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Phenotypic and molecular characterization of ESBLs producing Escherichia coli in bovine faecal and milk samples of North Gujarat

Extended-spectrum β -lactamases (ESBLs) producing E. coli seems to be emerging in veterinary science impacting major threat to public health due to resistance to golden age antibiotics. In this study a total of 109 samples (42 faecal and 67 mastitis milk) of bovines were collected from different regions of North Gujarat. The samples were cultured and identified by standard procedures. The screening for ESBLs production was performed by using Cefotaxime and Cefotaxime+Clavulanate (Combination disc screening method). A total of 71 E. coli isolates were recovered from 109 samples processed, out of which thirty (42.25%) isolates (17 from milk and 13 from faecal) were positive for ESBLs showing multiple resistance to the antibiotics used. The ESBL confirmed isolates were further processed for detection of bla CTX-M, bla TEM, and bla SHV genes. Major gene detected was bla TEM in 17 (23.94%) E. coli isolates. Antibiotic resistance pattern of E. coli isolates was studied against eleven commonly used antimicrobial drugs in the northern region of Gujarat. The results recorded resistance tofollowing antibiotics: tetracycline (100%), ampicillin/sulbactum (83.10%), amoxiclav and gentamicin (83.10%), chloramphenicol (57.74%), ceftriaxone (66.19%), cefoperazone (66.19%), ciprofloxacin (74.65%), amikacin (57.74%), enrofloxacin (74.65%) and, levofloxacin (74.65%).

in particular E. coli has emerged globally. Initially, ESBL producing bacteria were only observed in human medicine (Olowe et al. 2012, Ogbolu et al. 2013) but the occurrence of these bacteria in companion animals firstly and increasingly in livestock nowadays, has initiated monitoring studies more focused on livestock (Smet et al. 2010). It has been documented that food-producing animals are important reservoirs for ESBL-producing Enterobacteriaceae (Carattoli 2008, EFSA 2011, Smet et al. 2010. Recent studies raised severe concerns about the role of farm animals, especially dairy cattle, on dissemination of ESBLproducing Enterobacteriaceae and/or their mobile genetic elements via the food chain (Brinas et al. 2002, Aarestrup et al. 2006, Meunier et al. 2006, Dahmen et al. 2013, Liebana et al. 2013, Schmid et al. 2013. ESBL determinants have been detected not only in clinical isolates but also in commensal bacteria from humans and, animals and in isolates from products of the food chain and sewage, revealing distribution and suggesting the presence of environmental reservoirs for these resistance determinants (Brinas et al. 2002).
Currently, there is limited information available on ESBL producing E. coli in veterinary science and the possible contribution. The objective of the present study was to detect ESBL producing E. coli in the faecal and milk samples of bovines from different regions of North Gujarat.

MATERIALS AND METHODS
A total of 109 samples (42 faecal and 67 mastitic milk) were collected from bovines into sterile capped universal bottles with sterile spatula and were transported to the laboratory immediately. Isolation of E. coli isolates were carried out by culturing of samples on MacConkey's agar and incubated for 18-24 h at 37°C. The identification of E. coli was carried out by culturing on eosin methylene blue agar and incubated for 18-24 h at 37°C. Presumptive characteristic of E. coli isolates were identified and confirmed using arrays of biochemical tests (IMViC). The study was carried from August, 2017 to October, 2018 in the Department of Veterinary Public Health and Epidemiology, College of Veterinary Science and Animal Husbandry, SDAU, Sardarkrushinagar.
Phenotypic detection of ESBL production is done by combination disc screening method. Cefotaxime and cefotaxime+clavulanate (Hi Media, INDIA) discs were applied on freshly prepared Muller Hinton agar and the plates were further incubated aerobically at 37°C. A final measurement of the zone of inhibition was made after overnight incubation. ESBL producers were defined as having a differential zone diameter of ≥+5 mm (Cefotaxime+clavulanate zone -cefotaxime zone).
The DNA of the ESBL confirmed isolates by combination disc screening method was extracted separately using a DNA extraction kit (Qiagen, Germany). Molecular confirmation of ESBL producing E. coli was done by detection of blaCTX-M, blaTEM, and blaSHV genes by multiplex polymerase chain reaction (m-PCR) as described by Monstein et al. (2007) with some modifications using specific primers. The recommended oligonucleotide primers specific for the blaSHV, blaTEM, and blaCTX-M genes used in the m-PCR assay and expected amplicon sizes are depicted in Table 1.

RESULTS AND DISCUSSION
In the present study, out of 109 samples, 71 (65.13%) isolates were identified as E. coli, giving greenish metallic sheen on EMB agar ( Fig. 1) and through biochemical characterization. The highest prevalence of E. coli was observed in faecal samples (83.33%) followed by mastitic milk samples (53.73%).
All the 71 E. coli isolates that were subjected to antimicrobial testing (Fig. 2)  other antibiotics. Comparatively resistance to chloramphenicol (C) (57.74%) was recorded to be lower.
Antibiotic resistance is an emerging threat in human medicine as well as in veterinary medicine (Witte 1998, Lawson 2008. Antibiotic resistance has put deleterious effect on livestock disease management thus in turn on the productivity of the animals.
Recently, there has been increased resistance to beta lactam antibiotics due to plasmid mediated acquisition of ESBL producing genes predominantly by E. coli. Although most of the studies conducted on ESBL producers are confined to human beings within the hospital premises, focus should also be given on its occurrence and dissemination in food producing animals. Reports by several researchers also indicated its presence in chicken meat and raw milk (Geser et al. 2012, Lyhs et al. 2012.
Phenotypic detection of ESBL producing E. coli is detected in 30 (42.25%) isolates (17 from mastitis milk and 13 from faecal samples) out of the 71 isolates by combination disc screening method (Fig.4). Similarly, higher prevalence of 71% ESBLs was recorded by Olugbenga et al. (2015) from bovine faecal samples. All the 30 ESBL producers were resistant (100%) to ceftriaxone, ampicillin/sulbactum, amoxiclav, cefoperazone, ciprofloxacin, chloramphenicol, amikacin, enrofloxacin, levofloxacin, gentamicin and tetracycline as revealed by disc diffusion assay. These observations reiterate the findings in other studies that have reported increased antibiotic resistance amongst different bacterial species especially E. coli isolated from cattle and other animals at an alarming rate (Kozak et al. 2009, Ajayi et al. 2011. The conclusive finding in the present study is multiple drug resistant against commensal E. coli in bovines to most commonly used antibiotics such as tetracycline (100%), ampicillin/sulbactum (83.10%), amoxiclav (83.10%), and gentamicin (83.10%). This finding correlates with similar results obtained fromother studies that have reported some levels of multiple antibiotic resistance by E. coli from cattle, meat products and other animals (Saenz et al. 2001, Umolu et al. 2006. Chromosomal AmpC βlactamase hyperproduction by E. coli isolates (Livermore 1995) and plasmid-mediated β-lactamases (Sanders andSanders 1992, Zhou et al. 1994) are documented as the main cause for the increasing resistance to aminopenicillins, β-lactam-β-lactamase inhibitor combinations, narrowspectrum cephalosporins, and cephamycins to a greater extent and, resistance to carboxy-and ureidopenicillins and extended-spectrum cephalosporins to a lesser extent. Amongst, the plasmid-mediated β-lactamases, more often TEM-1 hyperproduction and its inhibitor-resistant variant (IRT enzymes) are found to be responsible for β-lactam-βlactamase inhibitor combination resistance, a major threat to therapeutic success and occasionally OXA type is reported for the same (Martinez et al. 1987, Zhou et al. 1994, Wu et al. 1994, Stapleton et al. 1995.
Out of 71 E. coli isolates, ceftriaxone, chloramphenicol, enrofloxacin, cefoperazone, levofloxacin, ciprofloxacin were found to be sensitive in 24 (33.81%), 24 (33.81%), 18 (25.35%), 6 (8.45%), 6 (8.45%), 6 (8.45%) isolates, respectively. Also, few isolates were found to be of intermediate resistance to ampicillin/sulbactum (16.90%), amoxiclav (16.90%), cefoperazone (25.35%), ciprofloxacin (16.90%), chloramphenicol (8.45%), amikacin (25.35%), enrofloxacin (8.45%), levofloxacin (16.90%) and All the 30 isolates were further subjected to m-PCR for the detection of beta-lactamase genes viz. blaSHV, blaTEM and blaCTX-M. The genes blaTEM were detected in 17 isolates. Similarly high prevalence of TEM-52 was detected in food animals by various researchers (Costa et al. 2004, Machado et al. 2008. CTX-M and SHV genes were not detected in any of the collected samples. Similar study carried out in China by Yuan et al. (2009) showed higher prevalence of blaTEM gene followed by blaCTX-M with no samples detected positive for blaSHV gene in chickens. These findings suggested that blaTEM was more common amongst the ESBL genes in these isolates. In this study, the occurrence of ESBL producing E. coli was as high as 23.94% as ESBL genes were detected in 17 out of 71 isolates screened. Several studies on high prevalence of ESBL-producing E. coli isolates in feacal samples from food producing animals has also been found in other European countries (Blanc et al. 2006, Moreno et al. 2007, Smet et al. 2008, Falgenhauer et al. 2019. The wide dissemination of ESBL-positive E. coli among faecal isolates of food-producing animals is a serious issue of food safety and it is important to analyze the factors that could contribute to this situation. One possible origin of this problem might be the use of broad-spectrum cephalosporins in animal production. Other possible factor would be overcrowding in herds, facilitating the transmission of resistant bacteria amongst the animals. It is difficult to know the specific contribution of each factor to the global problem of the wide dissemination of ESBL in the animal ecosystem, and all these factors should be further studied with larger sample size. Previous reports also refer to a relatively higher frequency of the presence of ESBL-producing E. coli isolates in samples of healthy humans (Miro et al. 2005, Moubareck et al. 2005, Pallecchi et al. 2007, Valverde et al. 2004). An important issue to be elucidated is whether presence of ESBL-producing bacteria in the intestinal tract of healthy animals and humans has occurred in an independent way, or by contrast, both ecosystems interrelated in their evolution.
In conclusion, increasing prevalence of ESBL producers in fecal and milk samples of normal animals is pointing towards the hypothesis of animals might be becoming carriers or even reservoirs of the same thus contributing to silent spread of these multidrug resistant bacteria amongst the animals at different places, borders and boundaries. Globally occurring climate changes had put effect on the genes causing mutations amongst organisms leading to resistance. These resistant traits call for gross surveillance in community settings because ESBLs gene is a growing threat worldwide for available antibiotics but its epidemiological effects and evaluation are still underestimated with low awareness.