From Dormancy to Viability: The Resuscitation Processes of Viable but Non-Culturable Bacteria—A Systematic Review

TL;DR

This systematic review explores methods to resuscitate viable but non-culturable bacteria, focusing on factors and protocols that help revive these metabolically active yet non-growing cells.

Contribution

The study systematically reviews in vitro resuscitation methods for VBNC Enterobacteriaceae and Pseudomonas aeruginosa, identifying key influencing factors and methodological gaps.

Findings

Resuscitation of VBNC bacteria often uses standard media with additives and moderate temperatures, with recovery typically occurring within 24–48 hours.

Pseudomonas aeruginosa requires less supplementation for resuscitation compared to Enterobacteriaceae.

Key resuscitation mechanisms include metabolic reactivation, oxidative stress modulation, and ribosome reactivation.

Abstract

Viable but non-culturable (VBNC) cells represent a reversible, metabolically active state that promotes the survival of bacteria under stressful conditions and their persistence in healthcare facilities and food industry. We conducted a systematic review following PRISMA 2020 guidelines to identify in vitro methodologies for inducing and resuscitating VBNC Enterobacteriaceae and Pseudomonas aeruginosa, and to determine key influencing factors. Eligible studies reported in vitro resuscitation of these species. Searches were performed in MEDLINE (PubMed), Scopus, and Google Scholar up to July 2025. Two independent reviewers screened and extracted data. Exclusion criteria included absence of original experimental data, focus on other species, or lack of clear VBNC definition. Risk of bias was qualitatively assessed. Analyses were descriptive without meta-analysis. Of the 1041 records, 24 articles (27 studies) were included. Resuscitation protocols typically employed standard culture media with additives and moderate incubation temperatures, with most successful recoveries occurring after 24–48 h. P. aeruginosa generally required less supplementation than Enterobacteriaceae. Reported mechanisms involved metabolic reactivation, oxidative stress modulation, nutrient sensing, and ribosome reactivation. The limitations of our study include protocol heterogeneity, lack of standardization, and selective reporting. While simple resuscitation methods were often effective, tailoring conditions to species-specific ecological preferences appears critical. Standardized approaches of VBNC cells will improve detection, risk assessment, and infection control.

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