Journal Article

Bacterial dormancy: A subpopulation of viable but non-culturable cells demonstrates better fitness for revival

Love, John, Kovacs-Simon, Andrea, Butler, Clive S., Titball, Richard W., Wagley, Sariqa, Tennant, Richard K., Power, Ann, Murray, Neil, Morcrette, Helen, Yang, Zheng R.

2021

Overview

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V . parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella . Our results showed that two subpopulations (P1 and P2) of V . parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:Δ lldD ) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:Δ lldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Bacteria / Bacterial Physiological Phenomena / Bacterial proteins / Bacterial Proteins - metabolism / Biology and Life Sciences / Catalase / Cell survival / Cells, Cultured / Cold shock / Culture Media / Dehydrogenases / Dormancy / E coli / Flow cytometry / Gastroenteritis / Gene expression / Gene Expression Regulation, Bacterial / Genetic aspects / Genetic transcription / Health aspects / Hydrogen peroxide / Investigations / Low temperature / Medicine and Health Sciences / Microbial Viability / Oxidative stress / Physical Sciences / Population / Proteins / Proteome - analysis / Proteome - metabolism / Proteomics / Research and Analysis Methods / Resuscitation / Seafood / Shellfish / Vibrio / Vibrio Infections - metabolism / Vibrio Infections - microbiology / Vibrio parahaemolyticus - growth & development / Vibrio parahaemolyticus - metabolism / Vibrio parahaemolyticus - pathogenicity / Virulence / Virulence (Microbiology)