Investigation Of Drug Resistance Of Main Pathogens From Bovine Mastitis And Study On The Inhibitory Effect Of Quercetin On The Biofilm Of Pseudomonas Aeruginosa

Mastitis seriously affect the health and economic value of dairy cows,generally caused by pathogenic microbial infections,and its etiology and influencing factors are more and complex,which brings difficulties to clinical treatment and prevention and control,and seriously hinders the economic development of China’s dairy industry.At present,the treatment of mastitis in dairy cows mainly uses antibiotic,but with the widespread use and abuse of antibacterial drugs,leading to the generation of drug-resistant strains of bacteria.Therefore,monitoring the pathogenic bacteria and drug resistance of mastitis on farms is important for the scientific prevention and control of mastitis in dairy cows.In this study,samples from 12 farms in Ningxia were collected for isolation and identification of cryptic and clinical mastitis and antimicrobial susceptibility testing,from which two strains of Pseudomonas aeruginosa resistant to gentamycin were screened to investigate the combined effect and anti-biofilm activity of quercetin(QEN)and gentamycin(GM).The main findings were as follows:1.360 mastitis samples were collected from 12 dairy farms,including 264 recessives and 96 clinical types,and the bacteria were isolated and identified by microbiological methods,Gram stain microscopy,flight mass spectrometry and 16S rDNA sequencing.Result indicates that we were isolated and identified 477 bacteria and 7 mycoplasma,containing 16 genera,37 species of bacteria and 1 mycoplasma.Among them,E.coli had the highest detection rate(20.66%),followed by coagulase-negative Staphylococcus,Pseudomonas aeruginosa,Enterococcus and Klebsiella,with detection rates of 20.50%,8.68%,6.20%and 4.75%,respectively.The main infectious pathogenic bacteria detected were Staphylococcus aureus,Streptococcus lactis and Mycoplasma bovis.The isolation rate of mastitis-causing bacteria varied widely among different dairy farms,which may be related to factors such as the feeding environment and management practices of dairy farms.2.Antimicrobial susceptibility testing of several important pathogenic bacteria isolated was performed using the micro broth dilution method.The results showed that the monitored bacteria were generally severely resistant to β-lactams and sulfonamides.Among them,Gram-positive bacteria had higher resistance rates to penicillin,benzocillin and sulfisoxazole;Gram-negative bacteria had higher resistance rates to ampicillin,sulfisoxazole and mucormycin.The multidrug-resistant(MDR)rates were all greater than 70%.3.The MIC of QEN and the combined drug sensitivity test by checkerboard method were performed on the isolated Pseudomonas aeruginosa(PA),and then the effect of QEN combined with gentamicin on PA biofilm formation was observed by crystalline violet staining,scanning electron microscopy and laser confocal electron microscopy.The results showed that there was no significant inhibitory effect of high concentration of QEN on PA alone,and the inhibitory effect was significantly enhanced when combined with GM,and the synergistic and additive effects were determined by FIC values.The results of crystalline violet staining revealed that the inhibitory effect of QEN and GM on PA biofilm formation was not significant when acting alone and was significantly enhanced when combined with 1/2FIC concentration,and the inhibitory effect increased as the combined concentration increased.The results of scanning electron microscopy and laser confocal microscopy showed that the combination of QEN and GM could significantly reduce the thickness of PA biofilm,resulting in a significant decrease in the proportion of viable bacteria in the membrane and serious damage to its spatial structure,further verifying that the combination of QEN and GM effectively increased the anti-biofilm activity.In summary,360 milk samples from 12 farms in Ningxia were collected for pathogenic bacteria isolation and identification and drug susceptibility analysis to determine the prevalence of mastitis pathogens and drug resistance of major pathogens in the region,providing a scientific basis for the prevention,control and treatment of mastitis in dairy cows.The antibacterial and anti-biofilm activities of QEN and GM alone and in combination against drug-resistant PA were also determined to provide new ideas for the clinical treatment of mastitis in dairy cows infected with PA.