title: High dynamic range detection of Chlamydia trachomatis growth by direct quantitative PCR of the infected cells.
abstract: Chlamydiae are obligate intracellular bacteria developing in an intracytoplasmic niche, the inclusion. Chlamydia growth measurement by inclusion counting is a key task in the development of novel antichlamydial antibiotics and in vaccine studies. Most of the current counting methods rely on the immunofluorescent staining of the inclusions and either manual or automatic microscopy detection and enumeration. The manual method is highly labor intensive, while the automatic methods are either medium-throughput or require automatic microscopy. The sensitive and specific PCR technology could be an effective method for growth related chlamydial DNA detection; however the currently described PCR approaches have a major limitation, the requirement of purification of DNA or RNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of Chlamydia trachomatis DNA directly from the infected HeLa cells. With our method we were able to detect the bacterial growth in a 4 log scale (multiplicity of infection (MOI): 64 to 0.0039), with high correlation between the biological and technical replicates. As a further proof of the method, we applied the direct qPCR for antibiotic minimum inhibitory concentration (MIC) measurements. The measured MICs of moxifloxacin, tetracycline, clarithromycin and compound PCC00213 were 0.031 μg/ml, 0.031 μg/ml, 0.0039 μg/ml and 6.2 μg/ml respectively, identical or close to the already published MIC values. Our direct qPCR method for chlamydial growth and antibiotic MIC determination is less time-consuming, more objective and more sensitive than the currently applied manual or automatic fluorescent microscopy- based methods.
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sqr. 10, 6720 Szeged, Szeged, Hungary.
authors.forename: Ildikó
authors.initials: I
authors.lastname: Eszik
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sqr. 10, 6720 Szeged, Szeged, Hungary.
authors.forename: Ildikó
authors.initials: I
authors.lastname: Lantos
authors.affiliation: Department of Dermatology, Paracelsus Medical University, Strubergasse 21, 5020, Salzburg, Salzburg, Austria.
authors.forename: Kamil
authors.initials: K
authors.lastname: Önder
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sqr. 10, 6720 Szeged, Szeged, Hungary.
authors.forename: Ferenc
authors.initials: F
authors.lastname: Somogyvári
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sqr. 10, 6720 Szeged, Szeged, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Burián
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sqr. 10, 6720 Szeged, Szeged, Hungary.
authors.forename: Valéria
authors.initials: V
authors.lastname: Endrész
authors.affiliation: Department of Medical Microbiology and Immunobiology, University of Szeged, Dóm sqr. 10, 6720 Szeged, Szeged, Hungary. Electronic address: virok.dezso.peter@med.u-szeged.hu.
authors.forename: Dezső P
authors.initials: DP
authors.lastname: Virok
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001431:Bacteriological Techniques; D002690:Chlamydia Infections / Q000175:diagnosis / Q000235:genetics / Q000382:microbiology*; D002692:Chlamydia trachomatis / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D017291:Clarithromycin / Q000494:pharmacology; D004269:DNA, Bacterial; D006367:HeLa Cells; D006801:Humans; D008826:Microbial Sensitivity Tests; D012329:RNA, Bacterial; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D013752:Tetracycline / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D013752:Tetracycline; D017291:Clarithromycin
keywords: Chlamydia; Chlamydophila; Direct PCR; Growth; MIC; MOI; QPCR