| Objective: (1) To determine the status of eight antimicrobial agents resistance among U.urealyticum strains in Jiangsu Province. (2) To study the mechanisms of U.urealyticum resistance to common used antimicrobial agents including tetracycline, fluoroquinolones and erythromycin. (3)To design and evaluate the PCR-RFLP genotyping method according to the difference of 23S rRNA between Biovar 1 and T960 types, and to further investigate the association of these biovars with gynecological coinfections and susceptibilities of U.urealyticum to antibiotics. (4)To study correlation of in vitro susceptibilities to fluoroquinolones of naturally occurring quinolone-resistant Neisseria gonorrhoeae strains with changes in GyrA and ParC. Methods:(1)The preliminary isolation and identification of mycoplasma strains including U.urealyticum and Mycoplasma hominis were conducted by Mycoplasma ID kit. The positive specimens of U.urealyticum were further identified by culture of filtered positive medium and specific PCR assay. (2)In-vitro susceptibilities of U.urealyticum (MICs) to eight antimicrobial agents (tetracycline, minocycline, erythromycin, clarythromycin, azithromycin, ciprofloxacin,ofloxaxin and levofloxacin) were determined by a broth microdilution method. (3)According to the susceptibilities of U.urealyticum,34 strains of U.urealyticum were randomly selected from 84 isolates to amplify the tetM gene, specific fragments of gyrA genes of randomly selected 28 strains were amplified and sequenced. Blastn program was used to analyze and compare the sequence of clinical isolates with the sequence of standard strain T3. (4) Primer premier5.0 software was used to design PCR assay and select appropriate restricted endonuclease according to the difference of 23S rRNA gene between Biovar 1 and T960 types, new genotyping method was designed and evaluated by comparing its genotyping results with standard strains and clinical strains identified by"golden standard". (5) 54 strains of N.gonorrhoeae were randomly selected from 95 clinical isolates, chromosomal DNA of which was used as a template in PCR to amplify the quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes. And PCR-amplified DNA was further sequenced. (6) In combination with epidemiological data,experimental results were analyzed by usingχ2 test, fisher's exact test and ANOVA analysis method.Results: (1)For U.urealyticum the MIC50 of minocycline, tetracycline, clarythromycin, azithromycin, erythromycin, ciprofloxacin, ofloxacin and levofloxacin were <0.0625, <0.125, 0.25, 1, 2, 8, 4 and 2mg/L respectively; the MIC90 of minocycline, tetracycline, clarythromycin, azithromycin, erythromycin, ciprofloxacin, ofloxacin and levofloxacin were 0.125, 1, 1, 4, 8, 64, 16 and 8mg/L respectively. (2) Significant difference of susceptibilities of U.urealyticum to macrolide were found between Mh coinfection group and non-Mh coinfection group, U.urealyticum from Mh coinfection group showed higher resistance to macrolide. (3) TetM gene was detected among 7 of 34 U.urealyticum strains, strains with tetM gene showed more resistance to tetracycline and minocycline in comparison with strains with non-tetM gene. Three alterations of GyrA in 28 U.urealyticum strains were found:①A alanine to valine substitution at position 101(corresponding to position 84 in E.coli);②A aspartic acid to glutamine substitution at position 112(corresponding to position 95 in E.coli);③An absence of T base at position 267 in gyrA gene. U.urealyticum with GyrA alterations was more resistant to fluoroquinolones and GyrA alterations were not found among all strains susceptible to fluoroquinolones. (4) PCR-RFLP typing method had excellent typeability(100%), specificity and repeatability among standard strains, the map of agarose gel electrophoresis was clear and easy to differentiate and interpret. Among clinical isolates identified by"golden standard", the sensitivity and specificity of PCR assay were 97% and 89% respectively,...
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