​Prevalence of Shigatoxigenic, Enteropathogenic and Antimicrobial Drug Resistant Escherichia coli from Captive Wild Animals of Alipore Zoo, Kolkata, India

The presence of large population of India’s wildlife needs attention in prioritizing research on zoonotic pathogens in wildlife, particularly in a multidisciplinary one-world one-health approach (Singh and Gajadhar, 2014). Echerichia coli is one of the commonest intestinal flora harboured by wildlife, domestic animals and humans; and it is considered one of the indicator pathogen for studying antimicrobial resistance. Currently, there are 186 different E. coli serotypes based on outer membrane lipopolysaccharide (O) antigen and 53 H-types. Several serotypes of E. coli are non-pathogenic and pathogenic strains of E. coli are detected by specific virulence factors and their effect in susceptible species.

Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC) strains belong to the category of diarrheagenic E. coli that are important food-borne pathogens associated with diarrhoeal disease in humans. Although ruminants especially cattle are the main reservoirs of STEC, wildlife including game animals also play an important role as carriers of these pathogens (Miko et al., 2009).

Antimicrobial resistance of E. coli isolates from wildlife origin was reported for the first time from Japanese wild birds in 1978. The production of extended spectrum beta-lactamases (ESBLs) by Enterobacteriaceae, specifically by E. coli, has caused a major concern in past few decades. The production of the enzyme confers resistance to a variety of beta-lactam antibiotics and monobactams (e.g. aztreonam), but usually not the carbapenems or the cephamycins (e.g. cefoxitin). ESBL-producing E. coli isolates from wild birds and animals was reported first time in Portugal in 2006 (Costa et al., 2006), it has now been observed all over the world. AmpC b-lactamase- producing organisms (ACBL) can produce resistance against cephalosporins, penicillins, cephamycins and monobactams in addition to b-lactamase inhibitors such as clavulanic acid.

Although STEC and EPEC E. coli were extensively studied in humans, farm animals, birds, food and the environment, it has been less frequently studied in captive wild animals of India (Mishra et al., 2016; Milton et al., 2019). Antimicrobial resistance of E. coli from wildlife also contributes eventually to the global problem of ESBL producing pathogens. The present study was designed to study the prevalence of STEC, EPEC and antimicrobial resistance of E.coli from captive wild animals in a zoo in Kolkata, India.

In the present study, seventy five fresh faecal samples were collected during March 2019 to June 2019 from birds and mammals housed at Alipore zoo, Kolkata, India (Table 1). These wildlife species were treated with both b- lactam and non-b- lactam antibiotics for therapeutic purpose individually as veterinary care when necessary. This study did not require ethical approval because faecal samples were collected only after defaecation but consent was taken from zoo authority for sampling.



The faecal samples of 2-10 g were collected in the morning using sterile swabs in peptone water (HiMedia, India), kept in chilled box at 4°C, transported to the laboratory of Department of Veterinary Microbiology at West Bengal University of Animal and Fishery Sciences, Kolkata and processed immediately. One gram of each sample was suspended in 9 ml of peptone water broth (HiMedia, India) and incubated overnight at 37°C. After enrichment, E.coli were isolated as per standard procedure and identified using Gram-staining and biochemical tests (Quinn et al., 1999). E.coli isolates were serogrouped at National Salmonella and Escherichia Centre, Central Research Institute, Kasauli, India. Pathotypes of E. coli isolates were detected by PCR assays for STEC (stx1, stx2 genes) and   EPEC (eae gene) respectively (Vidal et al., 2004).

For phenotypic confirmation of the presence of ESBL, cefotaxime (30 μg) and ceftazidime (30 μg) disks were used with and without clavulanate (HiMedia, India) in disc diffusion method (Clsi, 2015). Cefoxitin-cloxacillin double disc synergy test was used for AmpC b-lactamase production (ACBL) as per Tan et al., (2009).

Antimicrobial susceptibility was also determined by the disk-diffusion method (Clsi, 2015) using fourteen standard discs (Himedia, India). E. coli isolates showing resistance to three or more classes of antimicrobials were recognised as multidrug-resistant (MDR). Determination of multiple antibiotic resistance (MAR) index was performed as per the procedure described by Krumperman (1983).

From seventy five samples, sixty (80.00%) bacterial isolates were identified as E.coli, 33 isolates belonged to captive wild birds and 27 isolates to captive wild mammals (Table 1). Overall sixteen different O-serogroups of E.coli were detected. Twenty one E.coli isolates were untypeable. Some strains of E.coli were rough or autoagglutinating, making these cultures O-untypeable (DebRoy et al., 2016). Prevalent serogroups in captive wild birds seemed to partially differ from earlier studies in wildlife. Awadallah et al., (2013) detected E.coli serogroup O119 in cattle egrets birds which was observed in Blue and Yellow Macaw, White Ibis and Goffin’s Cockatoo in the current study. None of the serogroups was observed in Bengal tiger by the study of Satpute et al., (2010) who reported serogroups O17, O103, O147 in Maharastra. Four E. coli serogroups O26, O118, O121 and O157 isolated in the current study, were among the most clinically relevant STEC serotypes in human illness (Sanchez et al., 2015).

Overall, 7 isolates (11.67%) were found to be positive for stx1 gene and 21 isolates (35.0%) were found to be positive for eae gene (Table 1). No isolate was found positive for stx2 gene and most E. coli isolates from birds (n=20) and mammals (n= 18) harboured none of the three virulence genes studied. EPEC was found to be the predominant pathotype (Table 1). Similar study was done by Milton et al., (2019). In contrast, captive wild mammals were found as the main reservoir of STEC and no isolate from birds harboured STEC in Chile (Marchant et al., 2016). Prevalence of EPEC and STEC strains were low among captive wild birds in Sau Paulo, Bazil (Sanches et al. 2017) and among zoo animals and birds in Spain (Alonso et al., 2017). Prevalence of STEC at Alipore zoo was low compared to wild animals (23.87%) in Spain (Sanchez et al., 2009).  Prevalence of STEC in wildlife was found variable in different studies (Marchant et al., 2016; Sanches et al. 2017; Alonso et al., 2017), probably because of the difference in animal population studied and diagnostic methods used.

In the present study, STEC belonged to serogrup O157 and non-O157 serogroups such as O2, O26, O118 and O149. Similarly STEC serogroup O157 and non- O157 serogroup were also reported previously from wild birds (Foster et al., 2006), petting zoo animals such as jaguar (Hamzah et al., 2013) and from free ranging wild animals (Sanchez et al., 2009).

Overall, ESBL production by E. coli was found in 23 (38.33%) isolates and AmpC b-lactamase production in 35 (58.33%) isolates (Table 1). A similar observation was reported by Wang et al., (2012) from primates in six different zoos in China.  Dobiasova et al., (2013) reported a high percentage (71%) of wild birds and animals colonized by ESBL producing E. coli in a zoo of Czech Republic. Recently, prevalence of multi-drug resistant ESBL and AmpC producing E.coli was observed in domestic birds such as ducks and chicken (Banerjee et al., 2019; Samanta et al. 2015). Presence of antimicrobial resistance genes from clinically ill and farm animals might have impact in antimicrobial resistance of organisms harboured by wild animals.

The resistance of E. coli isolates from captive wild birds and mammals were observed against penicillins and cephasporins groups; and less frequently against gentamicin and amikacin (Table 2).Twenty (60.60%) isolates from birds and 17 (62.96%) isolates from mammals were detected exhibiting multiple drug resistance (MDR). Except the three isolates from wild birds (Eurasian spoonbill, Grey peacock-pheasant, Chinese silver pheasant), all 57 (95.0%) E.coli isolates exhibited MAR index value ≥ 0.2. Similar studies with high resistance was reported in E.coli isolates of leopard (Panthera pardus) and black buck (Vinodh Kumar et al., 2021), captive Nilgiri Langur (Trachypithecus johnii) (Balaji et al., 2018). The importance of this study lies with the fact that wild animals provide a biological source for dissemination of antibiotic resistance genes and zoonotic diseases (Radhouani et al., 2014).

Most E.coli isolates from captive birds and mammals that harboured none of the three virulence genes probably were commensals. Although limited samples were studied from each wildlife species, the occurrence of STEC and EPEC pathotypes in captive wildife warrants attention as they are potential reservoir. High antimicrobial resistance was also observed in captive wildlife in Alipore zoo, Kolkata, India.  Continous monitoring is required to assess their impact on animal and public health.

The authors thank honourable Vice Chancellor, WBUAFS for the infrastructural facilities and ICAR for partial financial assistance (Development Grant 7.1). We thank Director, Zoological garden for the assistance received during sampling and Director, Central Research Institute, Kasauli for O-serogrouping E. coli isolates.