Molecular and antibiogram analysis of Shiga toxin-producing E. coli in swine farms

Introduction

Shiga toxin-producing Escherichia coli (STEC) is responsible for numerous outbreaks of gastrointestinal diseases, including haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS), resulting in diarrhoea in both humans and animals, thus posing a significant threat to public health. Swine can serve as reservoirs for STEC and transmission to humans can occur through direct contact, environmental contamination, or consumption of contaminated products (Fratamico et al. 2008, Rajkhowa and Sarma 2014) [1,2] . Due to close interactions between farmers, livestock, and the farm environment, foodborne diseases caused by multidrug-resistant (MDR) STEC strains has a significant global public health concern. Limited information is available on epidemiology of STEC in pig farms, pig farmers/handlers and pig farm environment in India.

Materials and Methods

Sample collection

The study collected 550 samples from ten organized swine farms (Table-1), which included three samples each of feed and water, as well as 15 oral swabs, 15 rectal swabs, and 15 fecal samples representing three developmental stages: piglets, weaners, and adults from each farm. Additionally, one nasal swab, one stool sample, one urine sample, and one hand swab from pig farmers were collected from each farm. All samples were aseptically collected using cotton swabs and then transferred to the laboratory for analysis.

Isolation and identification of E. coli

The swabs were enriched in Tryptone Soy Broth (TSB) and incubated at 37 °C for 24 h. After enrichment, the samples were streaked onto MacConkey agar and incubated at 37 °C for 18-24 h, where lactose-fermenting colonies were subcultured onto Eosin Methylene Blue (EMB) agar to observe the characteristic metallic sheen. Pure colonies were selected, inoculated onto nutrient agar slants for biochemical tests and then prepared for DNA extraction through boil and snap chilling method. Presumptive colonies of E. coli were confirmed molecularly by E.

Detection of virulence genes

All the E. coli isolates were tested for haemolytic activity and biofilm formation on blood agar supplemented with 5% sheep blood and Congo red agar, respectively (Katongole et al., 2020) [4] . All the confirmed E. coli isolates were screened for STEC-associated virulence genes using four set of primers i.e., stx1, stx2, eaeA and hlyA (Table -2) by multiplex PCR (m-PCR). The cyclical conditions were followed according to Paton and Paton (1998) [5] . The PCR products were subjected for 2% agarose gel electrophoresis with ethidium bromide dye and visualized by gel documentation system (BIORAD).

Molecular detection of E. coli O157

The confirmed STEC isolates were cultured on CT-SMAC (Sorbitol Mac Conkey Agar supplemented with Cefixime Tellurite) agar and incubated at 37℃, 24h. The sorbitol nonfermenting E. coli O157 isolates were then subjected for PCR analysis (Desmarchelier et al. 1998) [6] .

Antimicrobial resistance profile

All the confirmed STEC isolates were subjected to in-vitro antibiotic sensitivity test, carried out by Kirby Bauer disc diffusion method to assess the resistance/sensitivity patterns against 12 different antimicrobial drugs like amikacin (30 µg), nitrofurantoin (5 µg), novobiocin (10 µg), cefazolin (30 µg), co-trimoxazole (25 µg), ciprofloxacin (5 µg), chloramphenicol (30 µg), colistin (10 µg), piperacillin/tazobactum (100/10 µg), imipenem (10 µg), tigecycline (15 µg) and tetracycline (30 µg). A standardized inoculum (0.5 Mc Farland) was spread on Mueller Hinton agar (MHA) and the diameter of the inhibition zones was measured and isolates were interpreted as sensitive, intermediate resistant and resistant based on Clinical Laboratory Standards Institute (CLSI 2021) [7] .

Molecular detection of β-lactamase genes

All the positive STEC isolates were subjected to a m-PCR assay (bla TEM , bla SHV and bla OXA ) and one uniplex PCR assay (bla AmpC ) which were standardized for the detection of βlactamase genes (Table-2) in STEC isolates. The cyclical conditions for m-PCR and uniplex PCR assay were followed according to Dallenne et al. 2010 [8] and Manoharan et al. 2012 [9] , respectively. The PCR products were subjected for 1.5% agarose gel electrophoresis with ethidium bromide dye and visualized by gel documentation system (BIORAD).

Results and Discussion

Based on cultural and biochemical characteristics, 65.81% (362/550) of samples were identified as E. coli, with 45.09% (248/550) confirmed by PCR (Fig. 1). This finding is consistent with earlier studies by Begum et al. (2014) [10] and VinodhKumar et al. (2019) [11] , which reported E. coli prevalence rates of 59.33% and 58.40%, respectively, in pigs.

In this study, we assessed two virulence factors such as haemolytic activity and biofilm formation, using blood agar and Congo red agar, respectively. Haemolysis, a key pathogenic feature of STEC, is linked to Shiga toxin production, contributing to conditions like bloody diarrhea and HUS. We found 1.61% (4/248) of E. coli isolates exhibited haemolytic activity on sheep RBC, which contrasts with Meng et al. (2014) [12] and Baranzoni et al. (2016) [13] who reported 15% and 7%, respectively. The discrepancy of results may be due to methodological variations, geographical and environmental factors. The ability to form biofilm was assessed using Congo red agar, where 33.21% (92 out of 248) of the E. coli isolates exhibited black crystalline colonies. In contrast, Dimitrova et al. (2021) [14] reported a significantly higher percentage 93.75% of E. coli isolates displaying biofilm activity when evaluated using the microtiter plate assay. This discrepancy could be attributed to the lower specificity of the Congo red agar method compared to the microtiter plate assay. Through m-PCR, 30 isolates (12.09%, 30/248) were positive for any one of the virulence genes (Fig. 2). Among the 30 STEC isolates, stx2 (93.33%, 28/30) was the predominant gene, followed by hlyA (13.33%, 4/30) and stx1 and eaeA (6.66%, 2/30 each). [10] and Kataria et al.

(2014) [15] . The Stx2 gene is more predominant because it is associated with higher virulence and is more cytotoxic to endothelial cells. STEC genes were exclusively identified in four swine farms, namely farms B, D, E and H. The occurence of STEC genes were highest in farm D (40%, 12/30) followed by farm E (33.33%, 10/30), farm B (16.66%, 5/30) and farm H (10%, 3/30). The high occurrence in some farms may be due to the lack of hygiene practices, biosecurity measures, manure management and high population density. Among the different samples, the occurence of STEC was highest in feed sample (100%, 1/1), followed by water sample (18.21%, 2/11), rectal swabs (16.88%, 13/77) and faecal samples (15.21%, 14/92) of E. coli isolates. In water samples, STEC showed a prevalence rate of 18.21%. However, lower prevalence rates were reported by Puii et al. (2019) [16] and Kobayashi et al. (2003) [17] , at 4.4% and 2.02%, respectively. This variation could be attributed to differences in environmental conditions, water treatment methods, and agricultural practices. Among the different developmental stages, the average rate of STEC prevalence was highest in adults (16.90%; 12/71) followed by weaners (12.98%; 10/77) and piglets (6.94%; 5/72) of E. coli isolates. These observations were coincided with Baldo et al. (2020) [18] who isolated more stx2e-producing E. coli isolates in healthy adult pigs rather than weaners and piglets. Similarly, Fratamico et al. (2004) [19] and Remfry et al. (2020) [20] isolated STEC in faecal samples at the rate of 70% and 29.7% in finisher pigs, respectively. The high prevalence in adults may be due to the influence of environment stressors, management practices and dietary factors in adults. STEC was not detected in swine oral swabs, pig farmer stool and urine sample, hand and nasal swab. Among the 30 STEC isolates, 10 (33.33%) were identified as E. coli O157 using PCR (Fig. 3). Overall E. coli O157 prevalence was 4.03% (10/248) which was in slight agreement with the results of Keen et al. (2006) [21] who reported the prevalence of 1.2%. On the contrary, Fratamico et al. (2008) [1] isolated none from finisher pigs. The variation in E. coli O157 prevalence in pigs across different locations could be attributed to disparities in surveillance and detection methods, as well as differences in husbandry practices and geographical locations. Highest resistance was observed against cefazolin and cotrimoxazole (100%; 30/30 each) followed by tetracycline, nitrofurantoin and novobiocin (93.3%; 28/30 each), colistin (90.0%; 27/30), chloramphenicol (70.0%; 21/30), ciprofloxacin (50.0%; 15/30), amikacin (33.3%; 10/30). STEC isolates showed highest sensitivity against imipenem and tigecycline (96.6%; 29/30 each) followed by piperacillin/tazobactum (66.6%; 20/30). In the present study, the resistance towards co-trimoxazole (100%) and tetracycline (93.3%) which were in slight agreement with the findings of Baldo et al. (2020) [18] who reported the resistance towards tetracycline (98.25%) and co-trimoxazole (82.17%) and Meng et al. (2014) [12] who reported the resistance rates of 79.57% and 73.12% to co-trimoxazole and tetracycline, respectively. Resistance rates of novobiocin (93.3%) and amikacin (33.3%) were in line with the observations of Kataria et al. (2014) [15] who reported the resistance of 100% and 40% to novobiocin and amikacin, respectively. It may be due to the excessive use of antibiotics in the swine industry for therapeutic and growth promoting purposes. All the 30 STEC isolates (100%) showed MDR. The high prevalence of MDR bacteria in pigs can be attributed to the fact that pigs are often reared in intensive farming environments, where antibiotics are extensively utilized for purposes such as promoting growth, preventing diseases and providing treatment. In the current investigation, the MAR index of all MDR STEC isolates (100%) exceeded 0.2, while Vinodh Kumar et al. (2019) reported that 13.75% of isolates revealed MAR index > 0.2 and 2.64% isolates had MAR of 1.0. The higher percentage of MDR STEC isolates with MAR index > 0.2 in the current study, might be due to non-judicious use of antibiotics for longer period in both animals and humans in the study area. All the eight confirmed ESBL producers were subjected to m-PCR for detection of ESBL genes, where blaSHV was the only gene detected in two isolates, one from faecal sample of weaner obtained from farm D (DFW1) and other from faecal sample of weaner obtained from farm E (EFW3) (Fig. 4). The findings align with Puii et al. (2019) [16] noted a higher incidence of bla SHV (6.03%) in piglets compared to other βlactamase genes. However, bla TEM was the predominant gene in many studies (Vinodh Kumar et al. 2019, Tamta et al. 2020 [22] and Naidu et al. 2021) [23] . In the current study, none of the STEC isolates were positive for blaTEM, blaOXA genes. In uniplex PCR assay, six isolates were positive for blaAmpC gene (Fig. 5). STEC infections in swine can lead to various clinical manifestations, including diarrhea, dehydration, edema disease and in severe cases, mortality. This not only compromises animal welfare but also raises economic implications for swine producers due to decreased productivity and increased veterinary costs. In the current study, the presence of STEC was confirmed in pig farms, farm environment and pork. Pigs can be a source of STEC which poses a potential public health risk as an emerging foodborne pathogen, with zoonotic potential. Moreover, our study revealed high antibiotic resistance patterns in STEC isolates and were MDR, a threat to both animal and public health which may provide a vector for the spread of these opportunistic pathogen.

Acknowledgement

Authors would like to express grattitude to the Sri Venkateswara Veterinary University, Tirupathi for conducting the research.

Financial Support Not available

References

1. Fratamico PM, Bhagwat A, Injaian L, Fedorka-Cray PJ. Characterization of Shiga toxin-producing Escherichia coli strains isolated from swine feces. Foodborne Pathogens and Disease. 2008;5(6):827-838.
2. Rajkhowa S, Sarma DK. Prevalence and antimicrobial resistance of porcine O157 and non-O157 Shiga toxinproducing Escherichia coli from India. Tropical Animal Health and Production. 2014;46:931-937.
3. Russell DW, Sambrook J. Molecular cloning: A laboratory manual. Vol. 1. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory; 2001. p. 112.
4. Katongole P, Nalubega F, Florence NC, Asiimwe B, Andia I. Biofilm formation, antimicrobial susceptibility and virulence genes of uropathogenic Escherichia coli isolated from clinical isolates in Uganda. BMC Infectious Diseases. 2020;20:1-6.
5. Paton AW, Paton JC. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfb O111 and rfb O157. Journal of Clinical Microbiology. 1998;36(2):598-602.
6. Desmarchelier PM, Bilge S, Fegan N, Mills L, Vary JC Jr, Tarr PI. A PCR specific for Escherichia coli O157 based on the rfb locus encoding O157 lipopolysaccharide. Journal of Clinical Microbiology. 1998;36(6):1801-1804.
7. Clinical and Laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing. CLSI supplement M100. 31st ed. Wayne (PA): CLSI; 2021.
8. Dallenne C, Da Costa A, Decre D, Favier C, Arlet G. Development of a set of multiplex PCR assays for the detection of genes encoding important beta-lactamases in Enterobacteriaceae. Journal of Antimicrobial Chemotherapy. 2010;65(3):490-495.
9. Manoharan A, Sugumar M, Kumar A, Jose H, Mathai D, ICMR-ESBL Study Group. Phenotypic and molecular characterization of AmpC beta-lactamases among Escherichia coli, Klebsiella spp and Enterobacter spp. from five Indian medical centres. Indian Journal of Medical Research. 2012;135(3):359-365.
10. Begum S, Hazarika GC, Rajkhowa S. Prevalence of Escherichia coli from pigs and cattle. Journal of Animal Health and Production. 2014;2(3):38-39.
11. VinodhKumar OR, Singh BR, Sinha DK, Pruthvishree BS, Tamta S, Dubal ZB, et al. Risk factor analysis, antimicrobial resistance and pathotyping of Escherichia coli associated with pre-and post-weaning piglet diarrhoea in organised farms, India. Epidemiology and Infection. 2019;147:e174.
12. Meng Q, Bai X, Zhao A, Lan R, Du H, Wang T, et al. Characterization of Shiga toxin-producing Escherichia coli isolated from healthy pigs in China. BMC Microbiology. 2014;14(1):1-14.
13. Baranzoni GM, Fratamico PM, Gangiredla J, Patel I, Bagi LK, Delannoy S, et al. Characterization of Shiga toxin subtypes and virulence genes in porcine Shiga toxin-producing Escherichia coli. Frontiers in Microbiology. 2016;7:574.
14. Dimitrova L, Kaleva M, Zaharieva M, Stoykova C, Tsvetkova I, Angelovska M, et al. Prevalence of antibiotic-resistant Escherichia coli isolated from swine faeces and lagoons in Bulgaria. Antibiotics. 2021;10(8):940.
15. Kataria JL, Dutta TK, Roychoudhury P, Tiwari JG. Detection and molecular characterization of Shiga toxin producing Escherichia coli and enteropathogenic Escherichia coli from piglets in Mizoram, India. Journal of Pure and Applied Microbiology. 2014;8:2383-2388.
16. Puii LH, Dutta TK, Roychoudhury P, Kylla H, Chakraborty S, Mandakini R. Extended spectrum betalactamase producing Shiga toxin-producing Escherichia coli in piglets, humans and water sources in North East region of India. Letters in Applied Microbiology. 2019;69(5):373-378.
17. Kobayashi H, Khai LTL, Yamasaki S, Taniguchi T. Prevalence of pathogenic Escherichia coli in a swine breeding environment in Can Tho province, Vietnam. Japan Agricultural Research Quarterly. 2003;37(1):5963.
18. Baldo V, Salogni C, Giovannini S, D'Incau M, Boniotti MB, Birbes L, et al. Pathogenicity of Shiga toxin type 2e Escherichia coli in pig colibacillosis. Frontiers in Veterinary Science. 2020;7:545818.
19. Fratamico PM, Bagi LK, Bush EJ, Solow BT. Prevalence and characterization of Shiga toxin-producing Escherichia coli in swine feces recovered in the National Animal Health Monitoring System's swine 2000 study. Applied and Environmental Microbiology. 2004;70:7173-7178.
20. Remfry SE, Amachawadi RG, Shi X, Bai J, Woodworth JC, Tokach MD, et al. Polymerase chain reaction-based prevalence of serogroups of Escherichia coli known to carry Shiga toxin genes in faeces of finisher pigs. Foodborne Pathogens and Disease. 2020;17(12):782-791.
21. Keen JE, Wittum TE, Dunn JR, Bono JL, Durso LM. Shiga-toxigenic Escherichia coli O157 in agricultural fair livestock, United States. Emerging Infectious Diseases. 2006;12(5):780-783.
22. Tamta S, VinodhKumar OR, Pruthvishree BS, Karthikeyan R, Rupner RN, Chethan GE, et al. Faecal carriage of extended spectrum beta-lactamase (ESBL) and New Delhi metallo beta-lactamase (NDM) producing Escherichia coli between piglets and pig farmworkers. Comparative Immunology, Microbiology and Infectious Diseases. 2020;73:101564.
23. Naidu ST, Bodempudi B, Chinnam BK, Pedada VC, Nelapati S, Tumati SR, et al. Prevalence of betalactamase producing Shiga toxigenic Escherichia coli (STEC) in retail meats and chicken cloacal swabs. Journal of Animal Research. 2021;11(2):263-271.