Risk Factors of Sub-Clinical Mastitis, Antibiogram and Genotypic Analysis of Staphylococcus Spp. And Enterobacteria Resistant Bacteria Isolated from Humans and Lactating Dairy Cows from Small-Holder Farms in Gatundu Sub-County, Kenya

Abstract

The emergence and subsequent widespread antimicrobial resistance significantly impact global health. The aim of the study was to determine risk factors of sub-clinical mastitis, antibiogram and genotypic analysis of Staphylococcus spp. and Enterobacteria against antibiotic resistant bacteria isolated from Humans and lactating dairy cows from small-holder farms in Gatundu Sub-County, Kenya. The cross-sectional field and laboratory study involved the collection of milk samples from one hundred and sixty-four lactating dairy cows, and skin (neck region) swabs from one hundred and twenty human respondents from same household. The milk samples were subjected to California Mastitis Test (CMT) and thereafter cultured and bacteria identified based on growth morphology, color on the media, biochemical tests and use of API 20E Kit. Further, the antimicrobial susceptibility testing was carried out using Kirby Bauer disk diffusion method against 11 antibiotics such as; gentamycin, clindamycin, tetracycline, chloramphenicol, amoyclav, ampicillin, sulphamethoxazole-trimethoprime, oxacillin, vancomycin, cefoxitin and ciprofloxacin. DNA was extracted from the isolated Staphylococci spp. and Enterobacteria spp. Polymerase chain reaction (PCR) amplification was used to determine isolates positive for the resistant genes, mecA and ESBLs (blaTEM, blaSHV, blaCTX, blaKPC). The amplicons were resolved in a 1.5% gel. The purified PCR samples were sequenced using a 3730xl DNA Analyzer sequencer and based on BigDye™ Terminator v3.1 Cycle Sequencing Kit. The phylogenetic tree was constructed using MEGA11. The evolutionary distances were calculated using the Maximum Likelihood method. The robustness of the tree was assessed with 1,000 bootstrap replicates. From the 164 lactating dairy cows, the prevalence of sub-clinical mastitis based on CMT was 39.6%. The bacteria isolated from the milk samples were Coagulase Negative Staphylococci (CoNS) (50.3%), S. aureus (31.8%) Pantoea spp. (1.9%), Enterobacter cloacae (1.3%), Citrobacter koseri (0.6%), Klebsiella oxytoca (0.6%) and Serratia spp (0.6%). The bacteria isolated from humans included. S. aureus (49.4%), CoNS (16.9%), Pantoea spp. (13%), Serratia spp. (13%), Bukholderia cepacian (3%), Enterobacter spp. (3%), Yersinia enterocolitica (1.3%) and Pasturella aerogenes (1.3%). Antimicrobial susceptibility testing showed CoNS (100%) and S. aureus (86.8%) were mostly resistant to gentamycin but highly susceptible to sulphamethoxazole-trimethoprim (95.5%). Similarly, age had higher burden antimicrobial resistance bacteria among respondents of >50 years (p = 0.011, OD=1.745) as well as antibiotic usage (p = 0.025, OD = 0.204). The study showed that the cows with previous history of sub-clinical mastitis had a higher prevalence of mastitis (p = 0.026, OD = 2.503) as compared to those without such a history. The findings in the current study showed lack of the mecA gene among the 50 Staphylococci spp. isolates screened. However, blaTEM was found in 17 isolates, (41.5%). Phylogenetic analysis showed a close relationship between the Staphylococci and enterobacteria isolates from human and dairy cows. The study recommends further research on differentiating the coagulase negative Staphylococci spp. (CoNS) into species level. There is need for involvement of One health approach in control and genomic surveillance in the occurrence of drug-resistant pathogens in the study area.