Isolation,Identification And Antibiotic Resistance Of Mastitis-Associated Streptococcus From Large-scale Dairy Farms

Mastitis is one of the most common infectious diseases in dairy cows.Pathogenic microorganisms,especially bacteria,are among the most important cause of the disease.The occurrence of mastitis has caused huge economic losses to the dairy farming industry and severely restricted the healthy development of the industry.In this study,mastitis-associated Streptococcus strains were isolated and identified from milk samples in large-scale dairy farms in China,and the resistance rates of the isolates to common antibiotics were further detected.The resistant Streptococcus were further investigated for the major resistance mechanisms to aminoglycosides,tetracyclines and pleuromutilins,which might.provide a theoretical basis for the prevention and treatment of mastitis caused by Streptococcus.1.Isolation of mastitis-associated Streptococcus and detection of their antimicrobial susceptibility to commonly used antibacterial agentsDuring the period from January to December 2018,a total of 312 milk samples of clinical and recessive mastitis were collected from six large-scale dairy farms in China.Among them,273 samples were positive for seven major bacterial mastitis pathogens.In this experiment,56 mastitis-associated Streptococcus were identified.Among them,there were 30,22,and 4 strains of S.uberis,S.agalactiae,and S.dysgalactiae,respectively.In particular,S.uberis were more frequently isolated from farm SHGM(44.4%)of Shanghai and farm YCHF(43.8%)of Jiangsu province in East China,while S.agalactiae accounted for the highest from farm HNHX(80.0%)of Henan province and farm HBCZ(66.7%)of Hebei province in North China.The distribution of Streptococcus species varied between farms,presenting a regional epidemic characteristic.The microbroth dilution method was used to test the antimicrobial susceptibility of 56 Streptococcus isolates to 15 commonly used antibacterial agents recommended by CLSI.The results showed that the highest resistance rate was gentamicin(89.3%);followed by doxycycline(51.8%),lincomycin(37.5%),clindamycin(32.1%),erythromycin(28.6%),tiamulin(21.4%),amoxicillin(17.9%);penicillin(7.1%),ceftiofur(5.4%),rifampicin(5.4%)and vancomycin(5.4%);The lowest resistance was linezolid(3.6%).The results showed that the resistant rates varied between antibiotics,with gentamicin and doxycycline much more resistant.2.Detection of the major antibiotic resistance mechanisms to high-level aminoglycosides,tetracyclines and pleuromutilinsThe PCR was used to detect the presence of high-level aminoglycoside resistance genes ant6-Ib aac(6')-aph(2"),aph(2)-Ib,aph(2)-Ic,aph(2)-Id aph(3)-?a,ant(6')-Ia(aadE),the tetracycline resistance genes tet(K),tet(O),tet(L),tet(M),and the pleuromutilin resistance genes lsa(A)?lsa(B)?lsa(C)?lsa(E)?cfr?vga(A)?vga(B)?vga(C)?vga(E),in 56 Streptococcus isolates.The results showed that the highest detection rate of antibiotic resistance genes was high-level aminoglycoside resistance gene aph(3')-?a,accounting for 90%.The detection rates of tetracycline tet(M)and tet(K)were 38%and 24.1%,respectively.The detection rate of the pleuromutilin-resistant gene lsa(E)was 28.6%.Further,the resistance phenotype and genotype of the strains was analyzed for consistency,and the correlation between the two was clarified.Nine strains of the high level aminoglycoside resistant strains were positive for aph(3')-IIIa and one resistant strain was not.Among the tetracycline-resistant strains,18 resistant strains detected the antibiotic resistance genes tet(M)and tet(K),and 11 strains did not detect the corresponding antibiotic resistance genes.Among the strains resistant to pleuromutilin,4 of them detected the antibiotic resistance gene lsa(E),and 8 of them did not.The results showed that in the strains with consistent drug phenotype and genotype,the antibiotic resistance genes are the main mechanisms that mediate the resistance of these strains to such antibiotics.The emergence of strains that did not detect the corresponding antibiotic resistance gene indicates that there may be other drug resistance mechanisms that need to be further explored.In summary,this study provides certain epidemiological data for the prevention and treatment of mastitis in these farms,and may guide further study on antibiotic resistance level and resistance mechanism of Streptococcus isolates in dairy cows.