Sample collection
The present study was carried out during the year 2021-2023, at Department of Veterinary Pharmacology and Toxicology, VCRI, Tirunelveli, Tamil Nadu. A total of 540 caecum samples, 180 each from broilers, desi chicken and Japanese quail were collected randomly in aseptic polythene bag at different poultry meat retail outlets of Tirunelveli. Samples were processed at the laboratory or maintained at 4°C until further processing.
Isolation and phenotypic identification of ESBL E. coli
All the collected samples were processed for isolation of
E. coli on MacConkey agar, EMB agar (HiMedia, India) followed by biochemical characterization (IMViC test) as per the standard methods. Biochemically characterized isolates of
E. coli were cultured on MacConkey agar, which was supplemented with cefotaxime at 2 mg/L. Any growth on the plates was considered as ESBL producer and/or AmpC resistant
E. coli (Costa et al., 2009). Further, isolates shown growth on these plates was also subjected to a disk diffusion assay with commercially available antimicrobial disc (HiMedia, India): cefotaxime (CTX, 30 µg), ceftriaxone (CTR, 30 µg), ceftazidime (CAZ, 30 µg) and cefepime (CPM, 30 µg). The isolates showing resistant to any of the above antimicrobials were further subjected for combination disc method for detection of ESBL production, which is based on the principle that the ESBL producing isolate will exhibit an expanded zone of inhibition against third or fourth generation cephalosporin in the presence of beta-lactamase inhibitor like clavulanic acid (
CLSI, 2015).
Antimicrobial susceptibility testing
Phenotypically confirmed ESBL producing
E. coli isolates were tested for antimicrobial susceptibility using six classes of twelve different commercially available antimicrobial discs such as Cefpodoxime (CPD-10 µg), Cefepime (CPM-30 µg), Ceftazidime (CAZ-30 µg), Cefperazone (CPZ-75 µg), Cefotaxime (CTX-30 µg), Ceftriaxone (CTR-30 µg), Amoxycillin+Clavulanic acid (AMC-20/10 µg), Co-trimoxazole (COT-1.25/23.75 µg), Oxytetracycline (O-30 µg), Gentamicin (GEN-10 µg), Enrofloxacin (EX-5 µg) and Chloramphenicol (C-30 µg) by Kirby and Bauer disk diffusion method (
Hudzicki, 2009). The Clinical Laboratory Standards Institute (CLSI) guidelines were followed to measure the zone of inhibition and isolates were categorized accordingly as sensitive, intermediate and resistant.
Characterization of ESBL genes and virulence genes by PCR
All the phenotypically confirmed ESBL producing
E. coli isolates were further confirmed by PCR amplification of
uspA gene specific primers for
E. coli. The
E.coli isolates confirmed by PCR were further subjected to PCR amplification of important ESBL genes
viz.,
blaTEM, blaCTXM and
blaSHV and
blaOXA-1. Shiga toxin virulence genes
stx1 and
stx2 by using gene specific primers (Table 1). In brief, the bacterial DNA extraction from the isolates was done by Snap chill method
(Mandal et al., 2017). The uniplex PCR assays were performed with reaction mixture comprised of 12.5 µL of 2X Taq DNA Polymerase Master Mix RED (Ampliqon, Denmark), 1 µL (10 pM) of each primer, 2 µL of extracted DNA template and the final volume of 25 µL adjusted by addition of NFW and subjected to amplification in thermocycler (Eppendorf Mastercycler® nexus X2, Germany). The PCR cyclical conditions of
uspA, blaTEM, blaSHV, blaCTXM and
blaOXA-1 were set at an initial denaturation of 94°C for 5 min followed by 35 cycles each of denaturation at 94°C for 30 sec, annealing at 60°C for 15 sec, extension at 72°C for 30 sec and final extension at 72°C for 5 min whereas for
stx1 and
stx2 initial denaturation at 94°C for 5 min followed by 35 cycles each of denaturation at 94°C for 1.5 min, annealing at 62°C for 1.5 min, extension at 72°C for 1.5 min and final extension at 72°C for 7 min. Electrophoresis was carried out in 1.5% agarose gel to visualize the PCR products and the images were captured by gel documentation system (Gelstan-1312).
DNA sequencing and phylogenetic analysis
Among the ESBL genes, only the
blaTEM gene from broiler, desi chicken and Japanese quail was subjected for gene sequencing and phylogenetic analysis. GeneJET gel extraction kit (Thermo Fisher scientific, USA) was used for purification of the amplified products from the excised gel as per manufacturer’s directions. The purified DNA was sequenced using the same set of PCR primers at Eurofins Genomics India Pvt Ltd., Bangalore.
blaTEM gene sequences were submitted to NCBI database and obtained accession numbers. The present study sequences were compared with 11 distinct isolates of the
E. coli blaTEM gene from GenBank database. Multiple alignment and comparison of the study sequences with GenBank references were performed using clustal W. Further, phylogenetic tree construction and molecular evolutionary analysis were conducted using MEGA (Molecular Evolutionary Genetics Analysis) version 11.0 by neighbour joining method with maximum likelihood substitution model at 1000 boot straps replicates
(Tamura et al., 2021).
Statistical analysis
All the statistical analyses were performed using SPSS computer software version 22. Chi-square analysis was performed to compare the isolation of
E. coli and phenotypic confirmation of ESBL
E. coli by CDDT for broiler, desi chicken and Japanese quail.
