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      Current Perspectives on Viable but Non-Culturable (VBNC) Pathogenic Bacteria

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          Abstract

          Under stress conditions, many species of bacteria enter into starvation mode of metabolism or a physiologically viable but non-culturable (VBNC) state. Several human pathogenic bacteria have been reported to enter into the VBNC state under these conditions. The pathogenic VBNC bacteria cannot be grown using conventional culture media, although they continue to retain their viability and express their virulence. Though there have been debates on the VBNC concept in the past, several molecular studies have shown that not only can the VBNC state be induced under in vitro conditions but also that resuscitation from this state is possible under appropriate conditions. The most notable advance in resuscitating VBNC bacteria is the discovery of resuscitation-promoting factor (Rpf), which is a bacterial cytokines found in both Gram-positive and Gram-negative organisms. VBNC state is a survival strategy adopted by the bacteria, which has important implication in several fields, including environmental monitoring, food technology, and infectious disease management; and hence it is important to investigate the association of bacterial pathogens under VBNC state and the water/foodborne outbreaks. In this review, we describe various aspects of VBNC bacteria, which include their proteomic and genetic profiles under the VBNC state, conditions of resuscitation, methods of detection, antibiotic resistance, and observations on Rpf.

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          Recent findings on the viable but nonculturable state in pathogenic bacteria.

          Many bacteria, including a variety of important human pathogens, are known to respond to various environmental stresses by entry into a novel physiological state, where the cells remain viable, but are no longer culturable on standard laboratory media. On resuscitation from this 'viable but nonculturable' (VBNC) state, the cells regain culturability and the renewed ability to cause infection. It is likely that the VBNC state is a survival strategy, although several interesting alternative explanations have been suggested. This review describes the VBNC state, the various chemical and physical factors known to induce cells into this state, the cellular traits and gene expression exhibited by VBNC cells, their antibiotic resistance, retention of virulence and ability to attach and persist in the environment, and factors that have been found to allow resuscitation of VBNC cells. Along with simple reversal of the inducing stresses, a variety of interesting chemical and biological factors have been shown to allow resuscitation, including extracellular resuscitation-promoting proteins, a novel quorum-sensing system (AI-3) and interactions with amoeba. Finally, the central role of catalase in the VBNC response of some bacteria, including its genetic regulation, is described.
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            The viable but nonculturable state in bacteria.

            It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the "viable but nonculturable" (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.
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              A tentative direct microscopic method for counting living marine bacteria.

              Yeast extract (0.025%) and nalidixic acid (0.002%) were added to seawater samples and the samples were incubated for 6 h at 20 degrees C in the dark. Under these conditions, bacterial cells did not divide but grew to form elongated cells that are easily recognized by a direct microscopic method and epifluorescent microscopic technique. The number of cells thus obtained is proposed as a direct cound of viable bacterial cells (DVC). With open ocean samples, DVC was higher than 'viable' plate counts by up to three orders of magnitude and lower than the direct counts by about one order.
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                Author and article information

                Contributors
                URI : http://frontiersin.org/people/u/106445
                URI : http://frontiersin.org/people/u/140025
                URI : http://frontiersin.org/people/u/143493
                Journal
                Front Public Health
                Front Public Health
                Front. Public Health
                Frontiers in Public Health
                Frontiers Media S.A.
                2296-2565
                31 July 2014
                2014
                : 2
                : 103
                Affiliations
                [1] 1National Institute of Cholera and Enteric Diseases (NICED) , Kolkata, India
                [2] 2Collaborative Research Center of Okayama University for Infectious Diseases in India, NICED , Kolkata, India
                Author notes

                Edited by: Carla Pruzzo, University of Genova, Italy

                Reviewed by: Gian Marco Luna, National Research Council – Institute of Marine Sciences (ISMAR-CNR), Italy; Tanvira Afroze Sultana, Bangladesh Institute for Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders, Bangladesh

                *Correspondence: Thandavarayan Ramamurthy, National Institute of Cholera and Enteric Diseases, P-33 CIT Road, Scheme XM, Beliaghata, Kolkata 700010, India e-mail: tramu@ 123456vsnl.net

                This article was submitted to Environmental Health, a section of the journal Frontiers in Public Health.

                Article
                10.3389/fpubh.2014.00103
                4116801
                25133139
                e97348a6-c543-4f44-a220-5378ed1a074c
                Copyright © 2014 Ramamurthy, Ghosh, Pazhani and Shinoda.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 19 March 2014
                : 15 July 2014
                Page count
                Figures: 0, Tables: 1, Equations: 0, References: 105, Pages: 9, Words: 9181
                Categories
                Public Health
                Review Article

                bacteria,viability,culture,vbnc,resuscitation
                bacteria, viability, culture, vbnc, resuscitation

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