Study area
The study was carried out in the Department of Veterinary Microbiology, College of Veterinary Science and Animal Husbandry, Acharya Narendra Deva University of Agriculture and Technology, Kumarganj, Ayodhya. The samples were collected from Ayodhya and Sultanpur districts of Awadh region of Uttar Pradesh, India. The study was conducted between January 2020 to March 2021.
Collection of samples
In this study total 240 faecal samples (160 apparently normal and 80 diarrhoeic faeces) were collected from cattle and buffaloes from 5 tehsils of Ayodhya and 3 tehsils of Sultanpur district. Sampling was done randomly and comprising of 10 apparently normal and 5 diarrhoeic faecal samples from each of animal from above mentioned tehsils. The samples were collected into a sterilized test tube by swab technique. All collected samples were immediately transferred to bacteriological laboratory of Department of Veterinary Microbiology, under cold chain for further processing.
Isolation and Identification
All samples were inoculated into 2 mL nutrient broth and incubated at 37°C for 24 hrs. A loopful inoculums were taken and directly streaked on MacConkey agar plates and incubated at 37°C for 24 hrs. Individual lactose fermenting colony (rose pink) colour was picked up and directly streaked on Eosine Methylene Blue agar plates and colonies showing metallic sheen were tentatively suspected as
E. coli. A pure individual colony was taken onto sterilized nutrient agar slant and further identification was done by Gram’s staining and biochemical tests using KBM001 HiMotilityTMBiochemical kit for
E. coli (Hi-media) having combination of 12 tests. The standard test procedure was followed as mentioned in the kit and results were interpreted as per provided interpretation chart by the manufactures of the kit.
Confirmation of the E. coli isolates by PCR analysis
Extraction of genomic DNA
The genomic DNA was extracted using the snap-chill method described by
Franco et al., (2008).
Molecular confirmation of E. coli isolates
All presumptively positive
E. coli isolates were confirmed by PCR analysis using species specific uidA gene as per method described by Anbazhagan
et al. (2010) (Table 1). The PCR reaction was carried out in total volume of 25 µl volume comprising 12.5 µl of 2× EmeraldAmp GT Master Mixture, 8.5 µl nuclease free water, 1µl mixture of forward and reverse primers (0.5µl each primer, conc. 0.5µM each primer) and 3µl template DNA. Amplification was performed in thermo cycler (Bio-Rad, USA). The cycling conditions of the PCR are mentioned in Table 1.
Screening of ESBL and Carbapenemase producing isolates
All confirmed isolates of were subjected to 3
rd, 4
th generation cephalosprins and monobactam (Hi-Media, India) for screening of ESBLs and Carbapenem antibiotic discs (Hi-Media, India) for screening of Carbapenemase producing isolates. The result was interpreted as per
CLSI (2019) guidelines.
Phenotypic confirmation of ESBL and Carbapenemase producing isolates
Double disc synergy test (DDST)
Screened ESBL producing isolates were further confirmed by using ESBL kit 1 and Kit 3 (Hi-media) and similarly Carbapenemase production was confirmed by commercially available imipenem disc (10 µg) alone and imipenem + EDTA (10/750 µg) discs (HiMedia, India). These commercially available discs were placed on Muller Hinton agar (MHA) (HiMedia) plates, infused with1.5´108organisms/ml and incubated at 37°C for 24 hrs. The results were interpreted as per CLSI guidelines (2019).
Minimum inhibitory concentration (MIC) tests
The MIC of ESBL and Carbapenemase producing isolates were determined by using ESBL E-strip (Fig 3) and MBL E-strip (Fig 4) placing on MHA plates infused with 1.5×10
8 organisms/ml and incubated at 37°C for 24 hrs. The results were interpreted as per CLSI guidelines (2019).
Study of multi-drug resistance (MDR) pattern of ESBL and Carbapenemase producing isolates
All Phenotypically confirmed ESBL and Carbapenemase producing isolates were examined for their resistance against 20 antibiotics of 12 different classes (Hi-media) mentioned in Table 3. The antibiotic susceptibility test (ABST) was performed by disc diffusion method on MHA (Hi-media) plates seeded with 1.5×10
8 organisms/ml and incubated at 37°C for 24 hrs and isolates were classified as susceptible and resistant as per interpretation criteria of
CLSI (2019) guidelines and those organisms showing resistance to at least one antibiotic of three or more classes, were considered as MDR bacteria.
Plasmid profiling of ESBL and Carbapenemase producing isolates
Isolation of plasmid DNA from ESBL and Carbapenemase producing isolates
Plasmid DNA of each confirmed ESBL and Carbapenemase producing isolates were extracted using GeneJet plasmid Miniprep kit (Thermo Fisher Scientific, USA) as per manufacturer’s protocol.
Analysis of plasmid profile of ESBL and Carbapenemase producing isolates
To analyze the plasmid profile, each extracted plasmid DNA samples (5 µl) mixed with 2 µl of loading dye (6X) and electrophoresis was done on 0.8% (w/v) agarose gel mixed 1 µl ethidium bromide (conc. 5 µg/ml) at 80-100V for 1 to 1.5 hours using 1kb ladder as marker DNA. At the end of electrophoresis, the gel was visualized under UV trans-illuminator (EZ Gel Documentation system, Bio-Rad) fitted with high resolution digital camera. Plasmid profile was analyzed for the number and size of plasmids.