Exploration Of Culture Chlamydia Trachomatis In HaCaT Cell And Antibiotic Susceptibility Test In Vitro

Objective To find more suitable host cell for the growth of Chlamydia trachomatis (C.t), which is more coherent with the clinical results of susceptibility test in vitro. Method We cultured the C.t using HaCaT cells and passaged, we also tried to detect the clinical susceptibility of Chlamydia strains present in the genitourinary tract to macrolides, tetracyclines, quinolones, rifamycin using the HaCaT cells and compared this result of susceptibility test with using McCoy cells. The standard strains of serotype E of C.T and clinical specimens containing the C.t were inoculated in the HaCaT cells using the routine culture methods. The C.t inculation in the HaCaT cells was comformed by iodine staining, monoclonal fluorescent antibody testing and amplification of C.t endogenous plasmid using PCR technique. Inoculated the same amount of the standard strains of serotype E of C.t in HaCaT cells of24-well plates, and pretreated HaCaT cells using0,10,20,30,40,50μg/ml DEAE-D for30minutes and centrifuged after inoculum using0,1000,1500,2000,2451g centrifugal force1hour respectively. The best culture condition for the HaCaT cell culturing C.t was determined by counting the number of inclusion bodies of each well and ANOVA analysis by the software of SPSS17. The detection sensitivity test of34strains of clinical isolates of C.t to macrolides, tetracycline, quinolones, rifamycin in HaCaT cells and McCoy cells in vitro were detected at the same time. Whether there are difference of susceptibility test in the two types of cells was determined by Wilcoxon rank sum test analied by software of SPSS17. The ompl gene of34strains of clinical isolates was amplified by PCR and digested by restriction enzyme of Alu I, Msp I and the differences of the results of susceptibility test in different serotypes were compared. Results The HaCaT cells successfully cultured the standard strains of serotype E of C.T was identified by iodine staining, monoclonal fluorescent antibody testing and amplification of endogenous plasmid of C.t using PCR technique. The standard strains of serotype E of C.T in HaCaT cells was multiplied through the passage. It was proved by the number of inclusion bodies in each passage. The best DEAE-D concentration pretreating the HaCaT cells is30μg/ml. The best centrifugal force is 245lg. MIC values (mg/L) of34cases of clinical isolates of Chlamydia trachomatis was measured.0.125to1of erythromycin,0.004to0.016of clarithromycin,0.032to0.25of azithromycin,0.04to0.625of tetracline,0.016to0.5of Doxycycline,0.004to0.128of minocycline,0.125to1of levofloxacin,0.03to0.24of moxifloxacin,0.016to0.128of sparfloxacin,0.001to0.03of rifampin were MIC values detected in McCoy cells, measured by the MIC value (in mg/L):0.125to2of erythromycin,,0.004to0.064of clarithromycin,0.032to4of azithromycin,0.02to1.25of tetracline,0.016to1of Doxycycline,0.008to0.512of minocycline,0.25to4of levofloxacin,0.03to0.96of moxifloxacin,0.032to1.024of sparfloxacin,0.001to0.015of rifampicin were were MIC values detected in HaCaT cells. MIC values measured in HaCaT cells and McCoy cells were higher than previously reported and the MIC values detected in HaCaT cells were higher than in McCoy cells. There were significant difference in the result of susceptibility test to all antibiotics detected in the two kinds of cells through Wilcoxon rank sum test analysis. There were25strains of serotype E、8strains of serotype D、1strains of serotype F in the34clinical isolates. Vitro susceptibility testing of C.t using McCoy cells and HaCaT cells, the MIC values anti-Chlamydia trachomatis is basically the lowest F-type, D-center, E the highest, except rifampicin. The same serotype of C.t susceptibility MIC values in HaCaT cells and McCoy cells is different.Conclusions HaCaT cells can be used as host cells of C.t and these can proliferate in HaCaT cells. All these establishe good foundation for further laboratory research of C.t including pathogenesis, immunologic mechanism, drug susceptibility test, resistance mechanism, protective vaccine and so on. As the time goes by, antibiotic sensitivity showed different degreee of decline. In vitro to a variety of antibiotics against C.t MIC values in the different culture cells (HaCaT cells and McCoy cells), there are differences. It is need to detect more clinical strains to verify Whether HaCaT cells susceptibility test results are more in line with the clinical outcome and whether HaCaT cells are easier to select the drug-resistant strains.