Real-time PCR for pharmacodynamic studies of Chlamydia trachomatis.
Pharmacodynamic knowledge about Chlamydia trachomatis exposed to antibiotics is hampered due to methodological limitations. We have developed a quantitative real-time PCR method (qRT-PCR) for determination of viable C. trachomatis. The method measures specific RNA transcripts of omp2 (omcB) as an expression of viable C. trachomatis. Two clinical isolates (one strain derived from a patient with recurrent symptoms despite doxycycline treatment) were cultured in McCoy cells and exposed to doxycycline at concentrations of 0.0078-64 mg/L. MIC values were evaluated microscopically by immunofluorescence (IF) and by qRT-PCR performed on cDNA prepared from the total RNA. The MIC for two C. trachomatis strains were determined to 0.016 and 0.031 mg/L by both qRT-PCR and IF. The qRT-PCR assay enabled MIC determinations without subjective evaluation, which is a problem when visually evaluating inclusions. The presented qRT-PCR is a suitable method for MIC determination of C. trachomatis. It has the advantage of giving quantitative measurements of chlamydial RNA levels and the method is useful in pharmacodynamic studies of C. trachomatis.
Storm
Martin
M
Uppsala University, Department of Medical Sciences, Clinical Bacteriology, Uppsala, Sweden.
Gustafsson
Ingegerd
I
Herrmann
Björn
B
Engstrand
Lars
L
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001431:Bacteriological Techniques; D001483:Base Sequence; D002692:Chlamydia trachomatis / Q000187:drug effects* / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000706:statistics & numerical data; D008856:Microscopy, Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012680:Sensitivity and Specificity
D000900:Anti-Bacterial Agents; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D012333:RNA, Messenger