| Bovine mastitis caused by Staphylococcus aureus poses a threat to the healthy development of the dairy cow breeding industry and dairy industry.This study was aimed to clarify the infection rate,drug resistance and population characteristics of S.aureus strains causing clinical bovine mastitis from Hohhot area.257 milk samples from clinical bovine mastitis were collected from 13 dairy farms in 7 areas of Hohhot city.Herein,biochemical test combined real-time quantitative PCR(q PCR)with PCR based on nuc gene were used to conduct preliminary identification of S.aureus strains.Meanwhile,matrix-assisted laser desorption ionization time-of-flight mass spectrometry(MALDI-TOF-MS)was employed to identify the suspected S.aureus strains rapidly and accurately.At the same time,broth dilution and PCR methods were used to detect the drug sensitivity and 11 drug resistance genes of S.aureus strains,i.e.,erm A,erm B,bla Z,aac A-aph D,lnu A,dfr G,pc223,mec A,glr A,tet K and tet M.Pulsed field gel electrophoresis(PFGE)was employed to detect the molecular typings of S.aureus isolates.The main results are as follows:1.A total of 45 suspected S.aureus strains were isolated from 257 milk samples of clinical bovine mastitis and identified as positive biochemical test with detection rate of17.5%(45/257).While 43 of 45 suspected S.aureus strains were identified as S.aureus by PCR and q PCR amplification of nuc gene and MALDI-TOF MS with detection rate of 16.7%(43/257).2.Drug sensitivity of 43 strains of S.aureus were tested using 14 kinds of antibiotics,the result showed that 43 strains of S.aureus with different degrees were resistant to 12 kinds of antibiotics.All strains of S.aureus were resistant to penicillin and ampicillin of β-lactam with the resistance rate of 100%(43/43).Some strains of S.aureus were resistant to sulfamethoxazole(SMZ)with trimethoprim(TMP)(31%,13/43),ciprofloxacin(27.9%,12/43),and erythromycin(21%,9/43).The drug resistance of 43 strains to the other 7 kinds of antibiotics was in the order of gentamicin,clindamycin,chloramphenicol,tetracycline,daptomycin,oxacillin and cefoxitin from high to low with the resistance rate of 16.3%(7/43),11.6%(5/43),9.3%(4/43),6.9%(3/43),6.9%(3/43),6.9%(3/43),and 4.7%(2/43),respectively.All strains were sensitive to vancomycin and imipenem,exhibiting multiple drug resistance.3.A total of 9 drug resistance genes were detected in 43 strains of S.aureus,which were erm A,erm B,bla Z,aac A-aph D,lnu A,dfr G,pc223,glr A and tet K.The detection rates of lnu A,bla Z,and glr A genes was as high as 95.3%,93.1%,and 90.7%,respectively.The detection rates of the other six drug resistance genes pc223,erm B,erm A,aac A-aph D,dfr G,and tet K was 39.5%(17/43),16.3%(7/43),13.9%(6/43),13.9%(6/43),11.6%(5/43),and 6.9%(3/43),respectively.mec A gene of β-lactams and tet M gene of tetracycline were not detected in 43 strains of S.aureus.4.43 isolates of S.aureus from in Hohhot area were categorized into 11 molecular typings based on pulsed-field gel electrophoresis(PFGE).5 molecular typings(designated as HT1,HT2,HT3,HT4 and HT5)including 36 isolates of S.aureus accounted for 83.7% of all isolates(36/43).Herein,the typings HT1 and HT4 containing10 isolates of S.aureus accounted for the highest proportion of 27.8%(10/36),followed by HT5 19.4%(7/36),HT2 and HT3 13.9%(5/36),11.1%(4/36)respectively.The other types including 7 isolates of S.aureus accounted for 16.3%(7/43).Overall,HT1,HT4 and HT5 of all typings are the main prevalent in this area.In summary,MALDI-TOF MS technique can achieve rapid and accurate identification of S.aureus clinical isolates,providing a new idea for efficient detection of S.aureus in future.This research about drug resistance analysis and PFGE molecular typing on clinical isolates helps to understand the drug resistance and prevalence of local clinical isolates.These researches could provide technical reference for the prevention and treatment of dairy cow mastitis caused by Staphylococcus aureus. |
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