
G19 O87 stx 1C, stx2C, ehxA
G20 O2 stx 1C, stx2, ehxA, saa
G21 O85 stx 1, eae
G22 O60 stx 1C, stx2, ehxA, saa
G23 O87 stx 1C, eae, ehxA
G24 OUT eae, ehxA
G25 O158 stx 2d, saa
G26 O5 stx 2C, eae, ehxA
G27 O41 stx 1C, stx2C
G28 O113 stx 1C, stx2C, ehxA, saa
G29 OUT stx 1C, eae, ehxA
G30 O103 stx 1C, eae, ehxA
G31 O120 stx 1C, eae, ehxA
G32 O112 stx 1C, eae, ehxA
G33 O22 stx 2d
G34 O71 stx 2C, eae, ehxA
G35 O20 stx 1C, stx2C, saa
G36 O5 stx 1, stx2C, eae, ehxA
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The stx1 and stx2 were detected in 26 (72.2%) and 21 (58.3%) of STEC isolates, respectively. Further, E. coli harbouring eaeA only (Enteropathogenic E. coli) and ehxA was detected in 22 (61.1%) and 28 (77.7%) isolates, respectively. Whereas the saa was present in 8 (22.2%) E. coli isolates from goat in this study, out of which 5 (62.5%) were associated with the ehxA gene (Table 1, Fig. 1).

Fig. 1.

Fig. 1

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Phylogenetic analysis using RAPD-PCR profiles of the STEC and EPEC strains isolated from goats in West Bengal (India) using neighbour-joining method and dice similarity

Subtyping of stx 1 and stx 2

The subtyping of the 26 E. coli strains possessing stx1 showed that 73.% (19/26) of these isolates were positive for stx1C subtype. Most of the stx1C possessing strains occurred in combination with ehxA, saa or both. Of the 21 isolates with the stx2 gene, 42.8% (9/21) were positive for stx2C, and 38.1% (8/21) were positive for stx2d gene (Table 1, Fig. 1).

Molecular characterization of STEC isolates and phylogenetic analysis

All 36 STEC strains were characterized by RAPD- PCR to determine the genetic diversity among the strains. All the strains were typeable with primer 1247 and produced amplified fragment size ranging from 250 bp to 1900 bp (calculated by Doc-itLs image analysis software, UVP, UK). The phylogenetic analysis of STEC strains after RAPD reveals eight major clusters (clusters A to E, Fig. 1). No serogroup specific cluster was observed.

Antibiotic sensitivity test

Resistance was observed most frequently to erythromycin (80.5%), amikacin (52.7%), cephalothin (50%), kanamycin (41.6%), neomycin (36.1%) and gentamycin (36.1%) and less frequently to norfloxacin (2.7%), enrofloxacin (2.7%), and ciprofloxacin (2.7%). No resistance was observed in case of ceftazidine, levofloxacin, piperacillin along with tazobactam, cefepime along with tazobactam and ceftriaxone. Multidrug resistance was observed in eleven STEC isolates (Table 2).

Table 2.

Antibiotic resistance pattern of the STEC and EPEC from goats in West Bengal, India

Name of the antimicrobial agent (Conc. µg) Sensitive (%) Intermediate (%) Resistant (%)
Chloramphenicol (25 µg) 77.78 11.11 11.11
Co-trimoxazole (25 µg) 77.78 16.67 5.55
Ciprofloxacin (5 µg) 88.89 8.33 2.78
Gentamicin (10 µg) 5.56 58.33 36.11
Neomycin (30 µg) 0 63.89 36.11
Norfloxacin (10 µg) 88.89 8.33 2.78
Streptomycin (30 µg) 5.55 77.78 16.67
Oxytetracycline (30 µg) 19.44 55.56 25
Cephalothin (30 µg) 5.56 44.44 50
Amikacin (30 µg) 2.78 44.44 52.78
Ceftazidime (30 µg) 97.22 2.78 0
Kanamycin (30 µg) 5.55 52.78 41.67
Ceftriaxone (30 µg) 88.89 11.11 0
Levofloxacin (5 µg) 100 0 0
Amoxicillin + clavulanic acid (30 µg) 80.56 13.89 5.55
Cefaclor (30 µg) 36.11 38.89 25
Cefuroxime (30 µg) 72.23 19.44 8.33
Azithromycin (30 µg) 11.11 69.45 19.44
Piperacillin + tazobactam (100 + 10 µg) 52.78 47.22 0
Cefepime + tazobactam (30 + 10 µg) 100 0 0
Ampicillin + cloxacillin (10 µg) 69.45 22.22 8.33
Enrofloxacin (10 µg) 80.55 16.67 2.78
Amoxicillin (25 µg) 75 19.45 5.55
Erythromycin (10 µg) 0 19.45 80.55
Doxycycline hydrochloride (30 µg) 36.11 55.56 8.33
Pefloxacin (5 µg) 75 19.45 5.55
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Discussion

The study was aimed to detect the occurrence of STEC from healthy goats in West Bengal, India. Of the total 245 E. coli isolated during the present study 36 (14.7%) isolates harboured at least one marker gene for STEC. Similar occurrence of STEC was observed in previous studies conducted in local and migratory goats in Kashmir Valley, India (Wani et al., 2006 ▶) and from faecal samples, meat and milk of goats in other parts of the world (Orden et al., 2003 ▶; Islam et al., 2008 ▶; Momtaz et al., 2013 ▶). However, lower or higher occurrence of STEC was detected from faeces of healthy and diarrhoeic goats, chevon and goat milk cheese in other countries throughout the world (Orden et al., 2003 ▶; Horcajo et al., 2010 ▶; Ojo et al., 2010 ▶; Schilling et al., 2012 ▶). It seems that the occurrence of STEC varies with different geographical location and season.

All the STEC strains belonged to 22 different serogroups (O2, O5, O20, O21, O22, O25, O41, O44, O45, O60, O71, O76, O84, O85, O87, O91, O103, O112, O113, O120, O156, and O158) and three were untypeable. Similarly, the serogroups O2, O5, O22, O45, O60, O76, O87, O91, O103, O112, O113 and O156 were also previously isolated from faeces and milk product of goats (Wani et al., 2006 ▶; Vu-Khac and Cornick, 2008 ▶; Horcajo et al., 2010 ▶; Schilling et al., 2012 ▶; Jacob et al., 2013 ▶).

Out of the 36 STEC isolates of the present study, 26 (72.2%) and 21 (58.3%) isolates possessed the stx1 and stx2 gene, respectively. Similar occurrence of stx1 and higher occurrence of stx2 was observed in goats in previous studies (Oliveira et al., 2008 ▶; Vu-Khac and Cornick, 2008 ▶). Further characterization of the 26 stx1 positive isolates identified 19 (73%) strains positive for stx1C. Similarly, higher number of stx1C was observed in stx1 positive isolates from goats in Spain (Horcajo et al., 2010 ▶) and Central Vietnam (Vu-Khac and Cornick, 2008 ▶). In this study, we found that stx1C genes most commonly occurred with ehxA, saa or both which is in agreement with the previous finding by Vu-Khac and Cornick (2008) ▶. Subtyping of the isolates possessing stx2 gene, produced 9 (42.8%) and 8 (38.1%) isolates positive for stx2C and stx2d gene, respectively. Similarly, Zheng et al. (2005) found high proportion of isolates bearing stx2C genes in healthy goats in China which is consistent with the present finding. In contrast, higher recovery of stx2d gene has been observed in caprine isolates in Central Vietnam (Vu-Khac and Cornick, 2008 ▶). It seems that the prevalence of stx2C isolates is higher in this geographical region as China is nearer than Vietnam to the present study area.

The eaeA gene coding intimin protein was present in 22 (61.1%) isolates. Lower occurrence of eaeA was found in a study conducted by Vu-Khac and Cornick (2008) ▶, whereas Islam et al. (2008) ▶ could not isolate eaeA in non-O157 STEC from goats. Higher prevalence of eaeA in the present study may be a concern as LEE positive strains were considered highly virulent in human (Toma et al., 2004 ▶). A large number of isolates (77.7%) was found to possess enterohaemolysin (ehxA) in combination with other virulence genes in the present study which is in contrast with the previous study (Vu-Khac and Cornick, 2008 ▶). Association of saa gene in STEC isolates (22.2%) from healthy goats in the present study might be remarkable because in another study, saa was not detected in caprine STEC isolates (Cortes et al., 2005 ▶). Among the saa positive isolates 5 (62.5%) were concomitantly present with the ehxA, which is consistent with the previous studies (Paton et al., 2001 ▶; Paton and Paton, 2002 ▶).

The RAPD results showed genetic heterogenicity of the STEC isolates. No serogroupwise clustering of the STEC isolates in the present study was detected. This is in agreement with the previous observation by Mahanti et al. (2013) ▶ who found many RAPD banding patterns among the STEC isolates from buffalo belonging to a similar serogroup.

Resistance of STEC isolates against chloramphenicol, co-trimoxazole, ciprofloxacin, gentamicin, neomycin, norfloxacin, streptomycin, tetracycline, ampicillin, enrofloxacin, erythromycin as reported in the present study was also supported by earlier studies in goats (Ojo et al., 2010 ▶; Medina et al., 2011 ▶). However, higher occurrence of multidrug resistant STEC (69.5%) was reported in Nigerian goats than in the present study (Ojo et al., 2010 ▶). Isolation of enterovirulent E. coli such as STEC and EPEC is a serious public health concern in this area where the farmers stay in close proximity to goats with little concern for hygiene and people consume undercooked goat milk and meat products.

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