Early detection of viable Francisella tularensis in environmental matrices by culture-based PCR

Francisella tularensis is a fastidious, Gram-negative coccobacillus and is the causative agent of tularemia. To assess viability yet overcome lengthy incubation periods, a culture-based PCR method was used to detect early growth of the lowest possible number of F. tularensis cells. This method utilized a previously developed enhanced F. tularensis growth medium and is based on the change in PCR cycle threshold at the start and end of each incubation. To test method robustness, a virulent Type A1 (Schu4) and B (IN99) strain and the avirulent Live Vaccine Strain (LVS) were incubated with inactivated target cells, humic acid, drinking and well water, and test dust at targeted starting concentrations of 1, 10, and 100 CFU mL (low, mid, and high, respectively). After 48 h, LVS growth was detected at all targeted concentrations in the presence of 10 inactivated LVS cells; while Schu4 and IN99 growth was detected in the presence of 10 Schu4 or IN99 inactivated cells at the mid and high targets. Early detection of F. tularensis growth was strain and concentration dependent in the presence of fast-growing well water and test dust organisms. In contrast, growth was detected at each targeted concentration by 24 h in humic acid and drinking water for all strains. Results indicated that the culture-based PCR assay is quick, sensitive, and specific while still utilizing growth as a measure of pathogen viability. This method can circumvent lengthy incubations required for Francisella identification, especially when swift answers are needed during epidemiological investigations, remediation efforts, and decontamination verification.