Evaluation Of Antimicrobial Resistance And Treatment Failures For Chlamydia Trachomatis

Objective To explore the significance of multiple passage in wild strains of Chlamydia trachomatis with cell culture.To test the activity of tetracyclines, macrolides and quinolones against recent clinical isolates of urogenital Chlamydia trachomatis in vitro and explore the mechanism of resistance in gene molecular level.To evaluate the relationship between susceptibility test and clinical curative effects.Methods Afterl8～24h, McCoy cell cultured monolayer in the six well trays at 37℃and in CO2 of 5%, the standard strain and wild strain of Chlamydia trachomatis were inoculated and cultured for 48～72h. The existence of Chlamydia trachomatis was identified by Logus iodine dying, which the culture-negative strains cultured from the five passages. PCR has been taken as a reference, repeating subculture kit to be used in Chlamydia trachomatis detected. The diagnostic value of repeating subculture technique for detection of Chlamydia trachomatis in comparison with that of the polymerase chain reaction was established in a follow-up study of 299 samples. The PCR was performed with a set of primers of a Chlamydia trachomatis specific set directed against the common endogenous plasmid 42 strains of chlamydia tested demonstrated survival were cultured in McCoy cells. When more than 90% McCoy cells infected, the samples were collected to investigate susceptibility test to 9 Antimicrobials.To detect plasmids containing tetM associated with tetracyclines resistant by PCR amplification. Finding macrolide resistant Chlamydia trachomatis clinical isolates and studying them for possible mutations in 23S rRNA and L4 genes by RT-PCRand PCR amplification, respectively. To test the most frequent mutations of gyrA QRDR associated with quinolone resistance by RFLP.Additionally gene type were studied.Results 104 positive samples were detected by the culture technique in 299 cases. The positive rate of the initial culture (primary generations) was 5.69%, the positive rate of the passaging once (two generations) was 20.07%, the positive rate of the passaging twice (three generations) was 33.44%, the positive rate of the passaging three times (four generations) was 34.78%, the passaging four times (five generations) remained at 34.78%. With the passage of three generations ago, number of positive cases and the positive rate increased significantly, but four generations and five generations were in a basically stable trend. Results for the detection of Chlamydia trachomatis by the PCR were in complete agreement with the results by the culture method of detection, except for 24 culture-negative samples, which were found to be positive by the PCR. For all isolates, no isolates was observed resistant to other 8 antimicrobial. Two isolates were resistant to erythromycin, which of the MIC was 2 micrograms/ml and had the mutations C2452A and T2611C (Escherichia coli numbering) in the 23S rRNA gene. The studied region of gene L4 in resistant isolates showed no difference from that of sensitive isolates. All isolates had the double mutations found:Pro P113 (CCG)→Leu L (CTG) and Pro P156 (CCC)→Ala A (GCC) published GenBank sequence (NC000117.1). For 25 clinical isolates of Chlamydia trachomatis have been detected plasmids containing tetM. It was no discovery one point mutation (G→T) which caused Ser83-to-Ile change.Conclusions The detection rate of Chlamydia trachomatis can be significantly increased by culturing multiple passage; Chuan-blind testing standards once must have missed the diagnosis rate, clinical strains should be cultured three generations. Successful Chlamydia trachomatis culture establishes good foundation for further laboratory research of Chlamydia trachomatis including pathogenesis, immunologic mechanism, drug susceptibility test, resistance mechanism, and protective vaccine so on. As the time goes away, antibiotic sensitivity showed different degrees of declining. Clarithromycin showed best activities against C.t in vitro. Minocycline,moxifloxacin and sparfloxacin exhibited a markedly better in vitro activity against C.t, but the MICs of their exceeded the level of other reports. The low level erythromycin resistance is associated with the mutations C2452A and T2611C in the 23S rRNA gene. In the L4 gene of double mutations are not responsible for erythromycin resistance. Type E is still the main gene type. Further studies are needed for the elucidation of exact mechanism of Chlamydia trachomatis resistance to antimicrobial.