| Staphylococcus aureus is one of most common pathogenic bateria that being carried by human body and can cause vary infections. Recently, the increasing resistance of S. aureus to antibiotic made big problems to clinical therapies of their infections. Linezolid, as the first synthetic antibiotic belongs to oxazolidinone, was approved for clinical therapy use with several indications. However, the linezolid resistant isolates emerged with the widely use of this agent in the clinical setting, which has caused extensive concern. The thesis aims to study the linezolid resistant mechanisms and their molecular basis in S. aureus isolates from community-onset infectious patients in county hospitals in China to provide the basis for antibiotic-resistance control and clinical medication.Part Ⅰ:A total of475clinical isolates of S. aureus from community-onset infections were collected from34county hospitals in12provinces of China from August2010to December2011. The susceptibility testings of fosfomycin, oxacillin, amoxicillin/clavulanic acid, ciprofloxacin, gentamicin, cefazolin, cefuroxime, penicillin, clindamycin, erythromycin, tetracycline, linezolid, vancomycin and teicoplanin were determined. The isolates were divided into different groups according linezolid MICs. A certain number of isolates with different sensitivity were selected for further study. The mutations in23S rRNA allele gene (rrnl-rrn6), and the rplC, rplD and rplV genes, which encode the ribosomal proteins L3, L4, and L22were detected by PCR and sequencing. The cfr gene among475S. aureus isolates were screened by PCR.The results of susceptibility testings showed that all the isolates were sensitive to vancomycin and teicoplanin. The sensitive rate to fosfomycin, oxacillin, amoxicillin/clavulanic acid, ciprofloxacin, gentamicin, cefazolin, cefuroxime were98.7%,84.7%,85.9%,87.2%,86.6%,73.2%and78.2%respectively. No isolate met with the clinical resistant breakpoint of CLSI.1,4,10,10and11isolates of S. aureus with MICs of linezolid0.25,0.5,1,2and4μg/ml were selected respectively for further study. Nine point mutations on23S rRNA (T2238C, G2398A, C2263T, C2112T, C2129T, C2219T, G2127A, C2234T, G2416A) were detected in12isolates. And mutations on L3, L4and L22were detected in8isolates.14cfr-positive isolates were detected among475S. aureus isolates and the MIC of linezolid against those were4μg/ml and2μg/ml. All the isolates were susceptible to vancomycin and teicoplanin, but high resistant to chloromycetin clindamycin and erythromycin. Among the14isolates,3isolates had mutations on23S rRNA on domain V and5isolates had mutations had mutations on L protein.Further analysis showed that mutations in23S rRNA domain V and ribosomal protein L were not detected in isolates with MIC of0.5μg/ml, mutations in23S rRNA domain V were detected in4out of10isolates and no mutations in ribosomal protein L were detected in isolates with MIC of1μg/ml, mutations in23S rRNA domain V and ribosomal protein L were detected in6and4isolates respectively from10isolates with MIC of2μg/ml, mutations in23S rRNA domain V and ribosomal protein L were detected in3and5isolates respectively from11isolates with MIC of4μg/ml, and in which8/11isolates were cfr-positive. The analysis showed that, with the linezolid MIC value increasing, the numbers and the types of the mutations in23S rRNA domain V and ribosomal protein L were accumulated. This gradually accumulation of resisitant factors might contribute to the decreasment of susceptiblility to linezolid in the isolates.Part II:MLST typing was conducted and location of the cfr gene were determined by Southern blot; the cfr-carrying plasmid structure was determined by PCR walking. RNA secondary structure and three-dimensional structures of9mutations in23S rRNA domain V detected in part I of this study were built respectively with Mfold software and3dRNA software. The interaction between the mutations and linezolid were analyzed by Pymol software. Southern blotting revealed that the cfr genes in all14isolates resided on plasmids. Sequencing of the5.6kb cfr-carrying plasmid segment showed the multiresistance gene cfr was flanked by two copies of the IS256-like insertion sequence, with a downstream orf1gene. The genetic environment of cfr in these isolates were identical and showed99%identity to the corresponding sequences in plasmid pSS-01in Staphylococcus cohnii from animal in China. In addition, the plasmids pRM01and p7LC, which were detected in clinical Staphylococcus cohnii (S. cohnii) and Staphylococcus epidermidis (S. epidermidis) isolates, were also similar to the cfr-carrying plasmid segment in this study. Five isolates and three isolates belonged to sequence type (ST)188and ST965, respectively; the two ST types were previously reported in isolates of animal origin in some areas of China. These results indicate that S. aureus isolates or cfr-carrying plasmids in this study are likely of animal origin.RNA secondary structure and three-dimensional structures of9mutations in23S rRNA domain Ⅴ indicated that:(1) In MIC of1μg/ml group, cfr gene or mutations in ribosomal protein L were not detected. Thus, the slightly elevated linezolid MIC might be mainly due to T2238C mutation and G2398A mutation which were located at a distance of94.91A and102.8A away from linezolid binding site.(2) In MIC of2μg/ml group, the RNA secondary structure showed that the stable G:C base pairings were changed to the unstable G:U base pairing after the C2112T and C2129T mutations happened and G:U base pairing was changed to the relatively stable A:U base pairing after G2127A mutation happened. These changes in RNA secondary structure might loosen or tighten the stem structure and had further affects on the three-dimensional structures of23S rRNA. In addition, the5mutations (C2112T, G2127A, C2129T, C2219T, C2263T) in MIC of2μg/ml group were located at distances of56.06A,51.96A,54.07A,58.83A and52.02A away from linezolid binding site respectively. Hence, the5mutations might make contributions to the elevated linezolid MICs of the isoltes in this group. However, mutations in ribosomal protein L3or L22might make no contribution to the elevated linezolid MICs since the mutations were located far from peptidyl transferase center (PTC).(3) In MIC of4μg/ml group, the C2234T mutation and G2416A mutation in domain V of23S rRNA were located at distances of92.16A and72.68A away from linezolid binding site and cfr gene was detected in8isolates. Thus, the elevated linezolid MICs in this group might be due to the cfr gene presentation.Conclusions1. No linezolid-resistant clinical isolates was detected according to the CLSI breakpoints. However, the isolates in this study showed wide distributions of the MIC values and the highest MIC value is16times higher than the minimum value of MIC, which indicates that further work is needed to monitor the reduced susceptibility trends closely to prevent the emergence of resistant strains.2. With the linezolid MIC value increasing, the numbers and the types of the mutations in23S rRNA domain V and ribosomal protein L were increased. This gradually increasing in the numbers and the types of the mutations might contribute to the changing of susceptibility to linezolid in the isolates.3. Mutations in23S rRNA domain V in MIC of2μg/ml group are the main reason for the elevated linezolid MICs; cfr gene, not the mutations in23S rRNA domain V, make contributions to the elevated linezolid MICs in MIC of4μg/ml group.4. cfr-harboring S. aureus isolates have emerged in some areas of China and the results of the MLST analysis and genetic environment of the cfr gene indicates that cfr-carrying isolates may be transmitted between animals and humans. |
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