Assessment of physiological responses of bacteria to chlorine and UV disinfection using a plate count method, flow cytometry and viability PCR

Abstract

Aims: This study aimed to investigate the physiological responses of two gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa) and two gram-positive bacteria (Enterococcus faecalis and Bacillus sphaericus) to ultraviolet (UV) and chlorine disinfection.

Methods and results: Bacterial inactivation by UV and chlorine disinfection were evaluated with a plate count method for culturability, FCM and PMA-qPCR for membrane integrity and DyeTox13-qPCR for enzymatic activity, respectively. Both UV and chorine disinfection caused complete loss of culturability while membrane integrity remained intact after UV disinfection. Both DyeTox13-qPCR and PMA-qPCR showed high ΔC_t values up to 8.9 after chlorine disinfection, indicating that both methods were able to distinguish non-treated from chlorine-treated cells. Although PMA-qPCR could not differentiate membrane integrity of cells on UV exposure, DyeTox13-qPCR showed significant differences in ΔC_t values of 5.05 and 10.4 for gram-negative (E. coli) and gram-positive (Enterococcus) bacteria, respectively. However, DyeTox13-qPCR for gram-negative bacteria displayed relatively small differences in ΔC_t values compared with gram-positive bacteria.

Conclusion: UV and chlorine disinfection led to changes in physiological state of gram-negative and gram-positive bacteria. Particularly, UV disinfection could induce active but non-culturable (ABNC) for gram-negative bacteria and dormant cell for gram-positive bacteria where intact cells no longer showed the enzymatic activity.

Significance and impact of the study: UV and chlorine are commonly used to disinfect water, food and fomites to inactivate pathogenic bacteria. However, a viable but non-culturable (VBNC) state of bacteria induced by disinfection may underestimate the health risks because of the potential resuscitation of VBNC cells. This study highlighted that bacteria could undergo different physiological (ABNC or dormant) states during UV and chlorine disinfection. In addition, viability PCR techniques could provide insight into the changes in physiological states during disinfection processes.

Keywords: DyeTox13-qPCR; active but nonculturable (ABNC); disinfection; dormant cell; viability PCR.