A total of 150 cloacal swabs were collected from different villages in and around Tirupati, A.P. which include Chittoor, Venkatagiri, Tirupati, Nagalapuram, Pallam, Vampalli, B. Kandriga and Kalahasti.

The samples were subjected to bacterial isolation, Divya et al. biochemical characterization with reference to Enterobacteriaceae as per the standard protocols.

A total of 14 antibiotic discs were selected in the present study. The discs used include ampicillin, bacitracin, cefotaxime, chloramphenicol, ciprofloxacin, colistin, doxycycline HCL, gentamicin, enrofloxacin, vancomycin, furazolidone, nitrofurazone, virginomycin and tylosine. ABST was carried out using standard protocols. The selection of antibiotics was based on usage of antibiotics in commercial poultry farms and also based on priority of critically important antibiotics in humans. Inhibition zone diameters were interpreted according to CLSI (2014) guidelines (M100-S24).

DNA extraction was carried out by boiling and snap chilling method as described by Rao (2009) with minor modifications.

Multiplex PCR I and II were standardised and cycling conditions include initial denaturation at 94 ºC for 10 minutes. 30 cycles of denaturation at 94 ºC for 40 seconds, annealing at 60 ºC for 40 seconds, elongation at 72 ºC for 1 minute and final elongation at 72 ºC for 7 minutes and hold at 4ºC. Oligonucleotide primers used and their respective amplicon sizes were given in Table 1.

In this study, a total of 217 isolates with reference to the family Enterobacteriaceae were obtained from the faeces of desi chicken. 130 (59.9%) were characterized as E.coli, 42 (19.35%) were characterized as Salmonella spp. and 45 (20.73%) isolates were characterized as Klebsiella spp. Hundred percent resistance was recorded against bacitracin, colistin, furazolidone, nitrofurazone, vancomycin, virginomycin and tylosine. 70.96, 58.52, 30.8, 26.72, 22.5 17.5 and 16.1% resistance was observed against Doxycycline Hcl, ampicillin, ciprofloxacin, cefotaxime, chloramphenicol, enrofloxacin and gentamicin respectively.

Even though, antibiotics are not used in desi chicken, they found to harbour resistant gut microflora. Antibiotic resistance was also reported in desi chicken by other workers. Kakkar et al. (2017) tested samples from backyard poultry in New Delhi. High level of resistance was reported to chloramphenicol, ciprofloxacin, gentamicin, levofloxacin, norfloxacin and oxytetracycline. Ibrahim, (2017) tested poultry cloacal swabs from Sudan and reported 60% resistance to enrofloxacin, 40, 20, 10 and 5% resistance to tetracycline, ciprofloxacin, gentamicin and colistin respectively. The samples which were found to be resistant to ampicillin and cefotaxime in ABST were selected and further screened for the presence of beta lactamase genes.

A total of 127 isolates showing phenotypic resistance to ampicillin were selected. The DNA extraction was carried out and the isolates were tested for the presence of bla_TEM, bla_SHV and bla_OXA genes. Out of 127 (85 E.coli, 18 Klebsiella and 16 Salmonella), 103 (81.10%) isolates were found positive for the presence of bla_TEM gene and 2 (1.57%) samples were found to be positive for the presence of bla_SHV gene alone and 10 (7.87%) samples were found to possess both bla_TEM and bla_SHV genes (Fig. 2). None of the samples harboured bla_OXA gene. Out of 115 bla_TEM PCR positive samples, 85 (73.91%) belonged to E.coli, 14 (12.17%) were Klebsiella spp. and 16 (13.91%) were Salmonella spp. Out of 12 bla_SHV PCR positive samples, 8 (66.66%) belonged to E.coli and 4 samples (33.33%) belonged to Klebsiella spp (Fig. 1).

Lane M: Molecular weight marker (100bp); Lane 1 : Known DNA standard carrying bla_TEMand bla_SHVgenes; Lane 2 to 6 : Desi chicken microbiota carrying bla_TEM and bla_SHVgene; Lane 7: Negative control.

A total of 58 isolates showing phenotypic resistance to cefotaxime were selected. The DNA extraction was carried out and the isolates were tested for the presence of bla_CTXM Group 1 and Group 2 genes. Out of 58 (20 E.coli, 14 Klebsiella and 16 Salmonella), 15 (25.86%) samples were found to be positive for the presence of bla_CTXM group I gene (Fig 3). None of the samples harboured Group 2 gene. Out of 15bla_CTXM Group II PCR positive samples, 10 (66.66%) belonged to E.coli, 3 (20%) were Klebsiella spp. and 2 isolates (13.33%) were Salmonella spp (Table 2).

Presence of ESBL genes were reported from desi chicken in other studies. Hasan et al. (2012) isolated 66 E.coli from desi chicken in Bangladesh. Out of 66, 36 E.coli harboured ESBL genes. 34 of them belonged to the bla_CTX-M-1 group and 2 of them belonged to bla_CTXM-9 group. Combinations of bla_CTX-M-1 and bla_TEM-1 were detected in 50% of the isolates, whereas none of the isolates harboured SHV genes. In a similar study conducted by Hyati et al. (2019) from Indonesia, Klebsiella spp. was isolated from desi chicken. Isolates that showed phenotypic resistance to ampicillin and cefotaxime were screened by PCR for bla_TEM, bla_SHV, bla_OXA and bla_CTXM genes. The isolates harboured bla_SHV (9.1%), bla_TEM(100%), and bla_CTX-M (90.9%).

Lane M : Molecular weight marker (100bp); Lane 1 : Known DNA standard for bla_CTX-M group 1 gene (688bp); Lane 2 to 6 :Desi chicken microbiota carrying bla_CTX-M group 1 gene; Lane 7 : Negative control.

Samanta et al. (2015) tested 360 poultry samples from backyard poultry and120 samples from the farmed poultry in India. Phenotypic resistant ampicillin and cefotaxime samples were screened by PCR for ESBL genes. None of the E.coli isolates from the backyard poultry harboured any ESBL gene. 29.4% of isolates from the farmed poultry were found to possess the ESBL genes. These findings are contrary to our findings in the present study.