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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.

Vibrio fluvialis is a pathogen commonly found in coastal environs. Considering recent increase in numbers of diarrheal outbreaks and sporadic extraintestinal cases, V. fluvialis has been considered as an emerging pathogen. Though this pathogen can be easily isolated by existing culture methods, its identification is still a challenging problem due to close phenotypic resemblance either with Vibrio cholerae or Aeromonas spp. However, using molecular tools, it is easy to identify V. fluvialis from clinical and different environmental samples. Many putative virulence factors have been reported, but its mechanisms of pathogenesis and survival fitness in the environment are yet to be explored. This chapter covers some of the major discoveries that have been made to understand the importance of V. fluvialis.

In response to low iron availability, Vibrio parahaemolyticus synthesizes and secretes a polyhydroxycarboxylate-type siderophore vibrioferrin which is composed of 1 mol each of 2-ketoglutaric acid, L-alanine, ethanolamine, and citric acid. We have previously reported the cloning and characterization of the pvuA gene, which encodes the 78-kDa outer membrane receptor protein for ferric vibrioferrin. In this study, nine genes involved in the biosynthesis and transport of vibrioferrin have been identified in the genomic regions surrounding the pvuA gene. The genes were sequenced, and gene disruptants were constructed by insertion mutation for phenotype analysis. Five of the genes, named pvsABCDE, constitute an operon that is expressed under iron-limiting conditions. Homology searches of their predicted protein products suggested that the four genes pvsABDE are implicated in the biosynthesis of the siderophore. Another gene in the same operon, pvsC, encodes a putative exporter that is homologous to members of the major facilitator superfamily of multidrug efflux pumps. The remaining four genes, named pvuBCDE, encode proteins strongly homologous to Escherichia coli FecBCDE, respectively, which are components of the ATP-binding cassette transporter system for ferric dicitrate. Reverse transcriptase PCR analysis revealed that these transport genes are transcribed as a single mRNA with the upstream genes, psuA and pvuA. Phenotypic comparison between the wild-type strain and its targeted gene disruptants supported the biological functions for the respective operons that were expected on the basis of the homology search.

A protease was purified from a strain of Vibrio vulnificus isolated from the blood of a septicemic human. The vibrio was cultured in bacto peptone-yeast extract medium, and the protease was purified by a purification procedure including ultrafiltration of the culture supernatant with an Amicon YM 5 membrane, diethylaminoethyl-Sephacel column chromatography, Sephacryl S-200 column chromatography and fast protein liquid chromatography on Mono Q column. The protease preparation revealed homogeneity on polyacrylamide gel electrophoresis and about 30,000-fold purification was achieved, with a yield of about 30% . The isoelectric point of the purified V. vulnificus protease was about 5.80 and its molecular weight was ca. 45,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the protease activity was 8.0. The V. vulnificus protease was inhibited by a metalloprotease inhibitor and zinc ion and/or ferrous ion were essential for its enzyme activity. No cysteine residue was detected in the V. vulnificus protease. The protease had caseinolytic, elastolytic and collagenolytic activities.The illnesses caused by Vibrio vulnificus, a marine bacterium, are divided into two groups, wound-infection group and primary septicemia group (2, 3). In the former, edema and erythema are formed around a new wound in contact with sea water. In the latter group, cutaneous lesions, such as bullae, erythema or necrotic ulcer, often appear on one or more extremities.V. vulnificus is known to produce many toxic or pathogenic factors, such as cytolysin (8, 13, 23), protease (11, 13, 25), phospholipase A2 (27), and siderophores (24). However, in the pathogenesis of V. vulnificus infection, the actual role of any product has not been clarified yet. Kreger and Lockwood (13) reported that V. vulnificus cytolysin activity in the culture filtrate decreased in the stationary phase of the growth and the decrease correlated with the appearance of protease in the culture filtrate. We also observed significant excretion of protease from many strains of V. vulnificus isolated from patients. The pathogenic significance of proteases by some bacteria, such as pseudomonal protease (7,10,12) and serratial protease (9,16,19), has been reported. These proteases have vascular permeability-enhancing and hemorrhagic activities which are closely related to the above mentioned cutaneous lesions. We also found that the crude V. vulnificus protease preparation

In recent years, members belonging to the genus Vibrio of the family Vibrionaceae have acquired increasing importance because of the association of several of its members with human disease. The most feared of the Vibrio species is Vibrio cholerae, the causative agent of cholera, a devastating disease of global significance. Other important vibrios of medical importance are V. parahemolyticus, V. vulnificus, V. mimicus, and to a lesser extent V. fluvialis, V. furnissii, V. hollisae, and V. damsela. Recent studies have also implicated V. alginolyticus and V. metschnikovii in human disease, although their complete significance has not yet been established. The virulence of all medically important vibrios is aided by a variety of traits that help breach human defenses. In this review, we provide an overview of the environmental distribution of the pathogenic vibrios and the important virulence traits that enable them to cause disease.

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A new siderophore named vulnibactin has been isolated from low iron cultures of Vibrio vulnificus, a human pathogen. The structure was established as N-[3-(2,3-dihydroxybenzamido)propyl]-l,3-bis[2-(2-hydroxyphenyl)-trans.5-methyl.2-oxazoline-4.carboxamido]propane by a combination of acid hydrolysis, nuclear magnetic resonance spectroscopy and positive fast atom bombardment mass spectrometry. Vulnibactin is characterized as containing one residue of 2,3-dihydroxybenzoic acid as well as two residues of salicylic acid, both of which are involved in the formation of oxazoline rings with L-threonine bound to a norspermidine backbone. In addition, two other compounds with siderophore activity were purified and their structures were also determined. These two compounds provided further support for the structure of vulnibactin.