Discrimination of viable from non-viable Gram-negative bacterial pathogens in airborne particles using propidium monoazide-assisted qPCR

The presence of bacterial pathogens in airborne particulate matter (PM) has been of considerable concern from the public health standpoint. Conventional culture-based methods are tedious, time consuming and are unable to quantify stressed viable but non-culturable (VBNC) populations of these pathogens. This study reports the optimization, validation and application of a new and rapid quantitative method for enumeration of four live potential Gram-negative bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Aeromonas hydrophila) in PM of biomass burning origin. This method makes use of an intercalating dye (propidium monoazide, PMA) in conjunction with real-time PCR (qPCR) analysis following DNA extraction from PM samples for distinguishing viable from non-viable potential bacterial pathogens. This method was not affected by the complex matrix of the environmental samples, nor by any PCR inhibition effects. The number of viable pathogens ranged from 0 to 8 × 104 gene copies/m3 in PM. With the exception of A. hydrophilia, all the three pathogens were found to be present in PM. The correlation between the counts obtained using the PMA-qPCR (modified qPCR) and those from the culture-based method was very high with R2 ~ 1.0 and p value < 0.0001.

► Selective quantification of only live pathogens in airborne particles (PM) is reported.
► This method makes use of an intercalating dye-assisted real-time PCR technique.
► Viable potential bacterial pathogens were determined in PM of biomass burning origin.
► The method was not affected by the complex matrix of environmental samples.