In Vitro Activities Of Tedizolid And The Influence Of Its Subinhibitory Concentrations On Virulence Genes Transcription Of Staphlycoccus Aureus

Objective:To investigate the susceptibilities of a noveloxazolidinone antibiotic,tedizolid,against clinical gram-positive cocci from a tertiary hospital in Shanghai,and explore the molecularmechanisms mediated resistance to oxazolidinone in tedizolid-non-susceptible strains.Furthermore,we assessed the effects of subinhibitory concentration of tedizolid on transcription level of virulence genes ofStaphylococcus aurens Newman strains and N315 strains.Methods:A total of 1069 clinical non-duplicate gram-positive bacterial isolates were collected during January 2015 to December 2017.The isolates included methicillin-resistant Staphylococcus aureus?n=202?,methicillin-susceptible S.aureus?n=294?,coagulase-negative staphylococci?n=115?,Enterococcus faecalis?n=206?,Enterococcus faecium?n=55?,Streptococcus agalactiae?n=159?and Streptococcus anginosus group?n=38?.Antimicrobial susceptibility of each strain was detected by gram-positive cocci susceptibility card and Kirby-Bauer?K-B?disk diffusion test.The minimum inhibitory concentrations?MICs?of tedizolid and linezolid against the isolates tested were detected using the broth microdilutionmethod.The MICs of the two drugs were analyzed to clarify thedifferences of in vitro activities of them.Polymerase chain reaction?PCR?and sequencing were performed to detect the oxazolidinone-resistant genes cfr and optrA and the mutations of 23S rRNA V region in tedizolid non-susceptible strains.The MICs of tedizolid and linezolid against Newman strains and N315 strains were determined by broth microdilution method.RNAs of Newman srains and N315 strains exposed to differentsubinhibitory concentrations of tedizolid or linezolid?1/8 MIC,1/4 MIC and 1/2 MIC?were extracted.Real-time quantitative PCR?qRT-PCR?was used to measure the transcription levels of virulence genes,including?-hemolysin?hla?,?-hemolysin?hld??RNA ??,accessory gene regulator A?agrA?,autolysis-related locus S?arlS?,S.aureus exoprotein expression S?saeS?,S.aureus surface protein G?sasG?,staphylococcal protein A?spa?,clumping factor A?clfA?,fibronectin binding protein A?fnbA?,intercellular adhesion A?icaA?,sigma factor B?sigB?,staphylococcus accessory regulator A?sarA?,repressor of toxin?rot?,staphylococcal enterotoxins A?sea?,toxic shock syndrome toxin?tst?,phenol soluble modulins??psm??,elastin binding protein of S.aureus?ebpS?,staphylococcal serine protease A?sspA?,serine-aspartate repeat gene D?sdrD?,aureolysin?aur?,coagulase?coa?,nuclease?nuc?and leukocidin E?lukE?genes.Differences of expression levels of virulence genes among graded concentrations of antimicrobial agents were analyzed by Student's t-test.P value<0.05 was considered significant.Results:All Gram-positive cocci were sensitive to vancomycin,and the susceptibility of MRSA and E.faecium to antimicrobial agents was lower than that ofMSSA and E.faecalis.All isolates of staphylococci?MIC?0.5?g/ml?,E.faecium?MIC?0.5?g/ml?and streptococci?MIC?0.25?g/ml?weresusceptible to tedizolid.The sensitivity rate of E.faecalis isolates totedizolid was 94.7%.Eleven tedizolid/linezolid-non-susceptible?tedizolid MIC=1?g/ml,linezolid MIC=8?g/ml?were found,which were positive for the novel oxazolidinone resistance gene optrA.Based on MIC₉₀0 values,tedizolid revealed 4⁸-fold more effective than linezolid against allspecies detected.In comparison with the control group,only the level of fnbA and aur mRNA of Newman srains were suppressed by 1/2 MIC of tedizolid?P<0.05?.Also,tedizolid reduced the expression of hla and saeS mRNA of N315 strains at 1/8 MIC to 1/2 MIC?P<0.01?and at 1/8 MIC to1/4 MIC?P<0.01?,respectively.The transcriptions of the remainingvirulence genes studied were enhanced,when exposed to differentsubinhibitory concentration of tedizolid.Conclusions:Tedizolid is a promising new antibiotic for treating gram-positive cocci infections,however,its resistance mediated by the optrA in enterococci should be concerned.Subinhibitory concentrations of tedizolid are able to induce change of transctiption levels of multiple virulence genes,and havepotential effects on the pathogenic mechanisms of S.aureus.