Detection, characterization, and antibiogram of extended-spectrum beta-lactamase Escherichia coli isolated from bovine milk samples in West Bengal, India

Background: Milk is considered as complete food and an important part of human diet throughout the world including India. Bacterial contamination of milk such as Escherichia coli due to unhygienic condition and poor udder health can cause infections, especially in infants and elders or in immunocompromised persons. Possession of antimicrobial resistance genes by commensal bacteria present in milk makes the issue more serious. Aim: The study was aimed to isolate and characterize extended-spectrum beta-lactamase (ESBL)-producing E. coli from milk samples collected from different parts of West Bengal, India, to assess the potential risk associated with the food. Materials and Methods: Around 182 milk samples were collected from apparently healthy cows reared by organized dairy farms in West Bengal. E. coli was isolated from collected samples as per standard methods followed by serotyping. The detection of ESBL-producing E. coli was done both phenotypically and genotypically by detecting the presence of blaCTX-M gene. Antibiogram of the ESBL-positive isolates was done using common 12 antibiotics by disc diffusion method. Results: A total of 22 (12.1%) samples were found to be positive for E. coli in this study. Different serotypes such as O11, O20, O22, O34, O35, O128, O149, and UT were isolated from the collected samples. 12 (54.5%) E. coli strains showed the capability of producing ESBL, both phenotypically and genotypically with the presence of blaCTX-M gene. Antibiogram of these ESBL-positive isolates revealed the drugs such as colistin (100%), levofloxacin (83.33%), and imipenem (66.67%) to be highly sensitive against this pathogen but drugs such as cefotaxime (100%), ceftazidime (91.67%), amoxicillin/clavulanic acid (83.33%), tetracycline (75.00%), and gentamicin (58.33%) to be very much resistant. Conclusion: More than 50% of the E. coli strains prevalent in the bovine milk samples were positive for ESBL production and are resistant to most of the common antimicrobials which may be alarming for human health.


Introduction
India is one of the largest milk producing countries in the world with dairy industry playing an important role in the rural economy [1] generating huge self-employment. Bovine milk is generally considered to be a good source of protein and vitamins to human beings, particularly to the infants. However, due to faulty handling and storage of milk and poor management of the animal, milk may get spoiled due to rapid multiplication of bacteria due to milk's high nutritive value [2]. Escherichia coli is one dreadful pathogen, especially the "enterohemorrhagic E. coli" strains, causing infection through milk which has a great effect on human health [3,4].
The prevalence of extended-spectrum beta-lactamase (ESBL)-producing E. coli is increasing in the globe including India. These pathogens pose a major challenge for the treatment of general infections and cause a problem with the extensive use of second-or third-generation antibiotics for the treatment of bacterial infections [5]. ESBL E. coli is mostly insensitive to lots of commonly used antibiotics causing an increase in the use of last-resort antimicrobial drugs (i.e., carbapenems) during treatment. Again, E. coli strains carrying the resistance genes can easily transfer those genes to other pathogens leading to the spread of drug resistance [6]. Hence, the presence of ESBL-producing E. coli in the food processing chain or in the food of our daily consumption which is possibly coming from these healthy farm animals is the fact which has to be appropriately studied. The present study was aimed for the detection and characterization of ESBL-producing E. coli from raw milk samples (by detecting bla CTX-M gene in the isolates) from different dairy farms followed by further characterization and to know their antibiotic resistance patterns in vitro.

Ethical approval
As per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals, this study does not require any ethical approval from the University Animal Ethics Committee.

Collection of samples
Bovine milk samples (n=182) were collected from different unorganized dairy farms of West Bengal aseptically in sterile plastic containers (Table-1) during the period of April-June, 2018. Milk samples were taken at 15 ml (approximately) in sterile plastic containers directly from the teats of the cows. The cows were selected on the basis of the history of illness/ with decreased milk yield. The samples containers were kept in sample flask under ice and cooling pad cover and were transported through shortest route (for maximum 5-6 h approximately) to the Department of Veterinary Microbiology, Mohanpur, Nadia, for further study. All the collected samples were studied on the same day of receiving at the laboratory.

Isolation and characterization of E. coli
The collected milk samples were enriched adding sterile nutrient broth at 37°C for 6-8 h followed by streaking on to sterile MacConkey's agar (HiMedia, India) plates. The plates were incubated aerobically at 37°C for 10-12 h. The tentative pinkish single colonies (i.e., lactose fermenting) were selected for selective isolation by further streaking on sterile Eosin Methylene Blue (EMB) agar (HiMedia, India) plates followed by 10-12 h incubation again at 37°C. The single colonies showing characteristic greenish "metallic sheen" were picked up and were stored using sterile nutrient agar (HiMedia, India) slants for further morphological study by Gram's staining method and biochemical characterization with tests such as indole, methyl red, citrate utilization, Voges-Proskauer, catalase, and nitrate reduction [7,8]. All positive isolates showing typically to be E. coli were further confirmed for ESBL positivity later.

Serotyping of the E. coli isolates
All positive E. coli isolates were sent to the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, Himachal Pradesh, India, for serotyping. All E. coli strains were subcultured in small sterile glass vials and were properly packed in hardboard box under cotton cover followed by sending to the NSEC, Kasauli, by registered post.

Phenotypic detection of ESBL production in E. coli isolates
Phenotypic detection of the presence of ESBL in E. coli isolates was done in vitro by disc diffusion method [9] using both cefotaxime (30 µg) and ceftazidime disks (30 µg) with and without clavulanate (10 µg) as per the CLSI methods by Patel et al. [10]. A difference of >5 mm between the zone diameters of each disk and their respective clavulanate disk is measured to phenotypically confirm the ESBL production by the E. coli isolates under study [10].

Bacterial culture lysate preparation
Selective E. coli strains were inoculated into nutrient broth (HiMedia, India) followed by 18 h incubation at 37°C. 1 ml of young broth culture of each sample was taken in a sterile 1.5 ml microcentrifuge tube (Tarsons, India) followed by centrifugation at 6000 rpm for 5 min [11]. The obtained pellet was washed 3 times with TE buffer and was suspended again in TE buffer (1 ml). The microcentrifuge tube with culture was then boiled in water for 10 min followed by chilling in ice. Again each tube was centrifuged at 5000 rpm for 5 min followed by removal of cell debris and the supernatant with crude DNA was collected and stored at −20°C for further use as a template in PCR [11].

Detection of bla CTX-M gene (540 bp) in E. coli isolates
All phenotypically ESBL-positive E. coli isolates were considered for confirmation by PCR detection of the bla CTX-M gene in them as per the protocol followed by Weill et al. [12] with slight modifications. All standard reagents and primers (GCC Biotech, India) were used in this process. Amplification reaction mixture containing 3 µl DNA templates, 50 pmol the primer set [540 bp] (forward CTX-M -F 5'-CAATGTGCAGCACCAGTAA-3' and reverse CTX-M -R 5'-CGCGATATCATTGGTGGTG-3'), 1U GoTaq DNA polymerase (Promega, USA), 200 mM deoxynucleoside triphosphate, 10% dimethyl sulfoxide, and 2 mM MgCl 2 was prepared in a 25 µl reaction mixture and used in PCR amplification conducted in a thermocycler (Eppendorf, Germany). The PCR amplification was done in the following cycle condition with an initial denaturation at 94°C for 10 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 53°C for 30 s, and elongation at 72°C for 60 s with a 10 min final extension period at 72°C. The amplified PCR products were loaded onto a 1.5% w/v agarose gel (SRL, India), with ethidium bromide (0.5 µg/ml) (SRL, India) followed by agarose gel electrophoresis and were visualized by gel documentation system (UVP, UK). One ESBL-producing E. coli strain (O2) which is the departmental isolate and one Pseudomonas aeruginosa (ATCC 27853) were used as positive and negative controls in PCR assays.

In vitro antibiotic sensitivity test of ESBL E. coli isolates
Antibiogram of the ESBL-positive E. coli isolates was performed using 12 antimicrobials, i.e., amikacin, amoxicillin/clavulanic acid, azithromycin, colistin, cotrimoxazole, cefotaxime, ceftazidime, gentamicin, imipenem, levofloxacin, piperacillin-tazobactam, and tetracycline by Kirby-Bauer disc diffusion method [9]. Young broth cultures of all the ESBL-positive isolates were produced for the test. Separate and sterile Mueller-Hinton agar (HiMedia, India) plates were used for uniform spreading of each broth culture using sterile L-spreader, and standard discs (HiMedia, India) were placed with sterile forceps. All the plates were incubated at 37°C for 10-12 h, and the results were interpreted by measuring the inhibition zone diameter and comparing those with the standard chart [10].

Results
Out of 182 bovine milk samples tested, 22 (12.08%) samples were found to be identified as E. coli in this study. All the positive isolates showed typical characteristics during cultural, i.e., produced typical "metallic sheen" when grown on EMB agar plates (Figure-1) and morphological examinations (pink rods after Gram's staining). All showed typical results during their biochemical characterization, i.e., positive to indole, methyl red, catalase, and nitrate reduction whereas negative to VP and citrate utilization.
During ESBL detection, a total of 12 (54.54%) E. coli isolates were found to be phenotypically positive as ESBL producers by double disc method in this study. All phenotypically ESBL-positive E. coli isolates were detected to have the bla CTX-M gene (540 bp) by PCR (Figure-2) (Table-3).
In vitro, antibiotic sensitivity assay of the ESBLpositive E. coli isolates showed high-level resistance to cefotaxime, ceftazidime, amoxicillin-clavulanic acid, tetracycline, gentamicin, amikacin, etc. (with the range of 60-100%). Piperacillin-tazobactam (83.33%) was detected to be intermediately sensitive to these isolates (Table-4), and drugs such as colistin, levofloxacin, and imipenem were found to be sensitive against these pathogens.

Discussion
Approximately 12% of the total milk samples screened were found to yield E. coli isolates in this study which were also supported by Kamaruzzaman [13], Badri et al. [14], Geser et al. [15], and Ali et al. [16] who also reported 12.22-13.7% E. coli positivity in the bovine milk samples during their study. All positive E. coli isolates showed typical cultural, morphological, and biochemical nature in this study which was also supported by Carter and Wise [7], Samanta [17], and Quinn et al. [8].
The serotypes reported by the National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, were also supported by Osman et al. [18] who detected E. coli serogroups O26, O86, O111, and O127 from cattle milk in their study. Al-Zogibi et al. [19] reported the prevalence of E. coli serogroups, namely O22, O111, O113, and O172, from bovine milk samples in their study which also supports the current findings.
Approximately, 54.54% E. coli isolates were both phenotypically and genotypically positive to produce ESBL in this study which was also supported by Geser et al. [15] and Ibrahim et al. [20]. Kamaruzzaman [13] reported a high prevalence of ESBL-producing E. coli in milk (66.7%) followed by farm environment (27.8%) and cattle (5.5%) in his work. Ali et al. [16] reported 36 (23.53%) and Badri et al. [14] reported 29.3% ESBL-positive E. coli strains from bovine milk samples which may be of great concern as these pathogens may be carried out to the human consumers as well as calves leading to the spread of the antibiotic-resistant pathogens over human and animal population. Sharma et al. [21] also reported ESBL-positive E. coli serotypes in their study, matching the current findings.

Conclusion
The drug-resistant ESBL gene is significantly present in approximately 55% of the E. coli strains isolated from cattle milk samples which may be of great health concern for human beings. This drug resistance can easily be transferred between closely related pathogens in vivo which may result in risky and fatal health hazards due to unsuccessful treatment with common antimicrobials. Hence, proper care should be taken to combat these dreadful pathogens.

Author's Contributions
KB and SD designed the study. AB and ADS collected the samples. KB, AB, and SP carried out the