Biocide susceptibility and resistance genes in udder surface Staphylococcus from Indian peri-urban dairy farms

Poor udder hygiene practices are a risk factor for mastitis among unorganized peri-urban dairy farms in India. The common practice of washing the udder without wiping off excess water at the time of milking dislodges loosely bound pathogens from the skin, allowing them to accumulate at the teat tip as water runs off, creating a potential but often overlooked route for intramammary infection. We isolated Staphylococcus aureus (S. aureus) and non-aureus staphylococci and mammaliicocci (NASM) from udder washings and evaluated their susceptibility to chlorhexidine (CHX), cetyl trimethyl ammonium bromide (CTAB), and povidone iodine (PI). In farms that practiced teat disinfection, TVC decreased from 7 × 10³ CFU/mL to 1.89 × 10³ CFU/mL after milking. In contrast, among the farms that relied solely on water washing without disinfection, TVC increased from 7.28 × 10³ CFU/mL to 25.26 × 10³ CFU/mL (p < 0.01). Biocide susceptibility testing revealed higher MIC values among S. aureus isolates compared to NASM. The epidemiological cut-off values (MIC₉₅) for S. aureus were 4.88 microg/mL for CHX, 3.91 microg/mL for CTAB, and 62.5 microg/mL for PI, whereas for NASM, these values were 0.305 microg/mL, 0.977 microg/mL, and 39.06 microg/mL, respectively. A higher proportion of S. aureus isolates exhibited MIC values at or above the epidemiological cut-off. Mann-Whitney U test showed MIC distributions between S. aureus and NASM were significantly higher among S. aureus isolates for CHX and CTAB. A comparison of MIC values between isolates with and without the presence of the biocide tolerance genes qacA/B and smr genes revealed no statistically significant differences.