| Objectives:(1) To investigate the characteristics of drug resistance and current situation of methicillin-resistance in clinical isolates of staphylococcus aureus in Changsha, and to provide guidance for the proper use of antibiotics. (2) To evaluate the clinical value of oxacillin disk diffusion method, cefoxitin disk diffusion method, oxacillin agar dilution method, cefoxitin agar dilution method and chromogenic agars method for detection of methicillin-resistant staphylococcus aureus (MRSA). (3) To analysis the genetic background and investigate the molecular epidemiology profile of clinical isolates of staphylococcus aureus in Changsha. (4) To construct prokaryotic expression vector of femA gene (Factors essential for the expression of methicillin resistance gene), and express it in E.coli and establish foundation for further investigations.Methods:(1) 293 clinical isolates of staphylococcus aureus were collected from 11 hospitals in Changsha and then identified by Vitek-2 system. All the isolates were verified as staphylococcus aureus by PCR amplification of femA gene. K-B disk method was used to test drug sensitivity of staphylococcus aureus to 23 commonly used antibiotics. Chromogenic cephalosporin spot test was applied to detect beta-lactamase. The inducible resistance of erythromycin to clindamycin was checked by D-test. (2) MRSA was identified by oxacillin disk diffusion method, cefoxitin disk diffusion method, oxacillin agar dilution method, cefoxitin agar dilution method and chromogenic agars method separately. Detection of mecA gene by PCR was set as a standard assay to detect MRSA and comparing study were applied to evaluate results of other methods. (3) Pulsed-field gel electrophoresis (PFGE) was performed for genotypic and homologous analysis of clinical isolates of staphylococcus aureus.(4)The statistical analysis was performed by Whonet 5.4 and SPSS 11.3 software. (5) According to the sequence of femA gene in Genbank, PCR primers were designed using Primer Premier 5.0 software and femA gene fragment was amplified by PCR. Two restriction endonuclease recognition sites BamHl and Sal 1 were added to the 5'end of the primer. The DNA fragment and plasmid pQE30 were double-enzyme digested respectively, ligated and transferred into DH5a. The positive clones were selected and the recombinant plasmids pQE30-femA were purified and identified. Then the identified pQE30-femA was transformed into E.coli JM109 and the target protein expression was induced by IPTG. The expressed product was identified by SDS-PAGE and Western blot.Results:(1) Of the 293 S. aureus isolates,273(93.2%) wereβ-lactamase test positive. Of the 28 isolates resistant to erythromycin but susceptible to clindamycin,26(92.9%) showed a positive result of D-test. (2) The resistant rates to 14 of the 23 antibiotics tested were higher than 50%. Resistant rates to penicillin and ampicillin were the highest (both 96.6%), and the next were erythromycin and clindamycin with resistant rate of 77.1% and 67.2% respectively. The sensitive rates to 7 of the 23 antibiotics tested were higher than 50%. All the isolates were susceptible to tecoplanin, vancomycin and linezolid. The sensitive rate to Nitrofurantoin, chlormycetin and doxycycline was 97.6%,91.8% and 71.0% respectively. (3) Totally 293 S. aureus involved exhibited 93 antimicrobial-resistant profiles to the 23 antibiotics tested. The PX1 type included 47(16.0%) strains and the PX2 type included 35(11.9%) strains, which accounted 28.0%(82/293) of all the stains, were the primary epidemic antimicrobial-resistant profiles. (4) Of the 293 strains,190 (64.8%) were methicillin resistant staphylococcus aureus (MRSA) and 103 (35.2%) were methicillin sensitive staphylococcus aureus (MSSA). The resistant rates of MRSA to 18 antibiotics were higher than MSSA (P<0.05). (5) The resistant rate of strains to cefoxitin isolated from Xiangya hospital in 2006,2007 or 2008 was 66.7%,85.0% or 63.1% respectively and there are significantly different among them. While there was no significant difference among the resistant rate to other 22 antibiotics tested in these 3 years. (6) Resistant rate to 13 antibiotics of the 23 antibiotics tested were significant different among strains from different ward of Xiangya hospital (P<0.05). Resistant rate to 15 antibiotics tested were significant different among strains from different hospitals in Changsha at the same year (P<0.05). (7) The MIC range of OXA and FOX on staphylococcus aureus was 0.125μg/ml->256μg/ml and 2μg/ml->256μg/ml respectively. Both of their MIC50 and MIC90 were≥256μg/ml. (8) Using detection of mecA gene as the standard test, the sensitivity and specificity of the other 5 methods were all higher than 95.5%, without significant difference (P>0.05). (9) Of all the 115 clinical isolates of staphylococcus aureus,39 PFGE types were demonstrated. PFGE type A with the incidence of 48.7%(56/115) was the predominant genotype, and 55 of them were MRS A. The next was PFGE type L with the incidence of 4.3%(5/115). PFGE type A included 13 subtypes from A1 to A13, and the subtype A1 is the predominant type and epidemic clone with the incidence of 22.6%(26/115). PFGE type A strains were isolated from 8 hospitals (8/11), and both subtype A1 and A4 strains were isolated in the past 3 years in Xiangya hospital. (10) Verified by PCR, double-enzyme digested assessment and sequencing, recombinant plamid pQE30-femA was successfully constructed. SDS-PAGE and Western blot analysis revealed the recombinant plasmid pQE30-femA expressed a 53KD target protein in E.coli JM109. Bandscan software analysis showed that the expressed target protein at 4h accounted for about 27.5% of the total bacterial protein.Conclusions:(1) Staphylococcus aureus in Changsha is multiple resistant to commonly used antimicrobial agents and has a high resistant rate to methicillin. Positive rate of beta-lactamase, rate of inducible resistance of erythromycin to clindamycin, and isolating rate of MRSA were all high. (2) The major antimicrobial-resistant profiles of clinical isolates of staphylococcus aureus in Changsha were PX1 and PX2. The resistant rates to multiple antibiotics were significantly different in isolates from different wards and different hospitals. (3) All of the 5 phenotypic analysis methods can be used to detect MRSA. Cefoxitin disk diffusion method is the most convenient and reliable one, thus it can be used as a routine test for clinical laboratory to detect MRSA. (4) PFGE type A outbreak at clinical isolates of staphylococcus aureus occurred in Changsha and A1 subtype is the predominant epidemic clone. Dissemination of the same clone intra-and extra-hospitals was an important reason of the widely spread of MRSA. (5) Recombinant plamid pQE30-femA was successfully constructed and high-effective expressed in E.coli.
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