DEVELOPMENT OF A CELL CULTURE-QUANTITATIVE PCR BASED ASSAY FOR RAPID DETERMINATION OF THE MINIMAL INHIBITORY EXTRACELLULAR CONCENTRATION (MIEC) FOR THE INTRACELLULAR FORM OF FRANCISELLA TULARENSIS

Francisella tularensis is a highly virulent facultative intracellular bacterium. The lack of a safe and efficient vaccine makes antibiotics the preferred treatment. F. tularensis antibiotic susceptibility tests are based on the in vitro standard CLSI-approved microdilution method for determining the antibiotic minimal inhibitory concentration (MIC). However, limited data are available regarding the minimal inhibitory extracellular concentration (MIEC) needed to eradicate intracellular bacteria. Knowledge of MIC and MIEC values may provide a broad clinical view on the effectiveness of the suggested treatment. High MIEC values may indicate a low intracellular activity/concentration and may explain treatment failure. In order to facilitate the time-consuming growth based classical MIEC assay we set to develop a rapid 3-h quantitative PCR (qPCR) based intracellular antibiogram assay, which yields comparable MIEC values to those obtained by the classical 72-h colony forming assay. This rapid qPCR assay is highly advantageous in light of the relatively slow growth rates of F. tularensis. Using our assay we determined the MIECs for the WHO-recommended antibiotics for treating tularemia. Our results showed that the MIECs obtained for doxycycline, chloramphenicol and ciprofloxacin were indicative of intracellular activity. Although gentamicin is effective against the extracellular form of the bacteria, it demonstrated high MIEC values and was not effective against intracellular bacteria for at least 32 h post treatment, raising the question of whether slow-penetrating gentamicin should be used when prompt treatment is of need.

To date, our qPCR assay is the quickest F. tularensis MIEC determining assay reported. Moreover, the qPCR intracellular antibiogram assay may facilitate future standardization of intracellular antibiogram processes and may help to evaluate the effectiveness of new compounds against the intracellular form of F. tularensis.