Mechanism Of Vancomycin-mediated Tolerance Of Staphylococcus Aureus

Antibiotics can not only prevent diseases caused by pathogenic bacteria,but also be added to feed as growth promoters to promote the growth of livestock and poultry.In the process of dairy farming,antibiotics are widely used in the treatment of cow mastitis.However,due to the long-term nonstandard and unreasonable use of antibiotics,antibiotic resistant strains from domestic animals occur frequently.Staphylococcus aureus,as the main pathogen of cow mastitis,poses a great threat to the healthy breeding of dairy cows and the sustainable development of the dairy industry.In the process of antibiotic treatment for cow mastitis,it is found that the original non drug resistant Staphylococcus aureus will gradually evolve antibiotic resistance,which is considered to be the basis of bacterial resistance,and the resistant strains can survive longer in the process of high concentration antibiotic treatment,which will lead to the pathogen cannot be eradicated,leading to repeated attacks of the disease.At present,there have been many studies on the tolerance mechanism of Gram-negative bacteria,but there are few studies on the tolerance mechanism of Gram-positive bacteria.Until now,vancomycin is still used to treat severe gram-positive infections.Therefore,elucidating the antibiotic tolerance mechanism of Staphylococcus aureus has great reference value for rational drug use and the development of new antibiotics,and is very important for the prevention and treatment of cow mastitis and the healthy development of dairy industry.In the early stage of this experiment,we constructed an in vitro induction model of antibiotics.Staphylococcus aureus Newman was used as the model strain,which was repeatedly exposed to high concentrations of vancomycin(20 × MIC)for induction.The isolated VTSA strains were sequenced and the key genes with important functions were identified,and the phenotypes of VTSA strains were detected.The main results are as follows.1.101 vancomycin tolerant Staphylococcus aureus strains were induced by in vitro antibiotic induction assay and successfully isolated,9 VTSAs were identified by whole-genome sequencing,and all 101 VTSAs were identified by Sanger sequencing based on the results of whole-genome sequencing.According to the sequencing information,they were divided into 14 different genotypes(G0 ? G13).Among them,G0:8-17SC1 strain did not produce non synonymous mutation in the coding sequence.Among the 19 non synonymous mutations of all 18 genes,the mutation frequency of pbp4,mut L,stp1,htr A and pth genes was higher.2.In order to explore the key genes in the formation of vancomycin tolerance of Staphylococcus aureus further,plasmid complementation method was used to complement all the mutant genes separately,and vancomycin killing test was used to detect the antibiotic tolerance of Staphylococcus aureus.The results showed that the mutations of NWMN?1068,pbp4,stp1,pth and htr A genes played an important role in obtaining the tolerance phenotype of VTSA,and the rapid accumulation of mutated genes caused by mut L gene mutation also accelerated the formation of the tolerance phenotype of S.aureus under antibiotic pressure.However,assays in G0:8-17SC1 strain showed that mutation S in the non-coding region could not be ignored in the formation of vancomycin tolerance of S.aureus.3.Then the cell wall,growth ability and biofilm forming ability of VTSA were detected.The results showed that there was no significant change in the growth ability and biofilm formation ability of VTSA strains,but a series of changes in cell wall related phenotypes occurred,including decreased autolysis ability,decreased sensitivity of lysostaphin and thickened cell wall.These changes made VTSA strains obtain cross-tolerance to other antibiotics that target cell wall.In addition,pth gene mutation not only made Staphylococcus aureus tolerant to 3 antibiotics,but also enhanced the ability of biofilm formation.In conclusion,in this study,VTSA strains were screened and isolated by in vitro induction system.The genetic background of VTSA strains was analyzed by sequencing,and combined with in vitro antibiotic killing test,6 genes that play a key role in the formation of tolerance to S.aureus were found and the related phenotypes of the VTSA strain were further determined,and it was found that they have the characteristics of cross-tolerance to multiple antibiotics.These results are of great significance for the detection of clinical vancomycin tolerant isolates and the prevention of the development of antibiotic tolerance,providing a theoretical basis for the development of new antibiotics,and providing a new idea for the healthy breeding of dairy cows.