Study On The Relationship Between CRISPR System And MepA Multidrug Efflux Pump And Drug Resistance In Staphylococcus Aureus

Staphylococcus aureus is one of the most important strains of staphylococci and poses a great threat to public health.the CRISPR-Cas system is an immune defense system against foreign mobile genetic elements;the efflux protein MepA is a member of the multidrug and toxin efflux(MATE)family,and overexpression of MepA leads to the development of resistance to quinolones and disinfectants,based on this,this study will conduct related research on drug resistance of the CRISPR system and the efflux pump MepA.1.PCR was applied to detect and analyze CRISPR loci,drug resistance genes and virulence genes in 234 strains of S.aureus,and analyze the relationship between CRISPR and drug resistance genes and virulence genes.The results showed that the percentage of CRISPR loci present in 234 strains was 7.69%,and 13 drug resistance genes and 10 virulence genes were detected.The positive rate of resistance gene norA was higher in positive strains compared with CRISPR negative strains(P<0.01),and there was no association between CRISPR and the distribution of virulence genes(P>0.05).2.CRISPR prediction analysis of the whole genome of 14 sequenced S.aureus strains using online tools such as CRISPR Finder,CRISPR Target and Center for Genomic Epidemiology,the results showed that all 14 strains carried CRISPR loci,all 26 sets of consistent repetitive sequences could form conserved.The results showed that all 14 strains carried CRISPR loci,all 26 sets of concordant repeat sequences could form a conserved secondary structure,all 229 spacer sequences could be matched to the pre-spacer sequence,and most or all of the resistance and virulence genes carried by the 14 strains could be found in the genes carried by their corresponding homologous phage or plasmid.3.After applying the sub-inhibitory concentration ciprofloxacin multiplication concentration method to induce drug resistance in sensitive S.aureus as well as multi-drug resistant S.aureus without antibiotic stress in successive passages,the changes of CRISPR loci were analyzed by applying BioXM2.6 and CRISPR Finder.The results showed that the first repeat sequence and the first spacer sequence of the CRISPR loci of WLD19/YD and SAXL24/YD strains were lost,suggesting that S.aureus under antibiotic pressure may play a regulatory role in drug resistance through the loss of CRISPR loci.4.Homology modeling of the mepA gene detected in Ningxia region was performed by bioinformatics,and the mode of action of quinolones such as ciprofloxacin with MepA was analyzed by molecular docking method;the full length of mepA gene was cloned from wild strain S.aureus 4(mepA+)and electrotransformed into S.aureus RN4220 by shuttle vector,and the plasmid was extracted and then transformed into S.aureus 25(mepA-,ciprofloxacin MIC 0.25μg/mL)to obtain S.aureus 25-mepA(mepA+,ciprofloxacin MIC 0.5 μg/mL),and assayed S.aureus 4,S.aureus 25 and S.aureus 25-mepA in the planktonic state,the The susceptibility of S.aureus 4,S.aureus 25 and S.aureus 25-mepA to ciprofloxacin in the planktonic,resuspended and biofilm states was examined by real-time fluorescence quantitative PCR to determine the expression of mepA mRNA before and after the action of ciprofloxacin in S.aureus 4 and S.aureus 25-mepA planktonic and biofilm bacteria.The results showed that ciprofloxacin was the compound with the lowest binding energy to MepA protein among the quinolones.The susceptibility of the three S.aureus species to ciprofloxacin in different states was similar,and the results of real-time fluorescence quantitative PCR revealed that the expression of mepA in S.aureus 25-mepA biofilm bacteria did not show significant differences before and after the action of ciprofloxacin,suggesting that the increase of ciprofloxacin resistance in S.aureus 25-mepA biofilm bacteria may be related to the upregulation of the efflux pump gene mepA was not related.In conclusion,the CRISPR system is associated with the carriage of a few resistance genes and may regulate resistance through the loss of CRISPR loci.It was found that the increase of ciprofloxacin resistance in S.aureus biofilm bacteria was not associated with upregulation of the efflux pump mepA gene.This study provides an idea to address the propagation of drug resistance in S.aureus,and also provides a basis for further in-depth study on the mechanism of pump MepA resistance in S.aureus.