Evaluation of groundwater bacterial community composition to inform waterborne pathogen vulnerability assessments

Microbial water quality evaluations are essential for determining the vulnerability of subsurface drinking water sources to fecal pathogen intrusion. Rather than directly monitor waterborne pathogens using culture- or enumeration-based techniques, the potential of assessing bacterial community using 16S rRNA gene amplicon sequencing to support these evaluations was investigated. A framework for analyzing 16S rRNA gene amplicon sequencing results featuring negative-binomial generalized linear models is demonstrated, and applied to bacterial taxa sequences in purge water samples collected from a shallow, highly aerobic, unconfined aquifer. Bacterial taxa relevant as indicators of fecal source and surface connectivity were examined using this approach. Observed sequences of Escherichia, a genus suggestive of fecal source, were consistently detected but not confirmed by culture-based methods. On the other hand, episodic appearance of anaerobic taxa sequences in this highly aerobic environment, namely Clostridia and Bacteroides, warrants further investigation as potential indicators of fecal contamination. Betaproteobacteria sequences varied significantly on a seasonal basis, and therefore may be linked to understanding surface-water groundwater interactions at this site. However, sequences that are often encountered in surface water bodies (Cyanobacteria and Flavobacteriia) were notably absent or present at very low levels, suggesting that microbial transport from surface-derived sources may be rather limited. This work demonstrates the utility of 16S rRNA gene amplicon sequencing for contextualizing and complementing conventional microbial techniques, allowing for hypotheses about source and transport processes to be tested and refined.