Comparison of in vitro antibiotic susceptibility testing of R. typhi using plaque assay and quantitative real-time...

Murine typhus, caused by Rickettsia typhi, is a globally distributed flea-borne disease. Antimicrobial susceptibility data are largely based on reference strains, with little evaluation of variability across clinical isolates. The plaque assay, the gold standard for antibiotic susceptibility testing, requires up to 14 days to complete. Quantitative real-time PCR (qPCR) offers a rapid alternative for determining minimum inhibitory concentrations (MICs). Although qPCR-based susceptibility testing has been described, formal statistical assessment of agreement between methods remains limited. We estimated the MICs for azithromycin, doxycycline, and amoxicillin using qPCR and plaque assay on 24 R. typhi isolates (8 laboratory strains and 16 Lao clinical isolates) cultured in Vero cells. Heat-inactivated R. typhi (56°C, 30 min) served as a control. MIC was defined as the lowest concentration preventing plaque formation (plaque assay) or producing a Ct value greater than or equal to that of the heat-inactivated sample (qPCR). The mean MIC from three independent experiments was taken to represent each isolate. Agreement between methods was assessed using Bland-Altman analysis. Median qPCR MICs (MIC₅₀) of all isolates were 0.130 mg/L (IQR, 0.104-0.240 mg/L) for azithromycin, 0.130 mg/L (IQR, 0.065-0.224 mg/L) for doxycycline, and 256 mg/L (IQR, 234.7-341.3 mg/L) for amoxicillin. Corresponding plaque assay MICs₅₀ were 0.383 mg/L (IQR, 0.194-0.807 mg/L), 0.037 mg/L (IQR, 0.026-0.073 mg/L), and 170.7 mg/L (IQR, 112-256 mg/L), respectively. Bias was -0.3452 (-1.146, 0.4558) for azithromycin, 0.0969 (-0.2297, 0.4235) for doxycycline, and 85.33 (-187.3, 358) for amoxicillin. These findings demonstrate inter-isolate variability and quantitative agreement between methods.IMPORTANCEMurine typhus is a treatable febrile illness caused by Rickettsia typhi, transmitted to humans via fleas. The plaque assay, which detects bacterial-induced host cell death in the presence or absence of antibiotics, is the reference method for testing drug susceptibility. However, the method requires approximately 2 weeks to obtain results. Detection of bacterial nucleic acid provides an alternative laboratory approach that reduces the assay duration to approximately 1 week, thereby improving research efficiency and throughput when testing multiple isolates. Compared with plaque assays, quantitative real-time PCR allows downstream analysis following sample inactivation, reducing prolonged high-containment handling. In this study, we compared the antibiotic susceptibility of 24 R. typhi isolates using both methods. We tested antibiotics commonly used to treat R. typhi infection (doxycycline and azithromycin), along with amoxicillin as a negative control. Both showed comparable results for all antibiotics tested, further supporting nucleic acid detection as a reliable and faster alternative.