| Bovine mastitis is a common and multi-etiological cow breast disease caused by a wide range of pathogenic microbial.It not only causes a decline in milk production and milk quality,but is also one of the main causes for the elimination of lactating cows,leading to considerable economic losses to dairy cattle industry.During the past several decades,the predominant microbial flora of pathogens causing bovine mastitis have changed with the development of intensive dairy farming and the improvement of mastitis prevention and control mesures in China.And the reports of coagulase negative staphylococci(CNS)causing bovine mastitis are increasing and have become the main reason for outbreaks of bovine mastitis.Meanwhile,the continuous emergence of drug resistance in CNS has made it more difficult to treat.In this study,75 CNS strains obtained from 349 clinical mastitis milk samples from 15 dairy farms in China were investigated for biofilm,antibiotic resistance,resistance and virulence genes,to provide scientific data in clinical settings.1.An etiology survey was performed on 349 clinical bovine mastitis milk samples collected from 15 dairy farms in Gansu,Shaanxi,Shanxi,Ningxia and Qinghai provinces using the 16 S rDNA sequence method.A total of 298 strains were identified,mainly 88 strains of Escherichia coli,75 strains of CNS,26 strains of Staphylococcus aureus,14 strains of Serratia,13 strains of Klebsiella pneumoniae,13 strains of Enterococcus faecium and 9 strains of Enterococcus faecalis,etc.The frequency of 75 strains of CNS was 25.2%.Of which,40 strains of Staphylococcus chromogenes(53.3%),16 strains of Staphylococcus haemolyticus(21.3%),6 strains of Staphylococcus sciuri(8.0%),3 strains of Staphylococcus epidermidis(4.0%),3 strains of Staphylococcus xylosus(4.0 %),2 strains of Staphylococcus auricularis(2.7%),2 strains of Staphylococcus warneri(2.7%),2 strains of Staphylococcus equorum(2.7%),and 1 strain of Staphylococcus capitis(1.3%).2.The biofilm production of 75 CNS strains was detected by cell culture plate method.The results showed that 86.7%(65/75)of CNS was able to form biofilm,and the prevalence of strains that formed strong,medium and weak biofilm was 6.7%,16.0% and 64.0%,respectively.3.The antimicrobial susceptibility of 75 strains of CNS was determined by disk diffusion method and MIC test strip,and the antimicrobial resistance genes were detected by PCR method.The correlation between resistance phenotype and resistance genes was analyzed.All strains tested were highly resistant to penicillin(56.0%),followed by gentamicin,kanamycin and tetracycline,with a same drug resistance rate of 20.0%.However,more than 96.0% of the strains tested were susceptible to chloramphenicol,nitrofurantoin,and linezolid.The results obtained demonstrated that the detection rate of oxacillinresistant CNS was 45.3%(34/75)and the MIC value of oxacillin was mainly in the range of 0.5 to 1.5 μg/mL.Furthermore,the CN-K-E-TE-DA pattern(14.8%)was the main multi-resistant phenotype among oxacillin-resistant CNS isolates,and 82.4%(28/34)of oxacillin-resistant CNS were biofilm producers.The frequency of gene blaZ(46.7%)was the highest,followed by linA(25.3%)and aac6-aph2(21.3%).The frequencies of tetO,tetL,tetK and ermB in our study were 16.0%,12.0%,9.3%,and 10.7%,respectively.Penicillin resistance was significantly related to the blaZ gene(P <0.01),and the resistance to erythromycin was significantly related to the genes ermA and ermC(P <0.01).The tetracycline resistance phenotype was extremely significantly related to the gene tetL(P <0.01),and resistance to gentamicin and oxacillin was significantly correlated with aac6-aph2 and mecA,respectively(P <0.01).4.The virulence genes of 75 CNS were determined by PCR.The results suggested that the icaC(17.3%)gene was dominant in the operon icaADBC,and the frequencies of icaA and icaB genes were 1.3% and 2.7%,respectively.The detection rate of biofilm-associated protein gene bap was 4.0%,bhp gene was not found in any of the tested isolates.The sea(28.0%)was the predominant staphylococcal enterotoxin gene in the present study,followed by seg(2.7%)and sei(2.7%),the detection rates of selm,seln,selo and selu were 1.3% to each other.The genes tst and luk-PV were not detected in any of isolates analyzed.This study is of important guiding significance for the prudent use of antibiotics in bovine mastitis,the reduction of bacterial resistance,the improvement of clinical treatment effect,and the further research on the mechanism of antimicrobial resistance and pathogenesis of CNS of bovine origin. |
