Extended-spectrum beta-lactamase-producing Escherichia coli from pork in Muang district, Chiang Mai Province, Thailand

Background and Aim: Contaminated pork is one of the transmission routes for pathogens. Extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli is one of the critical threats to global public health. This study aimed to examine pork from different types of markets in Muang district, Chiangmai Province, Thailand, for a proportion of ESBL-producing E. coli, antibiotic resistance of ESBL-producing E. coli and ESBL-producing E. coli genotypes. Materials and Methods: Samples were collected from different market types; fresh markets, pork stores, and supermarkets, enriched and inoculated on selective media. Extended-spectrum beta-lactamases-producing E. coli was identified using double-disk diffusion method according to Clinical and Laboratory Standards Institute 2016. Antibiotic susceptibility test was performed through VITEK® System and ESBL-encoding genes were detected using a multiplex polymerase chain reaction. Results: About 69% of the samples were positive to ESBL-producing E. coli and showed high rates of resistance for ampicillin (100%), piperacillin (100%), cefalexin (100%), cefpodoxime (100%), cefovecin (100%) and ceftiofur (100%), gentamycin (89.86%), and tetracycline (TE) (84.06%). All isolates were multiple drug resistant; resistance patterns of beta-lactams, aminoglycosides, TEs, nitrobenzene derivatives, and sulfonamide groups were observed. The ESBL-producing E. coli-positive isolates carried blaCTX-M groups (100%), blaTEM (98.55%), and blaSHV (1.45%). None of blaOXA was found in this study. Conclusion: Extended-spectrum beta-lactamases-producing E. coli was found in various types of markets; all isolates were detected as multidrug-resistant. The dissemination of such strains can conceivably cause concerning public health, implying that supervised antimicrobial use in pork production and sanitary food preparation is recommended.


Introduction
Pork is one of the most consumed animal products in Thailand and worldwide. Department of Livestock Development (DLD) and National Institute of Development Administration of Thailand estimated that pigs slaughtered for consumption in 2020 to be over 6 million [1]. Consumers commonly come in contact with extended-spectrum beta-lactamases (ESBL)-producing Escherichia coli through contaminated meat, especially pork, from substandard production. The previous study in Chiangmai showed more than 40% of E. coli in pork [2] and swine production chains in the northern part of Thailand, including pigs, farmers, and environment represented more than 50% beta-lactamases-producing E. coli is resistant to various classes of antibiotics, including beta-lactams (except carbapenems and cephamycins), aminoglycosides, and fluoroquinolones, mainly due to the production of blaCTX-M, blaTEM, blaSHV, and blaOXA genes. These pathogens have evolved and increased the transmission of antibiotic-resistant genes among farmers, animals, and animal products in Thailand [12]. Such strains are also found in meat in the Netherlands and United Kingdom [13,14], vegetables in South Korea [15], and ready-to-eat foods in China [16].
Although pork is a common animal product in Thailand, there are relatively less data concerning ESBL-producing E. coli in pork from markets. Consequently, the objective of this study was to investigate the proportion of ESBL-producing E. coli, antibiotic resistance of ESBL-producing E. coli, and ESBL-producing E. coli genotypes. The study aims to raise awareness of antibiotic use in pork production and the dissemination of ESBL-producing E. coli throughout the food chain.

Ethical approval
Ethical approval was not required for this study.

Study period and location
This study was conducted from January 2019 to March 2020 at the biosecurity level-2 facilities of Virology and Molecular Diagnostic Laboratory, Faculty of Veterinary Medicine, Chiang Mai University, Chiang Mai, Thailand.

Sample collection
One hundred samples were collected from three types of markets in Muang, Chiang Mai, Thailand; 15 fresh markets (36 samples), ten pork stores (21 samples), and six supermarkets (43 samples). To illustrate, fresh markets supply fresh meat in community areas; pork stores particularly sell fresh meat from their own farms and supermarkets sell fresh meat from the suppliers with whom they signed contracts. A total of 100 samples were collected by 1-3 samples per shop, stored in a cooler box (4°C), and transported to the laboratory for bacteria isolation within 24 h. The sample collection was conducted from January 2019 to July 2019.

Bacterial isolation and identification
25 g of pork samples was mixed with 225 mL of Luria-Bertani broth (HiMedia, India) in a sterile stomacher bag at 37°C for 24 h. Then, a loopful of suspension was streaked onto MacConkey agar (Merck, Germany) supplemented with cefotaxime 1 mg/L and incubated at 37°C for 24 h [17]. Presumptive ESBLproducing E. coli colonies were selected from each plate and screened using biochemical tests [18]. After that, isolation from each sample was picked to be processed through phenotypic and genotypic tests for ESBL-producing E. coli and antimicrobial susceptibility test.
Phenotypic ESBL-producing E. coli testing Double-disk diffusion method was conducted for ESBL detection. Mueller Hinton agar (HiMedia) was swabbed using a suspension of a pure culture (0.5 McFarland Standard). Cefotaxime (30 µg) disks (Oxoid, Germany), cefotaxime with clavulanic acid (30 µg/10 µg) disks (HiMedia), ceftazidime (30 µg) disks (Oxoid), and ceftazidime with clavulanic acid (30 µg/10 µg) disks (HiMedia) were loaded on the agar. Each antimicrobial agent combined with clavulanate was tested in comparison with the same agent without clavulanate. The agent with clavulanate combination with the increase in zone diameter measurement of more than 5 mm compared to the one without clavulanate is confirmed ESBL phenotypic [19].

Statistical analysis
The data were analyzed using R software version 4.0.3 (R development Core Team, R Foundation for Statistical Computing, Vienna, Austria). The unit of analysis was ESBL-producing E. coli isolations. Descriptive statistics were used to explain the proportions of positive ESBL-producing E. coli, the patterns of antimicrobial susceptibility, and ESBL-producing E. coli genes. Chisquare statistics and Fisher's exact test were conducted to compare the proportions of positive ESBL-producing E. coli among three particular market types and represented positive ESBL-producing E. coli genes which were statistically significant test results (p ≤ 0.05).
All isolates were multiple drug resistant, showing resistance to at least six antibiotics. Patterns of antibiotic resistance were significantly detected in beta-lactams, aminoglycosides, TEs, nitrobenzene  Table-2). Considering all market types, the results showed the highest resistance (100%) to AM, PIP, CN, CPD, CFO, and CFT. Following high resistance rates were GM and TE (>80.0%). Supermarket and pork store isolates showed high resistance to GM, C, and trimethoprim/SXT (>70.00%). However, the isolates from supermarkets did not show resistance to TM and ones from pork stores did not show resistance to FT (Figure-2).   Since the results showed roughly the same proportions of blaCTX-M and blaTEM genotypes among the three market types, statistical significance was not different (p = 1). In addition, even though the proportion of blaCTX-M-9 was smaller than blaC-TX-M-1, the result did not represent a statistically significant difference between the three markets (p = 0.25 and 0.54, respectively). One isolation from fresh markets was found carrying blaSHV (1.45%), without blaOXA detection (Table-3).

Distribution of genotypic ESBL-producing E. coli
The distribution patterns of the ESBL-producing E. coli genotypes are shown in Table-4. The coexistence of blaCTX-M and blaTEM was highly observed (97.10%). blaCTX-M, blaTEM, and blaSHV showed the least common coexistence (1.45%). The frequency of the distribution patterns of blaCTX-M groups was high in blaCTX-M-1 (89.86%), followed by blaCTX-M-9 (5.80%). While, the coexistence of blaCTX-M-1 and blaCTX-M-9 was 10.15%. The distribution patterns of genotypic ESBL-producing E. coli among fresh markets, pork stores, and supermarkets did not show a statistically significant difference (p = 1).

Discussion
The study revealed that the proportion of ESBLproducing E. coli in pork from markets (69%), which was higher than the previous studies in China (11.76%) [16], United Kingdom (<7%) [14], and Thailand (>50%) [12]. Moreover, the previous studies concerning swine production chain, including pigs, farmers, and the environment in the northern Available at www.veterinaryworld.org/Vol.15/December-2022/19.pdf Table- The distribution patterns of the ESBL-producing E. coli genotypes from pork did not show statistically significant difference among fresh markets, pork stores, and supermarkets (p = 1). E. coli=Escherichia coli, ESBL=Extended-spectrum beta-lactamases part of Thailand represented more than 50% of ESBLproducing E. coli [3,4]. Foods from animal production are presented as a reservoir of ESBL-producing E. coli [22]. The results of the study emphasized the increasing risk of transmission in contaminated food chain and more outbreaks of the resistant pathogen. Extended-spectrum beta-lactamases-producing E. coli-positive results were found in supermarkets less than in fresh markets and pork stores due to more strict swine audit process. Suppliers have to be certified by DLD before providing pork products to supermarkets. The assessments include farm systems, slaughterhouse control, pork quality, and retail management [23][24][25][26]. In contrast, some shops in fresh markets are not required to obtain any certificates for the particular assessments. Besides, the sales pattern of pork meat in fresh markets basically involves middlemen who directly purchase pork at farms and sell it to vendors in markets, farmers who raise pigs, slaughter it for meat, and sell it on their own. In addition, pork stores supply fresh meat particularly produced by their own farms on their own slaughterhouse evaluation and selling process. This, as a result, can cause non-standardized pork assessment on the ground that consumers prefer buying pork at fresh markets to the other market types. It is evidently due to relatively cheaper prices, easier ways to buy, more accessible locations as well as acquaintanceship with the venders. In other words, pork quality is the secondary reason for most customers [27,28]. Local people in the north of Thailand, especially, those who prefer the traditional raw pork menu, take relatively high risks of exposure to resistant antibiotics. This can lead to more serious infections associated with the pathogen, more complicated antibiotic treatment, and higher medical expense. Due to the circumstances mentioned, it is important that we, as part of the food chain, take these facts into consideration for a health advantage. All ESBL-producing E. coli were detected as multidrug-resistant. The positive ESBL (100%) were resistant to beta-lactam antibiotic groups, followed by gentamicin which was widely used in animal production in Thailand [3]. Evidently, inappropriate use of antibiotics can lead to a high AMR rate. The resistance to beta-lactam antibiotic groups implies a challenge for treating diseases caused by ESBL-producing E. coli. Moreover, it results in critical public health as it is associated with morbidity, mortality, and limited alternatives of treatment. Over 50% of the bacteria with high resistance to TE and C groups were found, even though, interestingly, TE has been restricted since 2003 [29] and C has been prohibited since 2002 [30] in animal production. A previous study in Thailand also reported high levels of TE and C in swine farms, including pigs, farmers, and environment. In addition, coexistence of resistance between antibiotic classes was observed. Multiple drug resistance has become a major problem for clinical therapeutics for antibiotic-resistant genes that can potentially spread through horizontal gene transfer from commensal to environmental species [8]. However, this study showed susceptibility (100%) of the ESBL-producing E. coli to IPM. A previous study in China found ESBL-producing E. coli (95%) susceptive [16] and another study in Available at www.veterinaryworld.org/Vol. 15/December-2022/19.pdf Thailand also reported susceptibility (over 98%) of ESBL-producing E. coli [19]. Therefore, IPM could be a potential drug choice to cure ESBL-producing E. coli-associated infections.
blaCTX-M group was predominantly detected in this study, including blaCTX-M-1 with the highest rate, followed by blaCTX-M-9. Worldwide, previous studies also reported the particular group of genes in humans, animal farms, and foods [3,17,19,31]. However, a study in China found blaOXA (28.57%) in retailed fresh pork [16], which was not detected in this study. Evidently, genotypic ESBL-producing E. coli commonly vary in different countries and regions because of different antibiotic policies. The coexistence of different genes within the same isolation was noticed; the most common genotypic pattern was the combination of blaCTX-M and blaTEM. This can lead to enzyme combination issues. Moreover, single isolates with multiple blaC-TX-M can possibly cause more stubborn infections. Therefore, it is necessary that pork production should be responsibly monitored throughout the process because ESBL-producing E. coli are not only disseminated in animals but also in foods, humans, and the environment.

Conclusion
Pork meat can be prepared in various ways and consumed either cooked or raw. A high percentage of ESBL-producing E. coli was found in pork from various market types. In addition, all the isolates were multiple drug-resistant. The predominant genotype was blaCTX. Apparently, contaminated pork is one of the transmission routes for the pathogen to food chain. In summary, rational use of antimicrobial drugs, along with choosing pork from reliable sources and hygienic cooking process, are recommended to be practiced with stewardship by every involved unit, from producers to consumers, for the advantage of food safety as well as public health.