EMA- Versus PMA-Amplicon-Based Sequencing to Elucidate the Viable Bacterial Community in Rainwater

Ethidium monoazide bromide (EMA) and propidium monoazide (PMA), in combination with amplicon-based sequencing (ABS) and quantitative polymerase chain reaction (qPCR) assays, were compared for the detection of viable bacterial species in rainwater. The ABS α- and β-diversity indices indicated that, in comparison to the untreated samples, both EMA and PMA reduced the detection of non-viable bacteria in the rainwater samples. However, while comparable results were obtained for the detection of the most abundant bacterial families and genera in the rainwater samples for both the EMA and PMA pre-treatments; the EMA pre-treatment produced highly significant differences in the relative abundance of the dominant bacterial families and genera, and significantly decreased the detection of “unclassified” bacteria in comparison to the untreated samples (i.e. unclassified genera: untreated 46.7% versus EMA-treated 39.7%). Additionally, the inclusion of EMA and PMA allowed for the increased detection of less abundant pathogenic bacteria in the rainwater samples, such as Clostridium , Listeria and Streptococcus spp. The qPCR assays also indicated that the EMA and PMA pre-treatments were comparable for the detection of intact and potentially viable Acinetobacter , Legionella and Pseudomonas spp. The combination of viability pre-treatments with ABS thus offers a comprehensive monitoring approach to detect less abundant bacterial genera and/or pathogens, which may pose a health risk to the end-users (prioritise target organisms) of unpiped environmental water sources. Moreover, the subsequent combination of the viability pre-treatments with qPCR allows for the specific detection and quantification of these pathogenic genera, which increases analysis sensitivity and allows for the application of risk assessment and water safety strategies.