| In this study,the epidemiological characteristics of Staphylococcus aureus from yak farming were analyzed;the mechanism of resistance to methicillin-resistant S.aureus(MRSA)under antibiotics and disinfectants stress was explored.The yak nose swabs and anal swabs were sampled during the yak breeding process in Ganzi Prefecture.S.aureus were isolated and further identified by polymerase chain reaction amplification with using 16 S r DNA and nuc genes primers.S.aureus was subsequently tested for enterotoxin genes,virulence genes,disinfectant resistance genes,and heavy metal resistance genes.After that,MRSA strains were further selected from all the S.aureus isolates.SCCmec typing,MLST typing,and spa typing were performed to the identified MRSA strains.Also,the drug-resistant genes carried by MRSA strains were detected.The drug-resistant phenotypes of the strains were also detected at the same time.The minimum inhibitory concentration(MIC)of the strains to different disinfectants and antibiotics was determined by the micro-broth dilution method.The stress experiment was controlled by sub-inhibition concentrations,and the effects of antibiotic and disinfectant resistance on related genes were explored by fluorescent quantitative PCR.The details are as follows:(1)A total of 1281 samples(including 657 nasal swabs and 624 anal swabs)were collected from yaks in Ganzi Prefecture.91 strains of S.aureus were identified,with a total isolation rate of 7.10%(91/1281).Of which,anal swab isolation rate was 4.65%(29/624)and nasal swab isolation rate was 9.44%(62/657).Through the analysis of S.aureus contamination from the sampling of 18 counties in Ganzi Prefecture,we found the isolation rates of Shiqu County(25%),Daocheng County(18.18%),Batang County(18.18%),and Dege County(10%)were relatively high.Among 91 strains of S.aureus,the detection rates of enterotoxin genes selx(51.65%),set(13.19%),seo(16.48%),and sen(16.48%)were higher.The detection rates of other virulence genes hla(51.65%),hlb(57.14%),pvl(31.87%),and tsst-1(34.07%)were higher.In the disinfectant resistance gene test,we found the detection rates of qac C was 35.16%,qac A / B was 7.69%,qac G was 3.30%,and qac H was not detected.The strains were tested for 5 types of heavy metal resistance genes.Among them,the copper heavy metal resistance gene cop A was detected the highest,and the czr C zinc heavy metal resistance gene was not detected.The detection rate of each heavy metal resistance gene in descending order was: cop A(95.60%,78/91),ars B(94.51%,86/91),cad D(85.82%,69/91),ars A(50.55%,46/91).The carrying conditions of the enterotoxin genes,virulence genes,disinfectant resistance genes and heavy metal resistance genes of the isolated strains showed that S.aureus from yak farming in Ganzi Prefecture of Sichuan Province was potentially pathogenic and should be paid attention to.(2)The mec A gene was amplified by PCR,and a total of 24 MRSA strains were screened.Seven MRSA were isolated from anal swabs and 17 MRSA were isolated from nasal swabs.24 MRSA strains were identified by SCCmec,spa,and MLST typing,and 10 strains of them were identified as ST59-IVa-t437.4 strains of them were identified as ST59-III-t437.24 MRSA strains are multi-drug resistant strains,which are resistant to 2~8 antibiotics.The main types of resistance genes are tetracyclines resistance genes(teta,tet M),aminoglycosides resistance genes(aac6’),and macrolides resistance genes(msr A,erm C),fluoroquinolones resistance genes(grl A,nor A).The enterotoxin genes(seb,sek,seq and selx)were detected in MRSA strains.The main disinfectant resistance gene was qac C.There are differences in individual genes among the strains.(3)In the disinfectant resistance test,the MIC range of MRSA to benzalkonium bromide is 1.22 ~9.77 μg/m L;the MIC range to peroxyacetic acid is 14.65 ~ 117.19 μg/m L;and the MIC range to sodium hypochlorite is 343.75~2750 μg/m L.Compared with the three disinfectants,benzalkonium bromide has the best antibacterial effect.In the antibiotic resistance test,the MIC concentration range of MRSA to ampicillin sodium is 2.5 ~ 80 μg/m L;the MIC of oxacillin sodium is 2.5~20 μg/m L;the MIC value of cefoxitin is 10~160 μg/m L;the MIC value of ofloxacin is 2.5~10 μg/m L.All four antibiotics could inhibit MRSA bacteria,and the bacteriostatic effect of oxacillin sodium is better than others.(4)In this part,we selected two MRSA strains to test the stress experiment.After strains were stressed for 216 h,under 1/4 MIC oxacillin sodium,ofloxacin,and benzalkonium bromide,the expression levels of drug resistance genes mec A,grl A,and disinfectant resistance gene qac G were detected by Real-time fluorescence quantification.The phenotypes of the strains to oxacillin sodium,ofloxacin,benzalkonium bromide after 216 h stress were also detected at the same time.The results showed that after the MR-YB786(qacG-negative strain)was stressed by 1/4 MIC of oxacillin sodium,oxacillin sodium increased its MIC from 5.00 μg/m L to 20 μg/m L;ammonium bromide reduced its MIC from 4.88 μg/m L to 2.44 μg/m L.After being stressed by 1/4 MIC ofloxacin,ofloxacin increased its MIC value from 2.50 μg/m L to 80 μg/m L;benzalkonium bromide reduced its MIC from 4.88 μg/m L to 2.44 μg/m L.After 1/4 MIC benzalkonium bromide stress,oxacillin sodium increased its MIC from 5.00 μg/m L to 40 μg/m L;oxygen Fluoxacin increased from 2.50 μg/m L to 40 μg/m L;benzalkonium bromide decreased from 4.88 μg / m L to 2.44 μg/m L.Compared with the slaughtering strain MR-YS2(qac G positive strain)after 1/4 MIC oxacillin sodium stress,oxacillin sodium increased its MIC from 2.50 μg/m L to 10 μg/m L;benzalkonium bromide was increased from 0.04 μg/m L to 4.88 μg /m L;after 1/4 MIC ofloxacin stress,ofloxacin was increased from 20 μg/m L to 40 μg/m L;benzalkonium bromide increased its MIC from 0.04 μg/m L to 4.88 μg/m L;after 1/4 MIC benzalkonium bromide sodium stress,oxacillin sodium increased its MIC from 2.50 μg/m L to 40 μg/m L;ofloxacin increased its MIC from 20 μg/m L to 40 μg/m L;benzalkonium bromide increased its MIC from 0.04 μg/m L to 4.88 μg/m L.The above analysis showed that after being stressed by antibiotics or disinfectants with sub-suppressive concentrations,the antibiotics resistance of both strains was enhanced.However,whether under sub-level antibiotic or disinfectant stress environment,the resistance to benzalkonium bromide of qac G-positive strain MR-YS2 was increased,and the resistance to benzalkonium bromide of qac G-negative strain MR-YB786 was declined.(5)In view of the genes expression,though the resistance of MR-YS2 strain to oxacillin sodium and benzalkonium bromide increased after 216 h after 1/4 MIC oxacillin sodium stress,the relative expression level of mec A gene in strain MR-YS2 decreased.While the resistance of MR-YB786 strain to oxacillin sodium increased and the resistance to benzalkonium bromide decreased after 216 h after 1/4 MIC oxacillin sodium stress,the relative expression level of mec A gene in strain MR-YB786 did not increase significantly at the beginning,but decreased to a level close to the unstressed level in the later period.After long-term stress of 1/4 MIC of ofloxacin,the resistance of MR-YS2 to ofloxacin and benzalkonium bromide increased,and the expression of the grl A gene of strain MR-YS2 increased first and then decreased.After 1/4 MIC ofloxacin stress,MR-YB786 strain increased resistance to ofloxacin and decreased resistance to benzalkonium bromide.The expression of the grl A gene in MR-YB786 was the same as before stress.After prolonged stress with 1/4 MIC benzalkonium bromide,the resistance of qac G-positive strain MR-YS2 to benzalkonium bromide,oxacillin,and ofloxacin increased.The expression of qac G gene first increased and then decreased at 72 h.We speculated that the phenotypic changes of the two strains to benzalkonium bromide under stress might be related to the qac G gene.Induced by antibiotic or disinfectant stress,the resistance of the strains to antibiotics would be enhanced,indicating that long-term use of antibiotics and QACs disinfectants had a certain synergistic effect on the enhancement of MRSA strain resistance.In summary,this study investigated the molecular prevalence of S.aureus during yak farming in Ganzi Prefecture,Sichuan Province.Among them,24 MRSA strains were identified with a wide range of resistance to disinfectants and antibiotics,which posed a potential threat to yak breeding and yak meat processing.It was found that benzalkonium bromide and oxacillin sodium had better killing effect to these MRSA isolates.MRSA had different genes(mec A,grl A,qac G)expression levels during a stress environment.With subsuppressive levels of antibiotics or disinfectants,the resistance of MRSA strains to disinfectants and antibiotics was simultaneously increased.Our results provide effective information for the prevention and control of S.aureus during yak farming,and also provide a scientific basis for the safe use of disinfectants and antibiotics. |
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