Investigation On The Diversity And Antibiotic Resistance Of Bacteria Isolated From Teat Cup Liners In Dairy Farms In Some Areas Of Shandong Province

Milk and its products have high nutritional value and can provide humans with high-quality protein,biologically available amino acids,multiple vitamins,and minerals.Global milk consumption exceeds 800 million tonnes per year and is still growing.The teat cup is an important part of the milking machine,its the liner comes into direct contact with the skin of the udder surface and the raw cow’s milk during milking.In a proper milking process the udder is cleaned and disinfected prior to milking and it is the residual bacteria that contact with the liner,so the bacteria isolated on the teat cup liner would be the dominant flora on the cow’s udder surface.Understanding the bacterial diversity and antibiotic resistance of post-milking teat cup liners will help reflect the risk of bovine mastitis transmission through the cup and the probability of raw milk contamination,and will aid in the selection of clinically sensitive antibiotics with implications for dairy farming,food safety,and public health.In this study,swab samples were collected from the liners of teat cups after milking and the samples were tested for isolated bacteria and antibiotic resistance to analyse the diversity of teat cup liner bacteria and antibiotic resistance.Using sterile cotton swabs,248 samples were collected from the inner lining of milk cups from 6 dairy farms in 3 cities in Shandong Province after milking.Bacterial isolation and identification were conducted in the laboratory.The bacterial positive rate of the samples was 61.69%（153/248）.A total of 276 strains of bacteria were isolated from 153 positive samples,belonging to 73 different species and genera.Among them,Bacillus licheniformis（39/276,14.13%）had the highest isolation rate,followed by Bacillus pumilus（20/276,7.25%）,Bacillus cereus（17/276,6.16%）and Bacillus subtilis（16/276,5.78%）.The minimum inhibitory concentrations（MIC）of nine antibiotics,including ampicillin sodium（AMP）,streptomycin sulfate（STS）,neomycin sulfate（Nm）,lincomycin hydrochloride（LIN）,sulfadiazine（SD）,ceftriaxone sodium（CRO）,gentamicin sulfate（GM）,levofloxacin（LVX）,and doxycycline hydrochloride（DO）,were determined against 276 bacterial strains according to the criteria of the American Society for Clinical and Laboratory Standards guidelines.The results showed that the overall resistance rate ranged from 0%-87.37%,with the highest being lincomycin hydrochloride with 87.37%（166/190）,sulfadiazine,streptomycin sulfate,ampicillin,gentamicin sulfate,ceftriaxone and levofloxacin with 61.05%（116/190）,42.63%（81/190）,11.05%（21/190）,10%（19/190）,6.84%（13/190）and 1.05%（2/190）,and the lowest resistance rate was 0%for doxycycline.Among them,33.16%（63/190）of the strains showed multi-drug resistance,and 87.3%（55/63）of the multi-drug resistant strains were resistant to three to four antibiotics,with the strongest being resistant to six antibiotics at the same time.Subsequently,nine antibacterial genes,includingβ-lactams and polymyxins,were detected by designing primers and using PCR.The highest positive rate was for the sulfonamide resistance gene sul1（55.43%）,followed by the aminoglycoside resistance gene ant（4′）（51.09%）,the tetracycline resistance gene tet（M）（25.36%）,the quinolone（3.62%）,β-lactam resistance gene bla _KPC（3.62%）,polymyxin resistance gene mcr-1（0.72%）and lincomycin resistance gene Lnu B（0.72%）,andβ-lactam resistance gene bla _NDM-1 and aminoglycoside resistance gene Aph（2″）were not detected.In summary,the rate of positive bacteria in the post-milking cup liners of the dairy farms in Shandong province investigated in this study was high,and the presence of Bacillus cereus,which can lead to milk spoilage and food poisoning,and Staphylococcus haemolyticus,which can cause serious infections;the antibiotic resistance of the isolated bacteria was more common,with the rate of resistance to lincomycin reaching 87.37%and sulfonamide resistance also exceeding 50%;the detection rate of some antibiotic resistance genes was high,such as the detection rate of sul1,which was 55.43%.Farming enterprises should optimize the milking process and reasonably arrange or reduce the use of antibiotics to reduce the risk of raw milk contamination and mastitis transmission.