Foodborne illness caused by multidrug resistant STEC is one of the most important global public health problems in the world. So, the present study was undertaken to know the prevalence of β-lactamase (ESBL and AmpC) producing STEC in retail meats and chicken cloacal swabs by PCR. A total of 183 samples (135 foods of animal origin and 48 chicken cloacal swabs) collected from retail meat shops and poultry farms in and around Krishna district were subjected to cultural isolation and confirmation of β-lactamase producing STEC by different PCR assays. The overall prevalence of E. coli was found to be 37.15% (68/183) by species-specific PCR. The STEC specific virulence genes stx1, hlyA and stx2 were detected in 10.29%, 2.941% and 1.470%, of E. coli isolates, respectively and no eae A gene was identified. ESBL and/or AmpC-producing E. coli were found in 80.88% (55/68) of isolates with blaTEM being the predominant gene (87.27%) followed by blaCTX-M-2 (9.09%), blaOXA (7.27%), bla DHA (3.63%) and CIT (1.81%). β-lactamase activity was detected in 66.66% of STEC isolates. These findings revealed that retail meats are the potential source of ESBL and/or AmpC-producing E. coli.
HIGHLIGHTS
Prevalence of β-lactamase producing STEC in retail meats
Retail meats were found to be potential source for β-lactamase producing STEC
E. coliPCRSTECESBL and AmpC β-lactamases
E. coli is a Gram-negative, rod-shaped, flagellated nonsporulating and facultative anaerobic bacteria that belongs to Enterobacteriaceae family. Although, E. coli is a commensal that can be found in intestines of variety of animals including man, some pathogenic strains can cause debilitating fatal diseases in humans and animals (Jafari et al., 2012). These pathogenic E. coli are classified into Enterotoxigenic E. coli (ETEC), Enteropathogenic E. coli (EPEC), Enterohaemorrhagic E. coli (EHEC), Enteroinvasive E. coli (EIEC) and Enteroaggregative E. coli (EAgEC). Among these, EHEC which are also known as shiga toxin producing E. coli (STEC), are considered as the most dangerous group that cause a variety of diseases ranging from mild diarrhoea to severe bloody diarrhoea called haemorrhagic colitis (HC) and even leads to life-threatening sequelae such as haemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic Purpura (TTP) (Brett et al., 2003; Perera et al., 2015). As STEC is endemic in cattle and other domestic animals, undercooked meat and unpasteurized milk act as the main source of infection. STEC has also emerged as one of the most virulent groups associated with cases of food borne illness in humans. Retail meats derived from food animals could potentially serve as transmission vehicles for STEC and other diarrheagenic E. coli strains (Xia et al., 2010).
How to cite this article: Naidu, S.T., Bodempudi, B., Chinnam, B.K., Pedada, V.C., Nelapati, S., Tumati, S.R., Gottapu, C., Talluri, H.L., Puvvada, S., Chekuri, N., Kumar, G.N. and Susmitha, B. (2021). Prevalence of β-lactamase producing Shiga toxigenic E. coli (STEC) in retail meats and chicken cloacal swabs. J. Anim. Res.,11(2): 263-271. Source of Support: None; Conflict of Interest: None
STEC strains produce cytotoxins known as shiga like toxins (stx) that are classified into two major classes shiga like toxin1(stx1) and shiga like toxin2 (stx2) between which the amino acid profile is 56% similar. Stx1 is 98% analogous to the shiga toxin of Shigella dysenteriae (Jackson et al., 1987). In addition to stx 1 and stx 2, most disease causing STEC strains also produce a protein called intimin (eaeA) that is involved in the enterocyte attaching and effacing and enables the intimate adherence of STEC to the intestinal epithelium of the host (Yu and Kaper, 1992). Further a specific plasmid encoded haemolysin called EHEC haemolysin (hlyA) is involved in extraction of iron from the blood released into the intestine (Beutin et al., 1995).
In food producing animals, broad spectrum antibiotics are not only used to treat bacterial infections but also used for disease prevention (metaphylaxis and prophylaxis) and growth promotion. Nowadays, the antibiotics usage at subtherapeutic doses is increasing due to increased demand for livestock products globally, especially in South and Southeast Asia, due to rising incomes (Brower et al., 2017). This massive and indiscriminate use of broad-spectrum cephalosporins among animals could be an important reason for the occurrence of ESBLproducing bacteria among food producing animals and in meat products (Sabia et al., 2017). These resistant bacteria will be excreted through faeces and enter into environment that can contaminate the foods of animal origin during animal slaughter and carcass processing. The human gut could be colonized with these resistant bacteria particularly ESBL and AmpC β-lactamase producing strains through consumption of contaminated meat and cause fatal infections and treatment failure which results in serious consequences in patients. In case of β-lactamase producing STEC infections, antibiotic treatment results in higher occurrence of the infection due to liberation of toxins stimulated by the non-lethal concentration of antibiotics (Puii et al., 2019). A number of studies in various countries have proposed meat as a source of β-lactamase producing bacteria (Agerso et al., 2012). Recent studies in the Netherlands have reported that the predominant ESBL genes and plasmids in E. coli obtained from broiler meat are present even in human clinical isolates (Egervärn et al., 2014). In the past few decades, foodborne illness caused by E. coli mainly ESBL and/or AmpC β-lactamase producing E. coli was one of the most important global public health problems in the world since they were isolated from livestock, animal feed and water in increasing prevalence and pathogenicity (Gilliss et al., 2013). So, the aim of present study was to know the prevalence of β-lactamases producing STEC in meats of food animals collected from retail shops and also in chicken cloacal swabs.
MATERIALS AND METHODSStandard control and primers
ATCC (American Type Culture Collection) culture of E. coli (ATCC 25922) was used as standard positive control and Proteus mirabilis (ATCC 12453) was used as negative control. Oligonucleotide primers were custom synthesized from M/s. Bioserve Biotechnologies Pvt. Ltd.
(Hyderabad).
Sample collection
A total of 183 samples comprising 135 food samples of animal origin (17 chicken, 35 mutton, 15 beef, 20 pork and 48 fish) collected from retail meat shops and 48 poultry cloacal swabs collected from poultry farms in and around Krishna district, Andhra Pradesh. The study was carried out during the period of February 2019 to January 2020, in the Department of Veterinary Public Health and Epidemiology, N.T.R College of Veterinary Science, Gannavaram, India.
Ten grams of meat samples were inoculated into 90ml of Tryptose soya broth (TSB) and homogenized, while cloacal swabs were directly inoculated into 10 ml TSB broth test tubes and incubated at 37oC for 24h. After incubation, the samples were streaked on MacConkey and EMB agar plates and incubated at 37oC for 24h. Identification of isolates was done based on colony morphology (MacConkey – pink color colonies and EMB-greenish metallic sheen colonies) and the colonies were enriched in TSB for further confirmation by species-specific PCR.
DNA extraction from enriched broth samples
DNA was extracted by boiling and snap chilling method (Suresh et al., 2018). About 1.5 mL of enriched broth cultures were taken into microcentrifuge tubes and centrifuged at 8000 rpm for 10 min. Supernatant was discarded, 50 μL of nuclease-free water was added and placed in boiling water bath at 100°C for 10 min, then immediately snap chilled for 10 min and centrifuged at 10,000 rpm for 5 min. The supernatant was used for PCR assays as a DNA template.
Confirmation of E. Coli by species-specific-uniplex PCR
Presumptive colonies of E. coli were confirmed molecularly by species-specific genes (Table 1). PCR assay was optimized in 25 μL reaction mixture containing 2 μL of DNA template, 12.5 μL of 2x master mix (EmeraldAmp GT PCR Master Mix, Takara), 1.5 μL each of forward and reverse primers (10 pmol/μL) and the rest of the volume is made by adding nuclease-free water. The cycling conditions were as follows: initial denaturation at 94 °C for 5 min; 35 cycles of 95°C for 1 min, 50 °C for 50 sec and 72 °C for 1 min and a final extension step at 72 °C for 10 min. PCR products were subjected to gel electrophoresis using 1% agarose with ethidium bromide as fluorescent dye (Sambrook & Russell, 2001) and visualized using Gel Documentation unit (BIORAD).
Primer sequences for E. Coli species specific PCR (Sun et al., 2011)
Detection of virulence genes in E. Coli isolates
All the confirmed E. coli isolates were screened for the presence of virulence genes (Table 2). PCR assay was optimized in 25 μL reaction mixture containing 2.5 μL of DNA template, 12.5 μL of 2x master mix (EmeraldAmp GT PCR Master Mix, Takara), 0.3 μL each of forward and reverse primers (10 pmol/μL) and the rest of the volume is made by adding nuclease-free water. The cyclical conditions followed were according to the Paton and Paton (1998).
Primers used for detection of virulence genes in E. Coli (Paton and Paton, 1998)
Antimicrobial resistance profile of E. Coli isolates
All the confirmed E. coli isolates were subjected to antibiotic sensitivity test against 14 different antibiotics like cefepime, ceftriaxone, amikacin, tetracycline, ceftazidime, gentamicin, piperacillin/tazobactam, ceftazidime+clavulanic acid, ofloxacin, chloramphenicol, amoxicillin+clavulanic acid, ampicillin, cefotetan, imipenem on Muller Hinton agar by using Kirby Bauer disc diffusion method (Bauer et al., 1966). Direct colony of each isolate was suspended in PBS (pH 7.4) and the turbidity was adjusted to 0.5 McFarland units (equivalent to an approximate cell density of 1.5 × 108 CFU/ml having absorbance of 0.132 at wavelength of 600nm). The diameter of inhibition zones was measured and susceptibility patterns of E. coli species were interpreted according to Clinical and Laboratory Standards Institute guidelines (CLSI, 2018).
Detection of ESBL production in E. Coli isolates by phenotypic methods
Screening of E. coli isolates for resistance against thirdgeneration cephalosporins like cefotaxime (30 µg), ceftazidime (30 µg), ceftriaxone (30 µg) and monobactams like aztreonam (30 µg) was done by disc diffusion method (Bauer et al., 1966) on Mueller-Hinton agar using commercial discs (HiMedia, Mumbai). Resistance to at least one of the four antibiotics used was considered as a positive screening test (PST) for ESBL and AmpC β-lactamase production (Drieux et al., 2008; CLSI, 2018). PST positive isolates were further subjected to combination disc method (CDM) for phenotypic confirmation (PCT) of ESBL production, in which cephalosporin discs along with combination disc containing cephalosporin plus clavulanate/sulbactam were used i.e. ceftazidime (CAZ, 30 µg), ceftazidime + clavulanic acid (CAC, 30/10 µg), cefotaxime (CTX, 30 µg), cefotaxime + clavulanic acid (CEC, 30/10 µg) and ceftriaxone (CTR, 30 µg), ceftriaxone + sulbactam (CIS, 30/10 µg). ESBL production was confirmed by an increased inhibition zone diametre of ≥ 5 mm in case of combination discs (Drieux et al., 2008; CLSI, 2018).
Identification of ESBL genes by m-PCR
All PCT positive E. coli isolates DNA were subjected to two m-PCR assays to detect different ESBL genes (blaTEM, blaSHV, blaOXA, blaCTX-M-1, blaCTX-M-2 and blaCTX-M-9 groups). PCR assays were optimized in 25 μL reaction mixture containing 2 μL of DNA, 12.5 μL of 2X master mix (EmeraldAmp GT PCR Master Mix, Takara), 0.5 μL of forward and reverse primers (10 pmol/ μL) and the rest of the volume is made by adding nucleasefree water. The primers and PCR conditions were followed according to Dallenne et al. (2010).
Detection of AmpC β-lactamase genes in E. Coli by m-PCR
m-PCR assay for the detection of blaAmpC genes was performed to all the PST positive isolates since PST positive but PCT negative organisms may produce AmpC or both ESBL and AmpC β-lactamases. PCR was optimized in 25 μL reaction mixture containing 2 μL of DNA, 12.5 μL of 2X master mix (EmeraldAmp GT PCR Master Mix, Takara), 0.2 μL of forward and reverse primers (10 pmol/μL) and the rest of the volume is made by adding nuclease-free water, under standardized cycling conditions described by Manoharan et al. (2012).
RESULTS AND DISCUSSION
In the present study, E. coli were detected in 45.18% (61/135) of raw meat samples and 14.58% of chicken cloacal swabs (7/48) by PCR. The overall prevalence of E. coli was found to be 37.15% (68/183) which is almost similar to the findings of Prabhu Kishore (2017) who reported 34.03% of E. coli in the present study area. Among various raw meat samples, highest prevalence was recorded in fish (62.50%) followed by beef (60.00%), chicken (35.29%), pork (30.00%) and mutton (28.57%) (Table 3 & Fig. 1). Highest prevalence of E. coli was recorded in fish samples as water bodies can be easily prone to contamination with municipal sewage.
Prevalence of E. Coli in samples
Gel photograph of species-specific PCR for E. Coli. Lane 1: Positive control of E. coli (ATCC 25922) (231 bp); Lane 4: Negative control; Lane 5: DNA ladder (100 bp); Lane 2, 3, 6, 7, 8: E. coli (231 bp) isolated from different samples
Among the 61 E. coli isolates recovered from the raw meat samples, 14.75% (9/61) of isolates carried at least one of the STEC gene with stx1 being the predominant gene detected (77.77%, 7/9) followed by hlyA (22.22%, 2/9), stx2 (11.11%, 1/9) and no eaeA gene was detected. Shiga toxins were detected in 50.00% (3/6) of chicken isolates, 30.00% (3/10) of mutton isolates, 22.22% (2/9) of beef isolates and 3.33% (1/30) of fish isolates (Table 4 and Fig. 2). In our study, stx1 gene was recovered from six isolates (3-chicken, 1- beef, 1-fish and 1-mutton), stx2 from one beef isolate, hlyA from one mutton isolate and both stx1 and hlyA were found in one mutton isolate. In the present study, overall prevalence of STEC in different raw meats (14.75%) was comparable with the findings of Bai et al. (2015) who reporteded 19.39% prevalence of STEC in different raw meats in China. No isolate recovered from the chicken cloacal swabs was positive for STEC. However, 50.00% (3/6) of E. coli isolates recovered from the chicken meat samples were positive for shiga toxins. This might be due to the use of contaminated water during slaughter or from the unhygienic human handlers, knives and meat cutting boards.
Detection of shiga toxins in E. coli isolated from different samples
Antibiotic sensitivity test of E. coli isolates by phenotypic method
Gel photograph of PCR assay targeting shiga toxin genes. Lane 1: E. coli isolate with stx1 (180 bp) gene; Lane 2: E. coli isolate with stx2 (255 bp) gene; Lane 3: E. coli isolate with hlyA (534bp) gene; Lane 4: Negative control; Lane 5: DNA ladder (100 bp); Lane 6: E. coli isolate with stx1 (180bp) and hlyA (534bp)
All the E. coli isolates were subjected to antibiotic sensitivity test against fourteen antibiotics. Among these, highest levels of resistance was observed against tetracyclines identically detected in 59 (86.76%) isolates followed by ceftazidime/clavulanic acid in 38 (55.88%), amoxyclav in 34 (50.00%), ampicillin in 21 (30.88%), chloramphenicol in 16 (23.52%), gentamicin in 13 (19.11%), ceftazidime in 7 (10.29%), ceftriaxone in 6 (8.82%), ofloxacin in 4 (5.88%), amikacin and cefepime in 3 each (4.41%), cefotetan and piperacillin/ tazobactam in 2 (2.94%) each. None of the isolates were resistant to imipenem which is in accordance with Rasheed et al. (2014). Papich (2002) reported that resistance to carbapenems was extremely rare in veterinary medicine.
All the 68 E. coli isolates were subjected to PST, PCT and m-PCR for the detection of β-lactamases (ESBL’s and AmpC β-lactamases) out of which 80.88% (55/68) of isolates were positive for β-lactamases production by both phenotypic and molecular methods. Recent surveys have shown that 70–90% of Enterobacteriaceae in India are ESBL producers and colonization of this type of bacteria in humans is widespread (Brower et al., 2017). Heavy use of antibiotics could be one of the risk factors for the occurrence of ESBL-producing organisms (Paterson and Bonomo, 2005). Of the 55 β-lactamases producing E. coli isolates, 52 isolates were found positive for only ESBL’s (TEM, OXA and CTX-M-2), 2 isolates for AmpC β-lactamases (DHA and CIT) and 1 isolate for both ESBL’s and AmpC β-lactamases (TEM and DHA). Prevalence rate of β-lactamase E. coli among different samples was 100% in fish (30/30) and chicken cloacal swabs (7/7), 83.33% (5/6) in chicken, 80% (8/10) in mutton, 50% (3/6) in pork and 22.22% (2/9) in beef with blaTEM being the predominant gene detected (87.27%, 48/55) followed by blaCTX-M-2 (9.09%, 5/55), blaOXA (7.27%, 4/55), blaAmpC gene DHA (3.63%, 2/55) and CIT (1.81%, 1/55) among all the tested E. coli isolates (Table 6 and Fig. 3). A clear prevalence of only TEM-type β-lactamases were found in E. coli isolated from chicken (5 isolates), mutton (6 isolates), beef (2 isolates), pork (3 isolates), cloacal swabs (4 isolates) and fish (23 isolates). blaTEM was the predominant gene detected (87.27%) whereas Nadimpalli et al. (2019) and Nguyen et al. (2016) reported CTX-M to be the predominant gene among the foods of animal origin isolates. This variation might be due to differences in usage of different antibiotics in various geographical regions. TEM was also found in combination with OXA in two cloacal swab isolates, with CTX-M-2 in two fish isolates and with DHA in one fish isolate. Single CTX-M-2 gene was found in two fish isolates and in one cloacal swab isolate. OXA was found in two mutton isolates whereas AmpC β-lactamases were found in two fish isolates, with DHA in one and CIT in the other isolate. In the present study 66.66% (6/9) of STEC isolates harboured β-lactamase genes. Higher incidence of 59.38% β-lactamase producing STEC was reported from North East India (Puii et al., 2019).
Prevalence of β-lactamase genes in E. coli isolated from different samples
In the present study, prevalence of β-lactamases in fish and chicken cloacal swab isolates was 100% since nontherapeutic usage of antibiotics was particularly common in poultry production and aquaculture compared to other food animal production (Sivaraman, 2018). In aquaculture, application of antibiotics occurs en masse by inclusion of antibiotics in fish food. This wide application of antibiotics in fish food contributes to leaching of antibiotics into the water and pond sediments from unconsumed food and through faeces. Thus, it exerts selection pressures on the sediment and water micro flora and leads to development of resistance. In addition, fish farming is usually integrated with sewage or industrial waste water or with land agriculture. These unhygienic measures, overcrowding and other manipulations act as stressors to the fish and promote an increased use of antibiotics as prophylaxis. All the isolates (100%) from chicken cloacal swabs were carrying ESBLs which is in agreement with the studies reporting that healthy chickens frequently carry ESBL/AmpC-producing E. coli in their rectum (Dierikx et al., 2013; Reich et al., 2013). As healthy chickens are harbouring ESBLs, prevalence of ESBLs in chicken samples would be expected due to significant faecal contamination during slaughter (Nguyen et al., 2016). Less prevalence of β-lactamases in beef isolates was reported since the average annual consumption of antimicrobials to produce one kilogram of beef meat was low (45mg/1kg meat) when compared to chicken (148mg/1kg meat) and pork (172mg/1kg meat) (Van Boeckel et al., 2015).
Gel photograph showing ESBL and AmpC β-lactamase genes in E. Coli isolates. Lane 1: E. coli isolate with blaTEM (800 bp) gene; Lane 2: E. coli isolate with blaTEM (800 bp) and blaOXA (564 bp) gene; Lane 3: E. coli isolate with blaOXA (564 bp) gene; Lane 4: E. coli isolate with blaCTX-M-2 (404 bp) gene; Lane 5: Negative control; Lane 6: DNA ladder (100 bp); Lane 7: E. coli isolate with blaAmpC DHA (405 bp) gene; Lane 8: E. coli isolate with blaAmpC CIT (462 bp) gene
CONCLUSION
This study revealed the high prevalence β-lactamases especially in STEC isolates (66.66%) isolated from foods of animal origin which can be due to indiscriminate use of antibiotics in food animals. In developing countries like India, where antibiotics can be easily available due to poor monitoring, it is difficult to control the indiscriminate use of antibiotics in food producing animals. So, creating awareness among the livestock rearers about the use of antibiotic alternatives in growth promotion and disease treatment can decrease the use of antibiotics and ultimately results in decreased prevalence of virulent antimicrobial resistant strains.
ACKNOWLEDGMENTS
The authors are thankful to the Science and Engineering Research Board (SERB), Department of Science and Technology (DST), Govt. of India for funding the research project and Sri Venkateswara Veterinary University, Tirupati for providing the facilities to carry out the present study.
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