Quantitative Detection of VBNC State Pseudomonas aeruginosa Contributing to Accurate Assessment of Microbial Inactivation in Drinking Water Disinfection

Abstract

In harsh environments, bacteria often enter a viable but nonculturable (VBNC) state, which cannot be detected using heterotrophic plate counting (HPC). Importantly, VBNC bacteria can potentially resuscitate under favorable conditions, posing a risk to drinking water safety. This study introduces an innovative approach, combining improved quantitative polymerase chain reaction (qPCR) with propidium monoazide (PMA) dye and HPC to accurately quantify VBNC Pseudomonas aeruginosa (P. aeruginosa). The method was applied to assess the ability of various disinfection techniques to induce P. aeruginosa into the VBNC state. Different disinfection methods, including ultraviolet radiation (UV), sodium hypochlorite (NaClO), and peracetic acid (PAA), significantly reduced bacterial culturability (>99.9%), with the majority entering the VBNC state. Notably, under favorable conditions, UV-induced VBNC cells were resuscitated faster than those induced by NaClO. VBNC P. aeruginosa exhibited relatively high intracellular adenosine triphosphate (ATP) levels, indicating ongoing metabolic activity. Scanning electron microscopy (SEM) reveals that some bacteria maintained cellular integrity for UV and PAA treatment, while evident membrane disruption was observed after NaClO disinfection. This study represents a significant advancement in quantitatively detecting VBNC state P. aeruginosa, contributing to an accurate assessment of microbial inactivation during drinking water disinfection.

A Combination of UV and Disinfectant for Inactivating Viable but Nonculturable State Pseudomonas aeruginosa: Efficiency and Mechanisms

“…PMA-qPCR was employed to quantify the overall count of viable cells using the long primers (opr-L, Table S1). The validation of PMA-qPCR was elucidated in our previous publication [28]. During PMA-qPCR measurement, a stock solution of 2.5 mM PMA was first prepared and stored at −20 • C. A 10 µL PAA solution was added to a 1 mL sample containing P. aeruginosa, followed by thorough mixing and incubation in a centrifuge tube under dark conditions for 15 min.…”

“…Traditional methods rely on the culturable ability of bacteria to grow and form colonies on agar plates, which underestimates the presence of VBNC cells to grow under standard laboratory conditions. To address this challenge, we recently developed alternative techniques for quantifying VBNC P. aeruginosa via the integration of propidium monoazide (PMA) with a quantitative polymerase chain reaction (qPCR) approach [28]. PMA selectively penetrates cells with damaged membranes and binds to their DNA, preventing its amplification during qPCR.…”