16S rRNA chlorinated drinking water community with and without PMA treatment

Using an easy to construct, low-cost light box compatible with widely used Sterivex and flat nitrocellulose filters, we demonstrate that extracellular DNA (eDNA) removal is primarily driven by propidium monoazide (PMA) treatment rather than buffer controls (93.8% versus 16.7% removal). We further showed that PMA-mediated eDNA removal was highly effective but matrix-dependent, achieving 99.8% removal in tap, 86.1% in bottled and 99.8% in drinking water biofilms. We further related PMA treatment to cell viability by exposing Pseudomonas aeruginosa to experimental chemical kill gradients in pure culture and spiked into drinking water. PMA treatments showed close agreement with viable cell counts and intact cell counts. In contrast, total DNA and total cell counts failed to reflect loss of viability across the same gradients (p > 0.05). Having established effective eDNA removal in bottled water we explored the effect of chlorination on community composition with and without PMA. PMA increased community richness (from 214.6 +/- 24.4 to 314.8 +/- 92.6 ASVs) and evenness (Pielou's index from 0.50 +/- 0.04 to 0.66 +/- 0.09). Dominant genera, including Escherichia and Nitrotoga, were reduced (from 28.74 +/- 9.75% to 5.02 +/- 2.67%, and 23.34 +/- 4.69% to 7.06 +/- 4.32% respectively) while Mycoplasma and 'others' group increased in relative abundance (from 10.69 +/- 0.88% to 23.21 +/- 2.9% and 12.17 +/- 0.89% to 35.54 +/- 4.29% respectively). Together, these results support PMA treatment as a robust approach for drinking water microbiome analysis, reflecting viable and intact cells while also revealing hidden diversity under disinfectant stress by reducing DNA signal from eDNA and membrane compromised cells.