Detection and Molecular Characterization of Extended-spectrum β-lactamase Producing E. coli and Klebsiella spp. Isolates of Cattle Origin in Eastern Plain Zone of Uttar Pradesh

The emergence of antimicrobial resistance (AMR), especially among Enterobacteriaceae has been increasingly problematic and posses serious threat for both human and animal health (WHO, 2013). It is directly related with the use of these drugs in animal husbandry at insufficient doses and unprofessional choice which limit the treatment options. Resistant bacteria include both pathogenic and commensal organisms, with the later serving as a potential reservoir for mobile resistance elements (Khachatryan et al., 2004). Resistance among ESBL organism is mediated by enzyme Extended-spectrum b-lactamases (ESBLs) that hydrolyses most of the β-lactam antibiotics and mediates resistance against penicillins, 3^rd and 4^th generation cephalosporins (Saravanan et al., 2018). Among Enterobacteriaceae, E. coli and Klebsiella spp. are major ESBL producers and have been identified as global threat due to their increasing prevalence in livestock in last few years (Reuland et al., 2013). ESBL has been also reported in various niches as commensal in human, animals and environment. Such ecological niches may serve as reservoir and vehicle for transmission and dissemination in production animals due to their direct contact with food chain (Madec et al., 2017).
 
Since no in depth study has been done on distribution of ESBL among cattle in this area of study and also their possible role in development of resistance to other species or pathogens. The present study will provide database to help the field veterinarians, pharmaceuticals and policy makers in proper selection of antibiotics for treatment.

Study area
 
This study was carried out in the Department of Veterinary Microbiology, C.V. Sc. and A.H. Kumarganj, Ayodhya. The samples were collected from Ayodhya and Sultanpur district of Eastern Plain Zone of Uttar Pradesh, India. The study was conducted between August 2019 and December 2020.
 
Sample collection
 
Total 240 samples (120 milk samples and 120 faecal samples) were collected from 5 tehsils of Ayodhya and 3 tehsils of Sultanpur district. Samples were collected randomly and sampling consisted of 10 normal and 5 mastitic milk samples from each of the tehsil. Likewise, 10 normal and 5 diarrhoeic faecal samples from above mentioned regions. California mastitis test was used for screening of mastitic milk samples. Approximately 5 ml of milk was collected into sterilized test tubes and faecal samples were collected by swab technique. All collected samples were immediately transported to bacteriology laboratory in an icebox.
 
Isolation and Identification
 
All samples were enriched with 2 ml nutrient broth and incubated for 24 hrs at 37^oC. A loopful of inoculum was directly streaked on MacConkey agar plates added with 2 mg/L cefotaxime and incubated at 37^oC for 24 hr. Plates showing pink colonies were picked up and streaked on Eosine Methylene Blue agar plates. Colonies showing greenish metallic sheen were tentatively considered as E. coli while dark centred light purple colonies with mucoid appearance were suspected as Klebsiella spp. A single pure colony was picked up and transferred to nutrient agar slant. Further identification of the isolates was done by various biochemical tests viz. IMViC pattern, catalase test, nitrate reduction, urease test, triple sugar iron agar and sugar fermentation reaction as per the method of Edward and Ewing (1972) and PCR analysis.
 
Extraction of genomic DNA
 
The DNA templates were prepared by using snap-chill method as described by Franco et al., (2008).
 
Molecular identification of E. coli and Klebsiella spp.
 
All presumptively positive E. coli isolates were confirmed by PCR amplification using species specific uidA and Klebsiella spp. by bacteria specific 16S rRNA gene as per method described by Anbazhagan et al., (2010) and Andersson et al., (2008) respectively (Table 1). PCR reaction was carried out in total 25 µl volume constituted 12.5 µl of 2X EmeraldAmp GT Master Mix, 8.5 µl nuclease free water, 1 µl mixture of the forward and reverse primers (0.5 µl each primer, conc. 0.5 µM each primer) and 3.0 µl of template DNA. Amplification was performed using thermal cycler (Bio-Rad, USA). The cycling conditions of PCR are mentioned in Table 1.
 

 
Antibiotic sensitivity testing
 
All the confirmed isolates (151) were subjected to in vitroantibiotic sensitivity testing against 13 antibiotics of HiMedia mentioned in Table 4. It was performed by disc diffusion method (Bauer et al., 1966) on Muller Hinton agar (MHA) (HiMedia) plates inoculated with 1.5×10⁸ organism/ml and incubated at 37^oC for 24 hrs and isolates were classified as susceptible and resistant based on interpretation criteria of Clinical Standard Laboratory Institute (2019). The isolates showing reduced susceptibility towards cefotaxime, cefpodoxime, ceftazidime, ceftriaxone and aztreonam were screened as ESBL producers.
 
Confirmation of ESBL producing E. coli and Klebsiella spp. by phenotypic methods
 
Double disc synergy test (DDST)
 
The screened isolates were further confirmed by DDST using ESBL kit 1 and Kit 3 (Hi-media) (Fig 3). The commercially available discs were placed at 25 mm apart on MHA plates inoculated with1.5×10⁸ organism/ml and incubated at 37^oC for 24 hrs. The results were interpreted as per CLSI guidelines (2019).
 

 
Minimum inhibitory concentration (MIC) ESBL E-test
 
This test was done by placing E-strip on MHA plates inoculated with 1.5´108 organism/ml and incubated at 37oC for 24 hrs. The result was interpreted as per CLSI guidelines (2019) (Fig 4).
 

 
Detection of ESBL genes by polymerase chain reaction
 
Extraction of plasmid DNA
 
Single pure colony of ESBL positive isolates were inoculated into 10 ml of Luria-Bertani (LB) broth medium (HiMedia, India) and incubated at 37^oC for 18 hrs in shaking incubator. After that plasmid DNA was isolated using GeneJet plasmid Miniprep kit (Thermo Scientific) as per the instruction of the manufacturers.
 
Detection of CTX-M genes (bla-_CTX-M-1, bla-_CTX-M-9), bla-_TEM and bla-_SHV genes
 
Genotypic confirmation of ESBL genes was done in a total reaction volume of 25 µl for CTX-M and bla-_TEM as per method described by Dallenne et al., (2010) and bla-_SHV genes by Bhattacharjee et al., (2007). Amplicon size, primer sequence of targeted genes and cyclic conditions of PCR are mentioned in Table 2. Multiplex PCR was performed for CTX-M genes and simplex PCR was performed for bla-_TEM and bla_-SHV genes.  Amplified products (5 µl) were mixed with 3 µl of bromophenol blue dye (6X) and electrophoresis was done in 2% agarose gel for CTX-M genes using 50bp ladder and in 0.8% gel for bla-_TEM and bla-_SHV genes using 1 kb ladder at 60-70 mA for 40 min and gel was visualized using the UV illuminator (GeNei Bangalore, India).
 

A total 240 samples of milk and faeces were collected, out of which 152 (63.33%) isolates were presumptively identified as E. coli and 24 (10.0%) as Klebsiella spp. on the basis of morphological and biochemical characteristics. Further PCR analysis of these isolates confirmed 135 (56.25%) as E. coli and 16 (6.67%) as Klebsiella spp. (Table 3, Fig 1 and 2). In the present study, E. coli was predominant among isolates recovered from both milk and faecal samples. These finding were in concordance with the findings of Ibrahim et al., (2018) and Kotsoana et al., (2019). Higher isolation rate of E. coli in this study might be attributed to high prevalence of E. coli in the gut flora of ruminants.
 

 

 

       
The results of in vitro antimicrobial susceptibility test (AST) are mentioned in Table 4. The AST revealed that most resistant antibiotics were ampicillin (89.40%) followed by cefotaxime (72.18%), cefpodoxime (71.52%), ceftriaxone (66.9%), aztreonam (61.0%) and ceftazidime (54.30%) but drugs such as gentamicin, amikacin and chloramphenicol were found to be 100% sensitive. There are several reports that corroborate with the finding of this study (Ramasamy et al., 2021; Batabyal et al., 2018; Badri et al., 2017). In this study, carbapenem antibiotics like imipenem and meropenem also showed resistance against these isolates, 18.75% to 12.50% for Klebsiella spp. and 8.89 to 3.7% for E. coli respectively (Table 4). Although these antibiotics are not allowed to use in animal husbandry practices anywhere in the country, even then resistance in animal isolates may emerged and propagated as a result of clinical use in human medicine and transfer of these resistant genes to zoonotic pathogens (Bhardwaj, 2015). In this study 81.52% (75/92) isolates were found to be multidrug resistant (MDR), which highlighted a potential threat to human health and thereby limiting the therapeutic options. 
 

       
The present study also aimed to determine the proportion of ESBL phenotypes among clinical and apparently healthy samples. Total 151 isolates (135 E. coli and 16 Klebsiella spp.) were subjected to screening and confirmatory phenotypic tests. Out of 151 isolates, 129 (85.43%) were presumed as ESBL producer by AST. In phenotypic confirmatory testing, 101 (66.88%) isolates were confirmed as ESBL positive by DDST and 92 (60.92% by ESBL-E strip test. Final confirmation was done by PCR analysis which revealed 87 (57.61%) ESBL positive isolates (Table 5). There was little difference in the sensitivity of both phenotypic confirmatory tests and PCR analysis and this observation corroborated with the findings of Badri et al., (2017) and Olowe et al., (2015).
       

 
Gene distribution study of ESBL positive isolates was carried out using PCR by targeting CTX-M (bla-_CTX-M-1, bla-_CTX-M-9), bla-_TEM and bla-_SHV genes (Fig 5, 6, 7). Over all prevalence of ESBL gene was found to be 65.21% for bla-_CTX-M-1 followed by bla-_CTX-M-9, bla-TEM and bla-_SHV with 41.30%, 31.52% and 27.17%, respectively. The present finding revealed predominance of bla-_CTX-M-1 gene in this area. Similarly various co-workers across the world have also reported high frequency of bla-_CTX-M gene in different sample sources (Badri et al., 2017; Ibrahim et al., 2018; Paghdar et al., 2020; Olowe et al., 2015; Yadav et al., 2019; Schmid et al., 2013). Multiple co-existences of bla genes were also observed which has been mentioned in Table 6. Similar to this finding, co-existence of bla genes was also reported by various workers (Yadav et al., 2019; Tekinar and Ozpinar 2016).
 

 

 

 

This study highlighted, higher occurrence of ESBL producing E. coli than Klebsiella spp. and most of the isolates showed resistance to ampicillin, 3^rd and 4^th generation cephalosporins, which is an alarming situation for this area. Despite this, some isolates of E. coli and Klebsiella spp. also exhibited resistance against carbapenems, even without its use in animal husbandry practices, which are not a good sign from public health point of view. Therefore, a specific study on rational use of antibiotics and continuous monitoring for resistance genes against these antibiotics in livestock is warranted.

The author is thankful to Dean, College of Veterinary Sciences and Animal Husbandry, Kumarganj and livestock owners of the Ayodhya and Sultanpur districts for their kind support during collection of samples.

None.