Study On Cell Culture And Antimicrobial Susceptibility Of Chlamydia Trachomatis Clinical Isolates

Chlamydia trachomatis is an obligate intracellular bacteria and can cause many diseases in human.Urogenital Ct infection, except NGU, it also causes many complications and serious psychoburden to the patient. According to the estimation of WHO, there are 93 million new cases every year and making severe economic burden to the society.Antimacrobials is the main drug for the treatment of Ct infection. It was thought that the microbiological cure rate of antimacrobials was above 90%, but the clinical treatment failure cases report more and more. There are many reasons associated with treat failure, factors like age of the patient and renewal of sexual intercouse, also the low compliance, drug pharmacolinetics and availability at the tissue level can dictate failue. And most of all the clinic drug resistant strains usually induce treatment failure; even forming persistence infection, resulting in infection unrecover for long term. As the antimacrobials' alternation and rational use strengthened, the antimicrobial susceptibility of Ct changes unceasing.Objective: To investigate the in vitro effects of various antimacrobials against the clinic isolates of Ct, which are now mostly used in clinical. To understand the susceptibilitis of Ct in our clinical, moreover to explore the diversify of the susceptibilitis of Ct and the relation between the clinic curative effect and the experimental result. Establish a maturated feasible screening culture method and antimicrobial susceptibility testing method for clinical Ct which is suitable for our laboratory. Reserve the Ct positive clinical strains and it will lay a foundation for further research.Methods: Collecting 194 urethral or cervix swabs from the patients who came to Tianjin sexually transmitted disease institute between April 2005 to March , 2007. All of the patients were fit to the collection standard and we recorded their clinical related data. We undertook cell culture of clinical samples with McCoy cell on the foundation of Ct E standard strain. All the clinic strains were been blind passage cultured. The postive strain of the second generation are kept on culturing. When above 90% inclusions formed, we amplified this strain and reseved in refrigerator of 80 temperature below. First we explore the experiment conditions of the antimicrobial susceptibilities of Ct with Ct E standard strain. Then decide the inoculum size of the Ct, which can cause cell infection rate beyond 90%. Making the recommend concentration be the middle concentration, the ten antimacrobials(erythromycin,clarithromycin,azithromycin,tetracycline,doxycycline,minocycline,levofloxacin,sparfloxacin,moxifloxacin,rifamoin) are perpared for bexakis geometic proportion. We do the antimicrobial susceptibility test with 96-well plate. MIC was determined as the lowest concentration under which there were no inclusion forming, explore the relation between the clinic curative effect and the experimental result and screening the persister for the reseach of the drug resistance gene.Results: There were 52 culture positive clinical samples and 23sample infection rate beyond 90%. Minimal inhibitory concentration (MIC) of the 23clinical chlamydia trachomatis ranges were as follows: erythromycin,0.125～2 microgram /ml;clarithromycin,0.004～0.064microgram/ml;azithromycin,0.0313～1 microgra m /ml ; tetracycline , 0.040～0.625 microgram/ml ; doxycycline , 0.015625～0.125 microgram/ml;minocycline,0.002～0.128 microgram/ml; levofloxacin , 0.0625～0.5 microgram/ml ; sparfloxacin , 0.016～0.128 microgram/ml ; moxifloxacin , 0.015～0.24 microgram/ml; rifamoin, 0.001875～0.03 microgram/ml. Under the microscope, as the increasing of the drug concentration, the conclusions generate alteration in morphology , missing nomal oval and their quantity decreased. The results demonstrated that 8 wild strains is resistant to erythromycin.Conclusions: 1,Cultured Ct clinical samples from STD clinics in our department successfully and raised the positive rate; reserved the positive strains and lay foundations for further researches. 2,We grope a method suitable for clinical Ct strains cell culture and antimicrobial susceptibility test and found that eight wild isolate of Ct is resistant to erythromycin. In tianjin the clinical wild isolates of Ct have already been resistant to erythromycin . 4. we found that there are much difference between the clinic curative effect and the experimental result.