Molecular Characterization and Antimicrobial Resistance Profiling of Extended Spectrum Beta-lactamase (ESBL) Producing Escherichia coli in Bovines from J and K, India

Antimicrobial resistance (AMR) is a growing problem in veterinary medicine because it involves many different species of animals and microorganisms, as well as different animal rearing environments and resistance mechanisms. Some of the most common pathogenic bacteria, such as E. coli and Staphylococcus, are becoming increasingly resistant to first-line antibiotics (Lewis et al., 2007; Pop and D’Agata, 2005). Extended spectrum beta-lactamases are the enzymes that mediate resistance to extended-spectrum (third generation) cephalosporins (e.g., ceftazidime, cefotaxime and ceftriaxone) and monobactams (e.g., aztreonam) but do not affect cephamycins (e.g., cefoxitin and cefotetan) or carbapenems (e.g., meropenem or imipenem). The majority are derivatives of the TEM and SHV β-lactamase families, while others, including CTX-M, OXA and KPC β-lactamases, have only recently been discovered. The main mechanism underlying the rise of antibiotic resistance is horizontal gene transfer by mobile genetics elements like plasmids and integrons (Correa et al., 2014). Integrons are DNA elements that allow bacteria to share antibiotic resistance genes (Kargar et al., 2014). Integrons, also known as gene cassettes, are genetic components that receive and exchange foreign DNA through a site-specific recombination mechanism (Stokes and Hall, 1989). The most well-known gene cassettes discovered within integrons are antibiotic resistance gene cassettes. The increased incidence of MDR bacteria has prompted a frenzy of research on the genetics and methods by which bacteria have evolved antimicrobial drug resistance. There are three types of integrons: intI1, intI2 and intI3, all of which have been linked to antibiotic resistance genes (Mazel et al., 2006). Over 130 resistance gene cassettes have been found to be encoded by Class 1 integrons. In class 2 integrons, however, only 6 cassettes have been identified. In the literature and the GenBank database, there is also a lack of variety in class 3 integrons (Correa et al., 2014). Accurate prevalence estimates of ESBL-producing E. coli are currently unavailable due to the lack of regional or nationwide surveillance programmes.

Sample collection
 
A total of 200 (of which 180 were E. coli) faecal samples were collected from healthy cattle and buffalo from different areas of Jammu region between the period from March 2019 to January 2021.
 
Phenotypic tests for the detection of ESBLs
 
Isolation of presumptive ESBL producing Escherichia coli
 
The faecal samples were inoculated into nutrient broth and incubated at 37°C until the suspension matching 0.5McFarland standard (1.5 × 10⁸ CFU/mL) and 10 µl of this suspension was spread on ESBL ChromoSelect Agar plates using sterile spreader. Two pink colonies were selected from each plate and streaked on the nutrient agar slant separately, for further screening.
 
Screening of presumptive ESBL producing E. coli for resistance to ceftazidime and cefotaxime by disk diffusion test
 
The ESBL isolates were subjected to screening for resistance to cefotaxime and ceftazidime by disk diffusion test as recommended by CLSI. A suspension of each isolate matching 0.5 McFarland standard was made in nutrient broth. Using sterile cotton swab, the bacteria were spread on Mueller Hinton agar to obtain a lawn culture. After allowing the plate to dry, the cefotaxime and ceftazidime antibiotic disks were placed on the surface and the plates were incubated at 37°C for 18-24 hours. Following growth, the diameter of the zone of inhibition around the disks were measured and recorded. Isolates showing resistance to at least one of the antibiotics were considered for further processing.
 
Confirmation of ESBL producing E. coli by cephalosporin/clavulanate combination disks
 
Isolates of E. coli that were resistant to cefotaxime and/or ceftazidime were subjected to  phenotypic confirmatory test by using Double Disks Synergy Test as recommended in 2010 by CLSI guidelines which advocates use of ceftazidime (30 μg) (CAZ), ceftazidime + clavulanic acid (30/10 μg) (CAC), cefotaxime (30ìg) (CTX), cefotaxime + clavulanic acid (30/10 μg) (CEC) discs. An increase in the zone diameter by ≥5 mm around the disks containing cephalosporin with clavulanic over the disks containing cephalosporin alone confirmed ESBL production.
 
Molecular characterization of ESBL producing E. coli isolates
 
Extraction of bacterial DNA
 
The DNA was isolated by snap and chill method which includes boiling of colonies suspended in distilled water for 10 min to release DNA, cooled on ice for 10 min and centrifuged at 10,000 × g for 1 min.
 
Detection of ESBL producing E. coli isolates
 
All the isolates found positive for ESBLs production phenotypically, were tested for the presence of bla_TEM, bla_CTX-M, bla_SHV and bla_OXA genes by PCR assay (Fang et al., 2008). About 10 microlitres of PCR product was electrophoresed in a 1% (w/v) agarose gel for 1 hr at 5 V/cm with a Standard molecular weight marker.
 
Antimicrobial susceptibility testing
 
Bacterial isolates found to be positive for ESBL genes by m-PCR, were tested for multidrug resistance by the disk diûusion method in accordance CLSI,2010 guidelines against 20 antibiotics (Table 1). Zone of inhibition were measured and the susceptibility (or resistance) of each isolate was determined.
 

 
Molecular detection of class 1, 2 and 3 Integrons
 
Multi drug resistant ESBL positive isolates were tested for the presence of intI 1, intI 2 and intI 3 genes by m- PCR (Machado et al., 2005).

Isolation of presumptive ESBL producing Escherichia coli
 
A total of 360 presumptive ESBL producing E. coli isolates (2 from each sample) were obtained from 180 faecal samples. Present study revealed that the total prevelance of ESBL producing E. coli in bovines of Jammu region is 42.77% and is reported for the first time in Jammu region. The higher prevalence rate recorded in the current study could be attributed to the indiscriminate use of 3^rd generation cephalosporins as a source of growth promoters and disease prevention in bovines, as well as the Plasmid-mediated horizontal transfer of the bla gene. This study revealed higher prevelance when compared to other studies, where it is recorded as 29.1% from Andhra Pradesh by Sharif et al., (2017) and 35% from Assam by Borah et al., (2014). As analyzed from the different reports, the prevalence rate of ESBL in bovine has increased systematically from 35% in 2014 to 42.77% in the present time in India. When the scenario in India is compared with the worldwide scenario, the frequency detected in present study is comparable to 43.6% from china (Zheng et al., 2018), 47.7% from Nepal (Subramanya et al., 2021), but higher than 4.8% from Malaysia (Kamaruzzaman et al., 2020) and 11.2% from Germany (Michael et al., 2017), However highest percentage (63.2%) was reported by Olowe et al., (2015) from Nigeria.
 
Screening of presumptive ESBL producing E. coli for resistance to ceftazidime and cefotaxime by disk diffusion test
 
A total of 154 (42.77%) isolates were found to be resistant. Resistance to cefotaxime and ceftazidime was observed in 94 isolates (61.03%) and 60 isolates (38.96%), respectively and 70 (45.45%) isolates showed resistance to both as depicted in Fig 1. In contrast to findings of Faruk et al., (2016) from Turkey, reported diminutive sensitivity of 11.11% and 2.22% of isolates against cefotaxime and ceftazidime, respectively. 70 (45.45%) isolates showed resistance to both the antibiotics. The increased sensitivity of E. coli isolates to ceftazidime, cefotaxime and ceftriaxone in this study could be attributed to the fact that third generation cephalosporins are more active against Gram negative organisms (Karchmer, 1995).
 

 
Screening for ESBL production by double discs synergy test
 
All the 154 isolates were confirmed as ESBL producers based on the Double Discs Synergy Test as shown in Fig 2.
 

 
 Detection of bla genes by PCR
 
Out of 154 ESBL isolates, only 120 (77.92%) isolates carried bla_TEM, bla_CTX-M and bla_SHV genes. Of these only 04 (3.33%) isolates carried bla_TEM gene alone, 65 (54.16%) isolates carried bla_CTX-M gene alone, 45 (37.50%) isolates carried both bla_TEM /bla_CTX-M genes and only 06 (5.0%) isolates carried bla_SHV /bla_TEM /bla_CTX-M. bla_OXA gene could not be detected in any of these 120 isolates. Fig 3 showed genes amplified from m-PCR assay. In contrast to this study, Borah et al., (2014) from Assam and Sharif et al., (2017) from Andhra Pradesh reported the bla_CTX-M, bla_SHV and/or bla_TEM type ESBLs in cattle. bla_CTX-M-15, bla_TEM-52 and bla_SHV-12 have been reported from Germany by Michael et al., (2017). According to the findings, bla_CTX-M is the most common ESBL type in cattle of Jammu region, with E. coli being the most common ESBL producer. The higher rate could be attributed to the widespread use of third-generation cephalosporins, particularly ceftriaxone and cefotaxime, or it could be linked to high encoding gene mobility.
 

 
Antimicrobial susceptibility testing
 
The prevalence of AMR among the ESBL positive strains isolated from cattle and buffalo is shown in Table 1. All 120 E. coli isolates showed resistance to at least 20 antibiotics. The isolates that were resistant to more than two classes were identified as multidrug resistant (MDR) isolates. In this study, the prevalence of Ampicillin resistance in ESBL positive isolates was high, which is in agreement with statement from Indonesia by Sudarwanto et al., (2016) and from China by Zheng et al., (2018). The major finding in the present is the presence of multi drug resistance commensal E. coli in bovines to commonly used antibiotics such as amoxicillin/clavulanic acid, aztreonam, ceftriaxone, cefexime, cefepime, enrofloxacin, kanamycin and neomycin, which is comparable to the findings of Borah et al., (2014) from Assam, India. The observation of present study was comparable to those of Zheng et al., (2018) from China, Ejaz et al., (2021) from Brazil and Subramanya et al., (2021) from Nepal. It reiterates the finding in other studies that have reported antibiotic resistance among bacteria especially E. coli isolated from cattle and other animals is increasing at an alarming rate. However, in our study most of the isolates were senisitive to Imipenem, which is in agreement with Ejaz et al., (2021) from Brazil.
 
Molecular detection of class 1, 2 and 3 integrons
 
Out of the 120 MDR isolates, 59 were tested for the presence of integron gene. Among these isolates, class 1 integron-encoded intI 1 integrase gene was detected in 52 (88.23%) isolates. While 2 (3.38%) isolates tested positive for class 2-encoded intI 2 integrase and five isolates harboured both intI 1 and intI 2. No class 3 integron was detected (Fig 4). The prevalence of intI 1 in cattle and buffalo was 95.65% and 100.0%, respectively and the prevalence of intI 2 for cattle and buffalo was 10.86% and 15.38%, respectively. The observations are comparable to the 50% in Australia (Barlow et al., 2004) but higher than 16.77 % from Korea (Hasan, 2010), 6% from Iran (Kheiri et al., 2016). However higher percentage (84.5%) was reported by Ejaz et al., (2021) from Brazil. Kheiri et al., (2016) from Iran detected class 2- integrase gene in less than 1.2% of isolates. While in our work this percentage was 3.38%, which is in agreement with 4% from Iran by Kheiri et al., (2016). ESBL harbour both intI 1 and intI 2 which is higher than (0.4%) by Kheiri et al., (2016) from Iran. Integrons are known to be primary source of transferable resistance genes and are suspected to serve as reservoirs of antimicrobial resistance genes within microbial populations (Collis et al., 2002). Because integrons have the ability to capture and collect gene cassettes, there is a chance that antibiotic-resistant genes will become common in nature. E. coli, which are deadly pathogens if they become antibiotic resistant, can be extremely hazardous to the environment.
 
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Antibiotic discovery and development was unquestionably one of the most significant advances in modern medicine. Antimicrobial drug resistance in Gram negative enteric bacteria has emerged as a significant issue in both human and veterinary medicine. The use of antibiotics in farm animals, which are critical in human medicine, has been linked to the emergence of new strains of multi-drug resistant (MDR) bacteria that infect humans. Thus there is an urgent need to focus on the antibiotic selection and to reduce the spread of these increasingly resistant pathogens.

None.