Study design
The different livestock species from unorganized animal herds were chosen following cluster sampling method in which Chikkaballapur district was selected as primary sampling unit. Within this district, four clusters designated as blocks were selected based on geographical distance (approximately 30 kilometers apart). Within each cluster, four epi units (village) were selected randomly (approximately 5-10 kilometers apart). In the second strata, the households (HH) were randomly selected based on the sample availability within each epi unit to collect 15 samples from all the animal species (cow/buffalo milk samples- 8, poultry cloacal swabs-3, sheep/goat rectal swabs-2, pig rectal swabs-2). Animal species was relaxed based on the availability and importance of animal domestication in that geographical area. The farmers were contacted before the study by key veterinary health care staff and informed consents were obtained from all the households.
Sample information sheet
A questionnaire was designed which contained the general information (Block name, village name, farmers name, contact number and date of sample collection), epidemiological metadata (latitude, longitude and disease outbreak history), sample source and nature (sample details from different food animals, animal handler details), management practices (rearing practices, feeding practices, milking practices, sanitary practices) and sample transport information (sample ID, date of transport) along with the consent for each sample was collected. The data was entered in M.S Excel software and blind folded data cleaning was done. The geographical indication coordinates were noted, and map (Fig.1) was prepared in QGIS software version 3.14.
Samples collected
As per study design out of 240, 236 samples (105 cow milk samples, 23 buffalo milk samples, 45 poultry cloacal swabs, 61 sheep/ goat rectal swabs and 2 pig rectal swabs) were collected from 200 households in sixteen epi units under four clusters of Chikkaballapur District, Karnataka (Chikkaballapur, Gauribidanur, Sidlaghatta, Chintamani) between January to December 2019 (Fig.1). The sample distribution details are annexed in Table 1.
Table 1
Isolation of Staphylococcus spp. and E. coli from different sampling sites
Block | Village | Households | Latitude | Longitude | Cow milk | Buffalo milk | Poultry rectal swab | Sheep/Goat rectal swab | Pig rectal swab | Total | Staphylococcus spp | E. coli |
Chikkaballapur | Kalavara | 9 | 13.416 | 77.668 | 6 | 2 | 3 | 4 | 0 | 15 | 6 | 7 |
| Manchanabale | 9 | 13.442 | 77.831 | 7 | 1 | 3 | 4 | 0 | 15 | 2 | 10 |
| Thumukalahalli | 12 | 13.376 | 77.803 | 6 | 2 | 3 | 4 | 0 | 15 | 9 | 11 |
| Gundalagurki | 8 | 13.432 | 77.689 | 4 | 4 | 3 | 2 | 2 | 15 | 7 | 8 |
Subtotal | 60 | 24 | 36 |
Gauribidanur | Haleupparahalli | 10 | 13.592 | 77.448 | 4 | 4 | 0 | 4 | 0 | 12 | 8 | 4 |
| Hosuru | 13 | 13.536 | 77.504 | 8 | 0 | 3 | 3 | 0 | 14 | 10 | 9 |
| Mudigere | 15 | 13.561 | 77.468 | 5 | 3 | 3 | 4 | 0 | 15 | 7 | 6 |
| Kadalaveni | 15 | 13.594 | 77.583 | 4 | 4 | 3 | 4 | 0 | 15 | 9 | 7 |
Subtotal | 56 | 34 | 26 |
Sidlaghatta | Kakachokkondahalli | 9 | 13.315 | 77.854 | 8 | 0 | 3 | 4 | 0 | 15 | 6 | 7 |
| Nagamangala | 15 | 13.256 | 77.947 | 8 | 0 | 3 | 4 | 0 | 15 | 6 | 8 |
| Gattamaranahalli | 15 | 13.258 | 77.858 | 8 | 0 | 3 | 4 | 0 | 15 | 7 | 8 |
| Jangamakote | 15 | 13.31 | 77.839 | 7 | 1 | 3 | 4 | 0 | 15 | 8 | 7 |
Subtotal | 60 | 27 | 30 |
Chintamani | Kendanahalli | 10 | 13.332 | 78.079 | 7 | 1 | 3 | 4 | 0 | 15 | 6 | 8 |
| Thimmasandra | 14 | 13.393 | 78.092 | 7 | 1 | 3 | 4 | 0 | 15 | 6 | 5 |
| Nayanahalli | 15 | 13.412 | 78.041 | 8 | 0 | 3 | 4 | 0 | 15 | 7 | 2 |
| Konapalli | 15 | 13.393 | 78.045 | 8 | 0 | 3 | 4 | 0 | 15 | 9 | 5 |
Subtotal | 60 | 28 | 20 |
Grand total | 200 | | 236 | 113 (88.28%) | 112 (47.45%) |
*value in parenthesis represents percentage. |
Isolation, phenotypic and genotypic identification of bacterial isolates
All the collected samples were transported to the Bacterial epidemiology laboratory, ICAR-NIVEDI, Bangalore, India. For isolation of Staphylococcus spp., 128 milk samples were inoculated into Mannitol salt broth (MSB) and incubated for 18-24 h at 37 0C for enrichment. A loopful of broth culture was streaked on to Mannitol salt agar (MSA) and incubated for 18-24 h at 37 0C. The characteristic colonies were purified on Brain Heart Infusion agar. Staphylococci were tentatively identified based on colony morphology, pigment production, Gram’s staining, catalase and oxidase tests as per the standard protocol. For the isolation of E. coli, all 236 samples were inoculated in MacConkey lactose broth (MLB) for enrichment and incubated for 18-24 h at 37 0C. A loopful of broth culture was streaked onto MacConkey lactose agar plates (MLA) and incubated at 37 °C for 18–24 h. Phenotypic identification of E. coli was carried out based on colony morphology, lactose fermentation, Gram’s staining, IMViC tests (indole methyl red; Voges–Proskauer; citrate utilization) and oxidase test (Source of media: Himedia Laboratories, Mumbai, India).
For molecular confirmation, DNA was extracted from pure culture using QIAamp DNA Mini kit as per the manufacturer’s protocol (Qiagen, Germany). The quantity and quality of DNA was evaluated by using a NanoDrop2000 (Thermo Scientific, Waltham, USA) and on 0.8% agarose gel electrophoresis respectively. Genus-specific PCR for the detection of Staphylococci (Shome et al. 2012) and E. coli-specific multiplex PCR (Modak et al. 2012) were performed. The ATCC 25922 and ATCC 33591 were used as positive controls for E. coli and S. aureus respectively. For identification of five major Staphylococcus spp. (S. epidermidis, S. sciuri, S. chromogenes, S. haemolyticus, and S. aureus) multiplex PCR was performed (Shome et al. 2011).
Antimicrobial Susceptibility Testing (AST)
AST was performed by Kirby–Bauer disk diffusion method (Bauer et al. 1996) against 10 antimicrobial agents including cefoxitin, a marker of methicillin resistance for Staphylococcus isolates. Staphylococcus aureus (ATCC-25923) was used as a quality control strain. Minimum inhibitory concentration (MIC) was determined only for vancomycin by broth microdilution method. The E. coli isolates were tested against 17 antibiotics representing different classes of antibiotics (penicillins and beta-lactams, cephems, carbapenams, aminoglycosides, tetracycline, quinolone, folate pathway inhibitors, phenicols and polymyxin). The results were entered in WHONET software (version 5.6) and were categorized as susceptible, intermediate, and resistant based on CLSI guidelines (2021). Isolates non-susceptible to at least one agent in ≥3 antimicrobial classes were categorized as Multidrug resistant (MDR) (Magiorakos et al. 2012). The ESBL and AmpC producing E. coli isolates were confirmed by combined disc assay method and for colistin assay, broth microdilution was performed.
Molecular detection of determinants of antibiotic resistance
The phenotypically resistant Staphylococcus isolates were characterized for the detection of methicillin resistant determinant mecA and mecC genes as previously described (Al Talib et al. 2009; Christiane et al. 2011). E. coli isolates resistant to third and fourth generation cephalosporins were investigated for the presence of CTX-M, SHV, TEM, and OXA. Further, cefoxitin and cefotetan resistant isolates were screened for AmpC, ACC, FOX, MOX, DHA, CIT and EBC genes. Additionally, the carbapenem resistant strains were screened for NDM, IMP, VIM and KPC (Dallenne et al. 2010; Koovapra et al. 2016).