Alarming multidrug resistance in Staphylococcus aureus isolated from raw milk of cows with subclinical mastitis: Antibiotic resistance patterns and occurrence of selected resistance genes.

Bovine mastitis is a widespread and costly disease that affects dairy farming globally, characterized by mammary gland inflammation. Bovine intramammary gland infection has been associated with more than 135 different pathogens of which Staphylococcus aureus is the main etiology of sub-clinical mastitis (SCM). The current study was designed to investigate the prevalence, antibiotic resistance pattern, and the presence of antibiotic resistance genes (mecA, tetK, aacA-aphD and blaZ) in S. aureus isolated from the raw milk of cows with subclinical mastitis. A total of 543 milk samples were collected from lactating cows such as Holstein Friesian (n = 79), Sahiwal (n = 175), Cholistani (n = 107), and Red Sindhi (n = 182) from different dairy farms in Pakistan. From the milk samples microscopic slides were prepared and the somatic cell count was assessed to find SCM. To isolate and identify S. aureus, milk was streaked on mannitol salt agar (MSA) plates. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). All the biochemically confirmed S. aureus isolates were molecularly identified using the thermonuclease (nuc) gene. The antibiotic resistance pattern of all the S. aureus isolates was evaluated through the disc diffusion method. Out of 543 milk samples, 310 (57.09%) were positive for SCM. Among the SCM-positive samples, S. aureus was detected in 30.32% (94/310) samples. Out of 94 isolates, 47 (50%) were determined to be multidrug resistant (MDR). Among these MDR isolates, 11 exhibited resistance to Cefoxitin, and hence were classified as methicillin-resistant Staphylococcus aureus (MRSA). The S. aureus isolates showed the highest resistance to Lincomycin (84.04%) followed by Ampicillin (45.74%), while the least resistance was shown to Sulfamethoxazole/Trimethoprim (3.19%) and Gentamycin (6.38%). Polymerase chain reaction (PCR) analysis revealed that 55.31% of the isolates carried blaZ gene, 46.80% carried tetK gene, 17.02% harbored the mecA gene, whereas, aacA-aphD gene was found in 13.82% samples. Our findings revealed a significant level of contamination of milk with S. aureus and half (50%) of the isolates were MDR. The isolated S. aureus harbored various antibiotic resistance genes responsible for the absorbed phenotypic resistance. The alarmingly high prevalence of MDR S. aureus isolates and MRSA strains in these cases possess a serious risk to public health, emphasizes the urgent need to address this issue to protect both human and animal health in Pakistan.

Epidemiological analysis of anaplasmosis in cattle from Khyber Pakhtunkhwa, Pakistan.

Background and Aim: Anaplasmosis, a tick-borne disease affecting livestock caused by the bacteria Anaplasma, poses a global concern. This study aimed to estimate the prevalence, spatiotemporal variation, and associated risk factors of anaplasmosis in cattle from the Bannu and Lakki Marwat districts of Khyber Pakhtunkhwa, Pakistan. Materials and Methods: This study used 197 cattle exhibiting clinical symptoms of anaplasmosis in natural settings. Microscopic examination was used to estimate the prevalence. Potential risk factors, such as sampling regions and months, gender, breed, and age were studied. Results: The study revealed an overall anaplasmosis prevalence of 19.79%. Bannu district exhibited a higher occurrence at 22.10%, compared to Lakki Marwat district at 17.64%. Young cattle (<2 years) demonstrated a notably higher incidence of anaplasmosis (26.78%) compared to adults (>5 years), which had a prevalence of 12.35% (p < 0.05). Female cattle (22.36%) were more susceptible than male cattle (11.11%). Prevalence peaked in June (45.71%) and was lowest in February (3.57%). Crossbred cattle had a higher prevalence (23.52%) than purebred cattle (11.47%). Conclusion: Anaplasmosis can be effectively controlled using a comprehensive approach encompassing selective breeding for resilience, targeted care of young calves and females, effective tick control during warmer months, consistent use of insecticides, and proactive risk factor management. Raising awareness among farmers through diverse channels, including media, is pivotal to bolster tick-borne disease management strategies.