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Characterisation of Escherichia coli Recovered from Wild Animals Kept at Bikaner Zoo for Their Antibiotic Resistance

In the present study, a total of 41 E. coli isolates obtained from Boselaphus tragocamelus (5), Antelope cervicapra (18) and Gazella gazelle (18) maintained at Bikaner (Rajasthan) zoo were subjected to antibiogram determination against 15 antibiotics belonging to four different classes and were also genotyped for detecting presence of blaTEM, sul-2, strA and aadA genes. Antibiogram study revealed highest efficacy of ciprofloxacin (90.2%) followed by nalidixic acid (75.6%) and chloramphenicol (68.4%) and high resistance to β lactam, Sulfamethoxazole and Aminoglycoside antibiotics. The overall presence of blaTEM, sul-2, strA and aadA genes was detected in 95.12%, 80.48%, 60.97% and 87.8% in isolates. Original Research Article Zaidi et al.; JPRI, 32(3): 1-8, 2020; Article no.JPRI.55265 2


INTRODUCTION
Antimicrobial resistance is a matter of great concern due to the reduced efficacy or failures of antibiotics to treat various bacterial infections [1]. There are many probable reasons for emergence of development of antimicrobial resistance of which one reason could be the secretion of antimicrobial agents by another bacterium such as Streptomyces mainly as a means of microbial competition for an ecological niche [2,3,4,5]. Another possible reason could be the excessive use of antibiotics which can exert a selection pressure on bacteria which results in accumulation of resistance genes in microbiota converting them into a pathogenic bacterium which are otherwise commensals [6,7,8,9]. Hence, the natural selection is one of the main causes which has supported the selection and evolution of multiresistant phenotypes of bacteria and their spread to different areas without direct use of antimicrobials [10]. Mutations, horizontal transfer of genes and transfer of already resistant bacteria found in the faecal matter, manure, sewage water, soil, clinical specimen of other animals and human could also be the other possible sources of emergence of resistant genes in bacteria [11,3].
Wild animals are rarely exposed to antimicrobial agents and there are very few reports available on the presence of antibiotic resistant bacteria from wild captive animals but sufficient data is available which depicts the presence of antibiotic resistant bacteria in the vicinity of captive wild animals [12,13].
Escherichia coli is a commensal bacterium commonly found in the intestinal gut of animals and humans [14,15] and is responsible for causing intestinal infections [16,17]. This organism is commonly used as a potential indicator of faecal contamination and is expected to be reservoir of antibiotic resistance genes [6] because of the regular use of antibiotics for treatment of infectious diseases. This is one of the major growing concerns in veterinary medicine as the resistant bacteria can enter into other ecological niches and can cause a potential risk to human health. Keeping this in view, study of antibiotic resistance situations in a particular area is prerequisite in order to access the possible risk of its transfer to other individuals. At present, there are few reports available on isolation and prevalence of antibiotic resistance genes on E. coli found in wild captive animal species. To our knowledge, there are very few data available in India which can clearly depicts the situation.
Hence the present study was conducted to investigate phenotypic and genotypic properties of E. coli isolates obtained from wild captive animals at Bikaner zoo in regard to their antibiotic resistance. Our studies identified the presence of bla TEM , sul2, strA and aadA resistance genes in E. coli from captive wild animal populations. bla TEM provides resistance against various β-lactam antibiotics like penicillin and Ampicillin. sul2 gene provides resistance against sulfamethoxazole antibiotics, strA work against streptomycin and aadA gene codes for aminoglycoside adenyl transferase enzyme which allows selection for resistance to aminoglycoside antibiotics such as spectinomycin and streptomycin.

Sampling
For isolation of E. coli, the faecal samples from wild captive animals at Bikaner (Rajasthan) zoo were collected after permission from appropriate authorities. A total of 46 freshly voided faecal samples were collected from Gazella bennettii, (18) Antelope cervicapra, (20) Boselaphus tragocamelus (8) in June 2018. All the samples were kept at ambient temperature, transported to the laboratory and incubated overnight in the nutrient broth for 24 hours.

Isolation and Identification of E. coli
The isolation and identification of E. coli from faecal samples was carried out by conventional methods as described by Cowan and Steel [18] and Quinn et al. [19]. Each sample was inoculated on to the EMB agar by streaking and incubated aerobically at 37ºC for 18 to 24 hours. Appearance of green metallic sheen on EMB agar revealed the presence of E. coli. Colonies with typical E. coli morphology were further subcultured until pure isolated colonies were obtained. The obtained pure E. coli colonies were gram stained and biochemical identification was done using Vitek 2 automated system (BioMerieux India private LTD). The colonies were also genotypically confirmed by PCR using 16 S rRNA as described by Khaled et al. [20].
The confirmed isolated colonies were streaked on agar slants and maintained for further use.

Antimicrobial Susceptibility Testing
Antimicrobial susceptibility was performed by the agar disc diffusion method as described by Bauer et al. [21] and as per clinical and laboratory standards institute [22]. A total of 15 antimicrobial agents belonging to different classes and generations were tested in this study ( Table 1). Diameter of zone of inhibition was observed and results were interpreted as sensitive, intermediate and resistant isolates for different antimicrobial agents. The isolates with resistance to one or more antimicrobial agents were selected for detection of genes for antimicrobial resistance.

Detection of Genes for Antibiotic Resistance
Polymerase chain reaction was carried out with a Veriti 96 well thermocycler to detect presence of bla TEM, aadA, strA and sul-2 genes amplified through specific primers (Table 1) and using specific conditions (

RESULTS
A total of 41 E. coli isolates from G. bennettii,  Table 3. The Antibiogram against all the isolates revealed that most effective antibiotic was Ciprofloxacin followed by Nalidixic acid, Chloramphenicol and Gentamicin (Table 3) and no resistance was recorded against Ciprofloxacin, Nalidixic acid and Gentamicin and little resistance was exhibited against Chloramphenicol.
Resistance spectrum of E. coli for 15 antibiotics tested in descending order was respectively polymyxin B, Cephalothin, Cefepime, Cefaclor, Piperacillin, Cefexime and Co-Trimoxazole, Ampicillin and Tetracycline, Colistin, Chloramphenicol, followed by no resistance in case of Ciprofloxacin, Gentamicin, and Nalidixic acid. It was shown that none of the isolates were found resistant against Ciprofloxacin, Gentamicin and Nalidixic acid and 80.5% isolates were found resistant against Polymyxin B ( Table 3). The highest sensitivity was recorded for the antibiotic Ciprofloxacin followed by Nalidixic acid. Least Sensitivity was found against Co-Trimoxazole followed by Tetracycline.
The detection of genes for antibiotic resistance in the isolates by PCR revealed that of the total 41 isolates, bla TEM gene was present in 39 (95.12%) isolates and one isolate each from Antelope cervicapra (5.5%) and Boselaphus tragocamelus (20%) was found not to possess this gene.
The sul2 gene was present in 25 (60.97%) isolates. All the Gazella bennettii samples were found positive for this gene, whereas 13 (72.22%) isolates from Antelope cervicapra and three (60%) from Boselaphus tragocamelus samples did not have sul2 gene.   The strA gene was detected in 33 (80.48%) isolates. None of the isolates from Gazella bennettii was found negative for strA gene whereas six (33%) isolates from Antelope cervicapra and two from Boselaphus tragocamelus (40%) were found negative for this gene.
The gene aadA was present in 36 (87.80%) isolates. All the Gazella bennettii samples were found positive for this gene whereas 3 (16.66%) isolates from Antelope cervicapra and two (40%) from Boselaphus tragocamelus samples did not have sul2 gene.

DISCUSSION
Increasing antimicrobial resistance in bacterial population is a global problem because it creates trouble in fighting infectious diseases. Improper and excessive use of antimicrobial agents along with poor environmental sanitation and low personnel hygiene have been attributed to such problems. There is paucity of data in the literature about the susceptibility to antibiotics in E. coli isolates of healthy wild captive animals. If data are available they are restricted to a low number of animals. There are strong evidences available in literature supporting a direct relationship between the frequency of antibiotic resistance observed in E. coli isolates of captive wild animals and the degree of contact of these animals with humans which suggest the anthropogenic nature of Antibiotic resistance in E. coli isolates obtained from faecal matter of wild animals [24].
We investigated the occurrence of antibiotic resistance in commensal E. coli from wild captive animals in Bikaner district of Rajasthan state in India. It is an area with limited study on antibiotic resistance and use of antimicrobial agents. In our study, a high resistance against piperacillin (65. 8 Kozak et al. [34] also observed high resistance to tetracycline among E. coli isolates obtained from small wild mammals. The high resistance against tetracycline among E. coli isolates is also in accordance with previous values obtained from similar reports [35,36,37,38]. Tetracycline is being used as first line antibiotic for treatment and growth promotion among food animals [39]. It is likely that resistance genes present on mobile genetic elements [40] got transferred from food animals to humans and then from humans to wild captive animals. Even tetracycline used in animal feed can create a selection pressure on bacteria harbouring resistant genes which give these bacteria an opportunity to grow in tetracycline susceptible environments.
The present findings also confirmed the earlier results of Andrasevic et al. [41] who found E. coli isolates resistant to ampicillin, co-trimoxazole, ciprofloxacin and gentamicin.
Similar to the observed pattern of phenotypic resistance, genes conferring resistance to βlactamase, aminoglycoside adenosyl transferase, streptomycin/spectinomycin, and sulfamethoxazole were also found to be common among the isolates. Our results were almost similar to Adelowo et al. [42] who also observed high prevalence of bla TEM (85%), sul2 (67%), aadA (65%) and strA (70%) in poultry animals. However, it seemed that local and geographical factors and the factors other than antibiotic use may be contributing to the selection of resistance among the present isolates. All the genes seem to have a widespread occurrence within the study area. The widespread occurrence of all four antibiotic resistance genes observed in this study may be a result of their co selection and transfer with other resistance determinants on mobile genetic elements.
Escherichia coli isolates from humans have been found to be important reservoirs of quinolone resistance genes. In our study detection of quinolone resistance in E. coli isolates may be a result of plasmid mediated transfer of quinolone resistance genes from humans to captive animals. This is of concern as fluoroquinolones were listed by the World Health Organization as critically important antimicrobials for human health [43]. Therefore, it seems very important to pay more attention to decrease the spread of resistance in the captive areas where the interaction between human and animals are more common.
Though we tested for the resistance genes most commonly found in E. coli., but the absence of resistance genes in some of the isolates resistant to a particular antibiotic may be because of presence of some known mechanism not under this study or some unknown mechanism.
Though a definite conclusion on the association between drug use and occurrence of antimicrobial resistance cannot be drawn through this study as we did not include areas without antimicrobial use or the areas without any human interference but the results of this study highlights the need of routine screening for antibiotic resistance using both phenotypic and genotypic methods in commensal bacteria from wild captive animals.

CONCLUSION
This study suggests that close proximity to humans, unhygienic practices, poor maintenance of wild captive areas, are the reasons which increase the chances of antimicrobial resistance in E. coli isolates in captive wild animals of small areas like Bikaner zoo. The data of our study reveals that wild captive animals may act as a reservoir of antibiotic resistant genes which may be mobilized into other ecological populations. The data from the present study accounts for the critical need for regulation of antimicrobial drug usage in wild captive animals and highlights for continuous monitoring of antibiotic resistance.

CONSENT
It is not applicable.

ETHICAL APPROVAL
As per international standard or university standard written ethical approval has been collected and preserved by the author(s).

ACKNOWLEDGEMENT
We thank Forest department of Rajasthan, Jaipur, for permitting to collect samples from wild captive animals from Bikaner zoo. Thanks, are also due to Council of scientific and Industrial research, New Delhi for providing SRF to first author.