A total 240 milk and faecal samples were collected from different tehsils of Ayodhya and Sultanpur districts. Out of them 147 (61.25%) isolates were identified as
E.
coli and 19 (7.91%) as
Klebsiella spp. on the basis of their morphology, cultural characteristics in selective media and biochemical tests (Table 2, Fig 1, Fig 2 and Fig 3). In this study, isolates of
E.
coli was found to be predominate among isolates recovered from both milk and faecal samples. This finding was also supported by findings of various workers
(Ibrahim et al., 2018; Kotsoana et al., 2019). Higher isolation rate of
E.
coli in faecal samples may be attributed to high prevalence of
E.
coli in GIT flora of ruminants.
To study the prevalence of ESBL producing isolates among clinical and apparently healthy isolates, total 166 (147
E.
coli and 19
Klebsiella spp.) were subjected to screening, confirmatory phenotypic tests and PCR analysis. On preliminary screening, 79.5% isolates presumed as ESBL producer. Using phenotypic confirmatory testing, 62.65% isolates were confirmed as ESBL by DDST and 59.63% by ESBL-E strip test and final confirmation was done by PCR analysis, which revealed 55.42% ESBL positive isolates (Table 3). Total 92 (38.33%) were ESBL positive comprising 05 (1.25%), 16 (40.0%), 51 (63.75%) and 20 (50.0%) from normal milk, mastitic milk, normal faecal and diarrhoeic faecal samples, respectively (Table 2). These results were in conformity with findings of various workers
(Kotsoana et al., 2019; Yadav et al., 2019). The prevalence of ESBL producers was found much higher in mastitic milk than normal milk irrespective of pathogen, which may be attributed to indiscriminate and irrational use of antibiotics for treating mastitis in Eastern Plain Zone of Uttar Pradesh.
Genotypic analysis of confirmed ESBL producing isolates (85
E.
coli and 07
Klebsiella spp.) was done by targeting
bla-CTX-M (
bla-
CTX-M-1,
bla-
CTX-M-2,
bla-
CTX-M-9,) bla-
TEM and
bla-
SHV genes (Fig 6, 7, 8, 9, 10). The present study showed the overall predominance of
bla-CTX-M-1 (69.56%) followed by
bla-CTX-M-9,
bla-SHV,
bla-TEM and
bla-CTX-M-2 with 40.21%, 33.69%, 32.60% and 4.34% respectively (Table 4). This study discloses the predominance of
bla-
CTX-M-1 gene in this area. Likewise various co-workers across the world have also reported the high frequency of this gene in different sample sources
(Ibrahim et al., 2018; Paghdar et al., 2020; Yadav et al., 2019). Overall distribution of genes according to organisms,
E.
coli revealed highest (69.41%)
bla-
CTX-M-1 followed by
bla-
CTX-M-9 (42.35%),
bla-TEM (32.94%)
bla-
SHV (32.94%) and
bla-
CTX-M-2 (4.71%) where as
Klebsiella spp. revealed highest (71.42%)
bla-
CTX-M-1 followed by
bla-
SHV (42.85%),
bla-
TEM (28.57%) and
bla-
CTX-M-9 (14.28%) (Table 5). It was notable in this study that
bla-
CTX-M-1 and
bla-
CTX-M-9 was present in both isolates but
bla-
CTX-M-2 gene was only present in
E.
coli isolates. Multiple co-existence of
bla genes were also reported which has been mentioned in Table 4. Similar to this finding, multiple co-existence of
bla genes were also noticed by various workers
(Yadav et al., 2019; Tekinar and Ozpinar, 2016).
Antimicrobial resistance is currently a most serious problem that received the attention of larger scientific community across the world. In present study, antimicrobial susceptibility test (AST) of all ESBL positive isolates was performed against 20 antibiotics of 12 different classes. All isolates of
E.
coli and
Klebsiella spp. were found (70-100%) resistant to 3
rd, 4
th generation cephalosporins and ampicillin (Table 6). The plausible factors for high degree of resistance against these antibiotics might be due to persistent antibiotic pressure or acquired from horizontal transmission. Susceptibility pattern of these isolates varied with different classes of non-β-lactam antibiotics.
E.
coli isolates were found 98.83% to 100% sensitive against chloramphenicol, polypeptides and aminoglycosides classes respectively while
Klebsiella spp. was found 85.7% to 100% against chloramphenicol, polypeptides and aminoglycosides (Table 6). There are abundant evidences that corroborate with this finding in India and abroad for both
E.
coli and
Klebsiella spp. isolated from bovine
(Batabyal et al., 2018 ;
Gupta et al., 2019; Ghatak et al., 2013; Ibrahim et al., 2018). In this study resistance to carbapenem antibiotics also reported, even though these antibiotics are not used in animal husbandry practices across the country, this may be attributed as a result of clinical use in human medicine and transfer of these resistant genes to zoonotic pathogens (
Bhardwaj et al., 2015).
Presently, Multi-drug resistant (MDR) isolates is a cause of concern, they may possess severe health complications by limiting the treatment options. In this study 81.52% isolates were found to be MDR
i.
e. resistant to at least one antibiotic of three or more classes of antimicrobials, which highlighted the potential threat by limiting the therapeutic options.
