| With the widespread use of antibiotics,bacteria have gradually developed resistance,and generated plenty of multidrug-resistant bacteria.Among them,MRSA,as a kind of superbug,which carries many kinds of drug-resistant genes and has been detected in animal husbandry and animal food for many times,posing a serious threat to public health.MRSA can improve the pathogenicity and adaptability to the host environment by expressing a great deal of virulence.Its infection often depends on the regulation of a variety of virulence factors,such as adhesion proteins,extracellular enzymes,bacterial toxin and iron uptake,which can lead bacteria to produce adhesion,damage host cells,escape the host immune system and produce virulence effects and do harm to animal health.Therefore,studying the mechanism of drug resistance and the regulation of virulence expression of MRSA can cointributed to the development of new antibiotics and the clinical treatment of infection.In this study,we studied the virulence related information of bovine MRSA and the mechanism of cefquinome resistance in Ningxia,which provided a reference for the virulence study of LA-MRSA and the clinical prevention and treatment of LA-MRSA in Ningxia,and laid a foundation for the study of resistance mechanism of LA-MRSA to cefquinome.1.Using micro broth dilution method to test the sensitivity of 97 strains of Staphylococcus aureus to 18 antimicrobial agents.Through the whole-genome sequencing,we focused on the analysis of drug resistance genes,virulence factors and other related information carried by LA-MRSA and MSSA.The results showed that 97 strains were resistant to 13 kinds of antimicrobial agents except temicoxin,amikacin,linezolid,vancomycin and dutamycin,especially penicillin and sulfamethoxazole.9 strains of LA-MRSA were resistant to penicillin and sulfamethoxazole.Drug resistance of 88 MSSA to penicillin and sulfamethoxazole was 100%and 88.6%respectively.The results of whole genome sequencing analysis showed that the basic sequence differences of Staphylococcus aureus from Ningxia were small,and some strains had high homology,carrying 21 kinds of resistance genes and a large number of virulence genes.Among them,LA-MRSA representative strains carry a large amount of drug-resistant genes,but the virulence difference between LA-MRSA and MSSA is small.The results of gene Island prediction showed that LA-MRSA carried more gene islands and virulence gene islands,but there was no significant difference between the number of drug resistance gene islands carried by LA-MRSA and MSSA.2.The biofilm formation ability of LA-MRSA was detected by micro semi-quantitative method,and the invasiveness of LA-MRSA was detected by bacterial invasiveness test in vitro,and screened out high virulence LA-MRSA.The virulence expression profiles of LA-MRSA and standard strain ATCC33591 were compared by transcriptome sequencing.The results showed that LA-MRSA D10 had strong biofilm forming ability and invasiveness.Transcriptome analysis showed that the expression of related genes sunch as immune escape ability,biofilm forming ability,invasiveness and methoxycillin resistance of LA-MRSA D10 was up-regulated compared with the standard strain.However,KEGG pathway annotation analysis showed that the difference in the expression of stress and survival related genes between LA-MRSA D10 and the standard strain was not significant.3.The MIC of LA-MRSA D10 was measured by microbroth dilution method.The high concentration resistant strains to 128 MIC were obtained by different concentration of drug containing plates and culture media.The important targets and regulatory network of LA-MRSA resistance to cefquinome were predicted by comparative genomic and transcriptome sequencing.The results showed that there were five mutations in high cefquinome resistant strains,one of which was a non synonymous mutation site coding domain of mecA penicillin binding protein transpeptidase domain,and the expression of important regulatory factors of biofilm was up-regulated,the expression of invasive virulence gene was down-regulated,and a large quantity of genes related to the basic metabolism of bacteria and the maintenance of the basic physiological process of bacteria were overexpressed,KEGG pathway annotation analysis showed that compared with the original wild strain,in high concentration resistant strains,there were significant difference in up-regulated genes which related to lysine biosynthesis,glyoxylic acid and dicarboxylic acid metabolism,and the down-regulated genes.were significantly different from the related genes of ribosome pathway.In conclusion,this study analyzed the drug resistance and virulence of bovine MRSA in Ningxia.The comparative genomics and transcriptome studies between a highly virulent LA-MRSA and its high cefquime resistant strain were carried out to reveal the drug resistance and virulence characteristics of bovine MRSA in Ningxia and the potential target of resistance of LA-MRSA to cefquinome and the change of its internal expression,which provided a theoretical basis for the treatment of drug-resistant bacteria after infection and the study of the mechanism of cefquime resistance.. |
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