Antimicrobial resistance pattern of extended-spectrum β-lactamase-producing Escherichia coli isolated from fecal samples of piglets and pig farm workers of selected organized farms of India

Background and Aim: Extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are gradually increasing worldwide and carry a serious public threat. This study aimed to determine the antimicrobial resistance pattern of ESBL-producing E. coli isolated from fecal samples of piglets and pig farm workers. Materials and Methods: Fecal samples from <3-month-old piglets (n=156) and farm workers (n=21) were processed for the isolation of ESBL-producing E. coli in MacConkey agar added with 1 µg/mL of cefotaxime. E. coli (piglets=124; farm workers=21) were tested for ESBL production by combined disk method and ESBL E-strip test. Each of the ESBL-positive isolate was subjected to antibiotic susceptibility testing. The ESBL-producing E. coli were further processed for genotypic confirmation to CTX-M gene. Results: A total of 55 (44.4%, 55/124) and nine (42.9%, 9/21) ESBL-producing E. coli were isolated from piglets and farm workers, respectively. Antibiotic susceptibility testing of the ESBL-positive E. coli isolates from piglets and farm workers showed 100% resistance to ceftazidime, cefotaxime, cefotaxime/clavulanic acid, ceftazidime/clavulanic acid, and cefpodoxime. A proportion of 100% (55/55) and 88.9% (8/9) ESBL-positive E. coli were multidrug resistance (MDR) in piglets and farm workers, respectively. On genotypic screening of the ESBL E. coli isolated from piglets (n=55), 15 were positive for the blaCTX-M gene and of the nine ESBL E. coli from farm workers, none were positive for the blaCTX-M gene. Conclusion: Although there was no significant difference in isolation of ESBL-producing E. coli between piglets and farm workers, the ESBL-positive E. coli from piglets showed relatively higher MDR than farm workers.


Introduction
Treating infections associated with extended-spectrum β-lactamase (ESBL)-producing Escherichia coli are becoming difficult since they are capable of hydrolyzing penicillin, broad-spectrum cephalosporins, and monobactams and are often resistant to other antimicrobial classes such as fluoroquinolones, aminoglycosides, and trimethoprim-sulfamethoxazole [1]. The general trend of antibiotic resistance around the world indicates that an overall decline in the total stock of antibiotic effectiveness [2].
In the livestock sector, antibiotics are not only used for the treatment of infections but also for disease prevention and growth promotion [3]. India is one of the largest consumers of antibiotics in the world with 13 billion standard units in 2010 [2]. In India, human and animals live in close proximity which increases the risk of contamination and resistance between them and 95% of adults in India shows β-lactam antimicrobials resistance in Gram-negative bacteria [4,5]. Reports suggest the possibility of transferring ESBL-producing E. coli from farm workers to food animal and vice versa [6][7][8][9][10]. Multidrug-resistant E. coli are reported in food animals and pet animals of India [11][12][13][14][15]. Besides, food animals may act as a reservoir for ESBLproducing strains and foods as a vehicle for the transfer of β-lactam-producing bacteria [16].
This study was conducted to determine the antimicrobial resistance (AMR) pattern of ESBLproducing E. coli isolated from fecal samples of piglets and pig farm workers.

Ethical approval
Ethical approval was not required for this study. Available at www.veterinaryworld.org/Vol.13/February-2020/19.pdf

Sample collection
A cross-sectional study was conducted between August 2016 and May 2017 to sample five government organized pig farms covering three states, namely, Uttar Pradesh, Karnataka, and Tamil Nadu. The sample was collected from five different organized pig farms, namely, Aligarh (41), Bareilly (30), Chennai farm 1 (28), Chennai farm 2 (21), and Hassan (36). The fecal samples were aseptically collected with the help of fecal swab (HiMedia, Mumbai, India) directly from piglet's rectum and in case of piglet farm workers, swabs were distributed to the farmers which were collected in the next morning (n=21) and transported to the laboratory under cold chain for the isolation of ESBL-producing E. coli.

Genotypic detection of bla CTX-M gene
The genomic DNA was extracted from ESBL-positive isolates using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and the polymerase chain reaction (PCR) for bla CTX-M (forward: CAATGTGCAGCACCAAGTAA; reverse: CGCGA TATCGTTGGTGGTG) was carried out in a final reaction volume of 25 μL [11]. The amplified PCR product was visualized by a gel documentation system (UVP, UK) after electrophoresis in 1.5% (w/v) agarose gel containing ethidium bromide (0.5 µg/mL, Loba Chemie, India). The positive PCR amplicons were sent to commercial sequencing services (Eurofins Ltd., Bangalore) for further purification and sequencing by Sanger method. The homology searches were made using the BLAST algorithm available at http://blast. ncbi.nlm.ni.gov/Blast.cgi, and the representative sequences were submitted to GenBank for accession number.

Statistical analysis
The association of the isolation of ESBL-producing E. coli between piglets and farm workers was tested by Chi-square test/Fisher's exact (two-tailed) using SPSS version 20.0 statistical software (IBM Corp., Armonk, NY, USA).

Discussion
The emergence and spread of ESBLs producing E. coli in food-producing animals is a major public health issue worldwide [11][12][13][14][15]. Piglets and farm workers harbored ESBL E. coli, the percentage of cefotaxime and ceftazidime resistance is higher among isolates as compared to ciprofloxacin and chloramphenicol indicates the abundant use of beta-lactam and cephalosporin antimicrobials in piglets and human [5,15]. A recent study documented that the occurrence of ESBL-positive E. coli in pigs was not related to total antimicrobial use, but associated with the presence or absence of cephalosporin use at the farm [10]. The current investigations showed that once the bacteria are on the farm or in the farm environment may be widely spread among animals (including insects and rodents) and can reach the environment through manure [19,20]. In this study, the ESBL-producing E. coli showed resistance to clinically important antibiotics such as cephalosporins, fluoroquinolones, and aminoglycosides and jeopardize the effective prevention and treatment of various bacterial infections [21]. The present study revealed that all the ESBL-producing E. coli were 100% resistant to cefotaxime and ceftazidime and were concordance with others [11][12][13]. This study reported that the entire ESBL-positive E. coli from piglets as MDR, similarly, in a study from China reported MDR phenotype in total ESBL-producing E. coli isolated from pigs [22]. AMR rate depends on the factors around the farm, human behavior, improper sanitation, and hygiene [23]; the improper antibiotic use with route of administration is important for multidrug-resistant bacteria. This study revealed lower resistance for ciprofloxacin, tetracycline, amikacin, and chloramphenicol compared with other antibiotics. The difference might be linked with the overall decline in the use of these antibiotics in India since 2000 [24]. It is also observed that ESBL producers also show resistance to non-β-lactam antimicrobials, such as fluoroquinolones, aminoglycosides, and sulfonamides [22]. The study noticed the CTX-M genotype in ESBL-positive isolates, similar findings were reported in ESBL-producing enterobacterials which might indicate rapid dissemination of bla CTX-M genes [25][26][27]. In ESBL E. coli, the most commonly identified enzymes are CTX-M family [28].

Conclusion
The study highlights the AMR pattern of ESBLproducing E. coli isolated from fecal samples of piglets and farm workers. The ESBL-positive E. coli from piglets showed relatively higher MDR than farm workers. Hence, necessary steps are needed to reduce the use of antimicrobials in pig farming to decrease the AMR.