| Methicillin-resistant Staphylococcus aureus(MRSA)has caused worldwide concern because of its high incidence and undesirable outcomes.Vancomycin is used as a gold standard for the treatment of MRSA infection.However,its role has been questioned and debated by researchers over the past 5 to 10 years,because of heteroresistance and reduced vancomycin susceptibility.Linezolid,teicoplanin,daptomycin and tigecycline are new antibiotics that came into use in China in last decade.Though still rare,the number of clinical MRSA isolates resistant to these antimicrobial agents is growing.In order to give some reference to clinicians on rational use of antibiotics,this study analyzed the epidemiological genetic background of hospital-associated methicillin-resistant Staphylococcus aureus in Zhejiang Province by molecular typing method,and further investigated their drug resistant mechanisms.In part Ⅰ,a total of 391 non-replicated HA-MRSA isolates were collected from 12 tertiary-care teaching hospitals in 5 cities in Zhejiang Province from January 2012 to May 2013,and tested their in vitro susceptibility to 5 antimicrobial agents(vancomycin,teicoplanin,linezolid,tigecycline and daptomycin)by microdilution broth method and Etest method.The results show that all five anti-MRSA agents exhibited good susceptibility;only five resistant isolates were detected sporadically,with one resistant to linezolid,two resistant to tigecycline,one resistant to daptomycin,one resistant to daptomycin and intermediate to teicoplanin(SA1089).Gene mutations reported to be associated with antimicrobial resistance were investigated.Several point mutations created amino acid changes were detected in SA1089,including the S10T substitution in vraS,A113Vsubstitution in vraR,P109A substitution in sigB,G203A and R200L substitutions in cls2,and M113T substitution in rpoB and R214S substitution in walk.However,the mutations above were not reported previously,and further studies should be conducted to confirm the relationship between these mutations and antimicrobial resistance.In part Ⅱ,10 isolates from each hospital were randomly chosen for molecular typing,including multilocus sequence typing,pulsed field gel electrophoresis,staphylococcal cassette chromosome mec typing and staphylococcal protein A gene typing.A total of 17 STs were identified in 120 MRS A isolates recovered from 12 hospitals.Six novel STs were found.ST5 was the most predominant ST type(55/120,45.83%)and followed by ST239(28/120,23.33%)and ST59(15/120,12.5%).After comprehensive analysis of the results of all typing methods,CC5/ST5-MRSA-Ⅱ-t311(39/120,32.5%)was found to be the most predominant HA-MRSA clone,PFGE showed clonal spread between the different hospitals in Hangzhou.CC5/ST5-MRSA-Ⅱ-t002 was the most prevalent clone in Ningbo.CC239/ST239-MRSA was epidemic in certain hospitals in Wenzhou and Shaoxing.Moreover,ST59 MRSA was revealed to have spread into hospitals.Forty-six staphylococcal virulence genes were screened and compared between the most prevalent clones.ST5 isolates were found to contain more virulence genes,especially toxin genes and immune evasion genes.In part Ⅲ,the resistance mechanism of linezolid-resistant Staphylococcus aureus were investigated.Three Staphylococcus aureus(SA777,SA779,SA783)were consecutive isolated from a patient with knee prosthetic joint infection and septicaemia,long-term,repeated linezolid were used.The MICs of linezolid were 2mg/L,4mg/L and 16mg/L,respectively.The 23 S rRNA gene was amplified and sequenced to investigate the resistant mechanism.G2576T mutation in 23S rRNA was identified in SA779(one copy)and SA783(two copies).No mutation was found in ribosomal proteins L3,L4 and L22,and none of the three isolates presented the cfr and optrA genes.The growth parameters and hemolysis assay were analyzed for fitness and virulence evaluation.The results show that with the increased number of mutated rRNA operons,the linezolid MIC increased while the fitness and virulence of isolates decreased.Cross resistance were found among chloramphenicol,clindamycin and linezolid.In summary,CC5/ST5-MRSA-Ⅱ-t311was the most predominant clone and was spread between different hospitals in Hangzhou.ST5 isolates exhibit characteristic virulence gene profiles.Though several resistant isolates were found,five anti-MRSA agents(vancomycin,teicoplanin,linezolid,daptomycin and tigecycline)exhibited good activities against MRSA in vitro.G2576T is the most common mutations found in clinical isolates.With the mutated number of rRNA operons increasing,the linezolid MIC increased while the fitness and virulence of isolates decreased.Cross resistance were found among chloramphenicol,clindamycin and linezolid. |
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