Staphylococcus Aureus And Study On Biological Function Of Virulence Gene Tst

Objective:Staphylococcus aureus?S.aureus?can obtain cassette chromosome mec?SCCmec?through parallel transfer.SCCmec contains mecA gene,which is responsible for the methicillin resistance of S.aureus.Previously,we performed the study on SCCmec classification of the clinical methicillin resistant Staphylococcus aureus?MRSA?,and found that there were various phenotypes of SCCmec in MRSA isolated from our hospital,among which type IV is the most one,suggesting that the phenotypes of S.aureus were abundant in our hospital.It has been reported that there is a certain correlation between the infection and epidemic of MRSA and the bacterial virulence gene tst,suggesting that virulence gene tst plays an important role in the pathogenesis of S.aureus.This study aimed to investigate the antibiotic resistance characteristics of MRSA in this region,detect the virulence gene tst in MRSA strains and analyze the antibiotic resistance of MRSA strains.In addition,we aimed to study the role of virulence gene tst in pathogenesis of S.aureus through comparing the intracellular survival ability of tst-positive S.aureus and tst-negative S.aureus by infecting monocyte-macrophage cell line THP-1 cells,and comparing the clinical laboratory indicators of the mice infected by tst positive S.aureus and negative strains.Taken together,the purpose of our study is to provide the basis for guiding the reasonable using of drugs and controlling the prevalence and dissemination of MRSA strains.Methods:1:Investigation of antibiotics resistance of clinical isolates of MRSA and detection of tst gene?1?Antimicrobial susceptibility testing S.aureus were inoculated on the culture plates according to the instructions and performed the identification and antibiotic sensitivity test by BD automatic microbial analysis system.?2?Extraction of bacterial DNA template Take 1.5mL fresh LB liquid culture of MRSA bacteria,centrifuge 5000 g for 10 min at 4?,discard the supernatant,suspend bacteria by 180?l 0.1mol/L Tris?PH 8.0?,add Staphylococcal enzyme 1?l?2 mg/ml?and mix.After incubation at 30°C for 30 min,extract the DNA according to the kit instruction and DNA template was stored at-20°C.?3?Detection of methicillin-resistant gene mecA and virulence gene tst PCR amplifications of methicillin-resistant gene mecA and tst gene encoding S.aureus toxic shock syndrome toxin 1?TSST-1?were performed,and PCR products were identified by electrophoresis and sequencing.2:Study on biological function of virulence gene tst?1?Cell culture Macrophage cell line THP-1 cells were cultured in medium containing RPMI1640 and 10%fetal bovine serum?FBS?,and the culture conditions were37?,5%CO2.Prior to the experiment,we separate THP-1 to the amount of 1 x 10⁵ cells/well in 24-well plates and induced differentiation using PMA for 48 hours.?2?Detection of intracellular survival ability in macrophages The tst positive and tst negative S.aureus were cultured and collected at the logarithmic growth phase.We used 2×10³ CFU to infect macrophages THP-1,and then compared the intracellular survival abilities of these bacteria.?3?Role of tst in S.aureu pathogenicity to the BALB/c mice Take bacteria liquid tst positive and tst negative S.aureu respectively,which containing 1×10⁸ CFU,and injected into the orbital venous of BALB/c mice.To observe the differences between mice infected by tst positive and tst negative S.aureu,we measured the weight changes of the mice infected with different strains,took venous blood by removing eyeball to detect liver function,renal function,myocardial enzymes and cytokines,took a certain amount of liver,spleen,kidney,heart tissue to spread LB plates after obtaining tissue homogenate.?4?Detection of the cytokines level by flow cytometry We detected the cytokines secreted by macrophages THP-1 infected by S.aureu for 24h,and detected the expression level of serum cytokines in BALB/c mice infected with S.aureu for 72h as well.Result:?1?Antibiotic resistance characteristic of MRSA isolates 90 MRSA isolates were sensitive to vancomycin and linezolid.The resistance rate to penicillin was 100%,and the resistance rate to oxacillin,aztreonam,piperacillin/tazobactam reached 90%.Moreover,the gene mecA is present in all these strains.?2?Detection of virulence gene tst in MRSA strain The tst positive rate was16.7%,and all strains were resistant to many antibiotics except for vancomycin and linezolid.?3?Detection of intracellular suvival in macrophages The intracellular survival ability of tst positive S.aureu in macrophage THP-1 was significantly higher than that of tst gene negative S.aureu.In addition,the concentrations of cytokines IL-2,IL-6 and IL-17A in macrophages infected by tst positive S.aureu for 24h were significantly higher than that infected by tst negative S.aureu.?4?Results of BALB/c mice infected by S.aureu The BALB/c mice infected by tst positive S.aureu compared to tst negative S.aureu,showed mental sluggishness,weight loss and in a bad state of action.The expression of serum cytokine IL-6 was significantly increased in the mice infected by tst positive S.aureu,and the livers and spleens appeared swelling.Furthermore,the tissue homogenate coating results showed that the amount of bacteria was obviously increased in mice infected by tst positive S.aureu for 72h.Conclusion:This study shows that the antibiotic resistances of tst positive S.aureu strains were more serious than tst negative S.aureu,and tst positive S.aureu had stronger intracellular survival ability in monocyte macrophage cell line THP-1 cell.These results suggested that the virulence gene tst may play an important role on intracellular survival for S.aureu.The significantly reduced weight of BALB/c mice infected with tst positive S.aureu and the significantly increased bacterial load and the secretion of serum cytokine IL-6 indicated that tst gene played an important role on the virulence of S.aureu.