title: Molecular Viability Testing of UV-Inactivated Bacteria.
abstract: PCR is effective in detecting bacterial DNA in samples, but it is unable to differentiate viable bacteria from inactivated cells or free DNA fragments. New PCR-based analytical strategies have been developed to address this limitation. Molecular viability testing (MVT) correlates bacterial viability with the ability to rapidly synthesize species-specific rRNA precursors (pre-rRNA) in response to brief nutritional stimulation. Previous studies demonstrated that MVT can assess bacterial inactivation by chlorine, serum, and low-temperature pasteurization. Here, we demonstrate that MVT can detect inactivation ofEscherichia coli,Aeromonas hydrophila, andEnterococcus faecaliscells by UV irradiation. Some UV-inactivatedE. colicells transiently retained the ability to synthesize pre-rRNA postirradiation (generating false-positive MVT results), but this activity ceased within 1 h following UV exposure. Viable but transiently undetectable (by culture)E. colicells were consistently detected by MVT. An alternative viability testing method, viability PCR (vPCR), correlates viability with cell envelope integrity. This method did not distinguish viable bacteria from UV-inactivated bacteria under some conditions, indicating that the inactivated cells retained intact cell envelopes. MVT holds promise as a means to rapidly assess microbial inactivation by UV treatment.IMPORTANCEUV irradiation is increasingly being used to disinfect water, food, and other materials for human use. Confirming the effectiveness of UV disinfection remains a challenging task. In particular, microbiological methods that rely on rapid detection of microbial DNA can yield misleading results, due to the detection of remnant DNA associated with dead microbial cells. This report describes a novel method that rapidly distinguishes living microbial cells from dead microbial cells after UV disinfection.
authors.affiliation: Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Kris M
authors.initials: KM
authors.lastname: Weigel
authors.affiliation: Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Felicia K
authors.initials: FK
authors.lastname: Nguyen
authors.affiliation: Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: Moira R
authors.initials: MR
authors.lastname: Kearney
authors.affiliation: Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA.
authors.forename: John S
authors.initials: JS
authors.lastname: Meschke
authors.affiliation: Department of Environmental and Occupational Health Sciences, University of Washington, Seattle, Washington, USA gcang@uw.edu.
authors.forename: Gerard A
authors.initials: GA
authors.lastname: Cangelosi
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000528:radiation effects*; D050296:Microbial Viability / Q000528:radiation effects*; D016133:Polymerase Chain Reaction / Q000379:methods*; D014466:Ultraviolet Rays
keywords: UV irradiation; bacterial inactivation; disinfection; drinking water; molecular viability testing (MVT); propidium monoazide (PMA); ratiometric pre-rRNA analysis; viability PCR (vPCR); viable but nonculturable (VBNC)