Quantification of viable bacteria in air by molecular assays

Abstract

Microbes in air has gained particular attention due to the adverse impact on human health. The studies on monitoring airborne microbes are usually based on the analysis with culture assay. However, airborne bacteria may be present as viable but not culturable state; therefore, the exposure level to airborne bacteria may be underestimated by culture assay. To deal with this issue, this study was initiated to develop a qPCR-based method coupled with nucleic acid dyes (ethidium monoazide, EMA, and propidium monoazide, PMA) that quantified total viable bacteria exclusively. Various types and concentrations of nucleic acid dyes were evaluated, and the range of the limit of detection was determined. Moreover, the developed method was validated in different environments. For live cells, our results showed no statistical significant difference in the DNA concentration between PMA-treated and -untreated samples. On the other hand, a significant decrease of DNA concentration was observed for the heated (dead) cells pretreated with PMA as compared to untreated ones. These results showed that the PMA penetrated dead bacteria and inhibited the DNA amplification in qPCR, indicating that PMA coupled with qPCR (PMA-qPCR) is applicable to quantify live bacteria exclusively. Moreover, the DNA concentrations measured in the group of heated cells were similar when pretreated with PMA of 1.5 ug/mL or greater. Thus, PMA at 1.5 ug/mL coupled with qPCR was considered as the most suitable for quantifying viable bacteria. As for testing on the limit of detection of the PMA-qPCR assay, a linear range (R2=0.993) was obtained for the cells between 1×105 and 1×108cfu/mL. The PMA-qPCR was further applied to analyze the air samples collected from hospital, poultry and swine housing, and the results showed that the concentrations of viable bacteria were always greater than those of culturable bacteria but lower than those of total bacteria. The present data demonstrate that the PMA-qPCR developed was suitable for quantification of viable bacteria in various environments.