| Bovine mastitis is the most common disease in dairy farming and causes serious economic losses to the dairy farming industry.The effective control of bovine mastitis is an urgent problem in dairy farming and a global hot research topic.There are few studies on the main pathogens of mastitis in Hubei,in this study,the isolation and identification the samples of Salmonella,E.coli,S.aureus,Coagulase-negative staphylococci,S.uberis,S.agalactiae,and S.dysgalactiae from 5 large-scale dairy farms in Hubei province were carried out,and the sensitivity of the isolates to antimicrobial sensibility was determined;the genotypes,serotypes,virulence genes,resistance genes and molecular characteristics of S.aureus isolates were studied by PCR and Whole-Genome Sequencing,it provides theoretical basis for precise prevention and control of clinical bovine mastitis and scientific basis for clinical rational drug use and reduction of bacterial resistance to antibiotics.1.Sample collection and isolation and identification of the main pathogensA total of 887 milk samples,nipple swabs,nipple swabs and environmental samples from healthy cows,subclinical mastitis cows and clinical mastitis of cows were collected,including 96 nipple milk of healthy cows,49 nipple milk of subclinical mastitis cows,138 nipple milk of clinical mastitis cows,95 nipple swabs of healthy cows,39 nipple swabs of subclinical mastitis cows,78 nipple swabs of clinical mastitis cows,97 anal swabs of healthy cows,33 anal swabs of subclinical mastitis cows,80 anal swabs from clinical mastitis cows,and feed,bedding,faeces,drinking water,spraying water,cleaning water,air,milk cup swab and so on 182 environmental samples.After multiplication of each sample,different selection media were used for pathogens isolated and identification.The strains were identified by PCR amplification with specific primers and 16 S r DNA universal primers.407 strains of E.coli were isolated and identified;100 strains of S.aureus;37 strains of S.uberis;3 strains of Streptococcus agalactiae;134 strains of CNS,0 strains of Salmonella and S.dysgalactia.E.coli was the most important pathogens causing mastitis infection in dairy cows,and the isolation rate of E.coli in milk of clinical-type dairy cows with mastitis(37.7%)was significantly higher than the isolation rate of other pathogens.The isolation rates of E.coli,S.aureus and CNS in healthy milk and SCM milk were higher than those in CM milk,which may be related to the use of antibiotics in dairy farms.The above pathogens were detected in various environmental samples,and the isolation rate of E.coli in the environment(46.2%)was higher than that of other pathogens.The isolation rate of pathogens in B and C dairy farm is generally high.The difference isolation rate of pathogens in different dairy farms related to factors such as the feeding and management methods and dairy farm environment.2.Antibiotics sensitivity test of pathogensIn this study,antibiotics susceptibility tests were performed on the isolates using the micro-broth dilution method.E.coli and S.aureus are both resistant to β-lactams and sulfonamides.E.coli strains to ciprofloxacin,colistin,rifaximin,gentamicin,kanamycin,streptomycin,meropenem,ceftiofur,amoxicillin-clavulanic acid sensitivity rates were greater than 50%,and resistance rates higher than 50% to ampicillin,cefoxitin,tetracycline,florfenicol,sulfamethoxazole,and trimethoprim-sulfamethoxazole;The MDR rate was79.4%,resistance to up to 9 kinds of drugs.The resistance of S.aureus was more than 50%susceptible to trimethoprim-sulfamethoxazole,vancomycin,ofloxacin,enrofloxacin,erythromycin,and more than 50% resistant to trimethoprim-sulfamethoxazole,vancomycin,ofloxacin,enrofloxacin,erythromycin.The highest MDR strains showed 4-fold resistance.CNS were multi-drug resistant strains,with the resistance to up to 11 antibiotics,with5-fold resistant as the dominant resistant group.Sensitive to tilmicosin,erythromycin,gentamicin,doxycycline,florfenicol,oxacillin,vancomycin,linezolid.Mainly resistant to sulfonamides,cephalosporins and fluoroquinolones.Streptococcus are sensitive to amoxicillin-clavulanic acid,ceftiofur,cephalothin,gentamicin,florfenicol,rifaximin,vancomycin,ciprofloxacin,ofloxacin.Resistance to oxacillin,doxycycline,penicillin,erythromycin,sulfamethoxazole,sulfamethoxazole/trimethoprim,lincomycin and clindamycin higher than 50%.The highest MDR strains showed 7-fold resistance,with 5-fold resistance as the dominant drug-resistant group.3.S.aureus MLST types and serotypesBased on the results of whole gene sequencing,a total of 6 ST types were detected in100 strains of S.aureus,ST 25,ST 2683,ST 389,ST 188,ST 705 and ST 7462,with ST2683(46%)and ST 25(35%)as the dominant and ST 7462 as a new ST identified in this study.42% of strains were cap5 serotypes,55% were cap8 serotypes,and 3% were neither cap5 nor cap8.All strains of ST 25 were cap5 serotypes,ST 2683 and ST 188 strains were both cap8 serotypes,and 80%(8/10)of ST 398 strains were cap5 serotypes and other ST398 were neither cap5 nor cap8.The 6 ST types of 100 S.aureus strains mainly constituted four clonal complexes CC25,CC151,CC479 and CC398.4.S.aureus resistance genes and virulence genesBased on the results of whole genome sequencing of S.aureus,a total of 45 resistance genes of fluoroquinolones,tetracyclines,penicillins,macrolides,glycopeptide,and aminoglycosides were screened.The compliance of Penicillin and sulfonamide resistance genes with the resistance phenotype was 100% and 59.1%,respectively.All strains detected macrolide resistance genes,but only 12% of the strains showed resistance to macrolide antibiotics,and the strains were completely sensitive to fluoroquinolones,tetracyclines,and aminoglycosides when the detection rate of these resistance genes was higher than 90%.This difference in resistance genes and resistance phenotypes suggests that the development of drug resistance may result from the interaction of multiple factors.Five categories 95 kinds of virulence genes were detected in S.aureus,including adherence,enzyme,immune evasion,secretory system and toxin.The highest detection rate(46.3%,44/95)was found for toxin virulence genes,including enterotoxin genes seb(20%),seg(81%),sec(1%)and sei(45%),and hemolysin genes hly/hla(99%),hlb(1%),hlg A(99%),hlg B(99%)and hlg C(99%).The S.aureus strains volutionary relationships suggest a link between ST types,serotypes and virulence genes of S.aureus.The homology of S.aureus isolated from milk,skin swabs,anal swabs,feed,air,bedding and faeces in the same dairy farm suggests that S.aureus infection of dairy milk may be related to skin and environmental of the dairy farm.In summary,the pathogens causing bovine mastitis in Hubei are mainly E.coli and Staphylococcus,of which E.coli is also the main pathogens causing milk infection in dairy cows.5 pathogens are mainly resistant to β-lactams and sulfonamides,and the multi-drug resistance rate is high,so the clinical selection of drugs for the treatment of bovine mastitis should be rational in order to reduce the development of resistance.The occurrence of bovine mastitis is closely related to the contamination of the teat skin and the breeding environment,and breeders should give focused attention to it.There is a link between the serotype,ST type and virulence genes of S.aureus,and the detection of drug resistance genes in S.aureus provides a preliminary basis for the development of drug resistance.This study provides a theoretical basis for the precise prevention and treatment of bovine mastitis,and provides a scientific basis for rational clinical use of drugs,reducing the development of bacterial resistance,and reducing infections with pathogens in dairy farming. |
