The Viable but Non-Culturable (VBNC) State in Vibrio Species: Why Studying the VBNC State Now Is More Exciting than Ever.

During periods that are not conducive for growth or when facing stressful conditions, Vibrios enter a dormant state called the Viable But Non-Culturable (VBNC) state. In this chapter, I will analyse the role of the VBNC state in Vibrio species survival and pathogenesis and the molecular mechanisms regulating this complex phenomenon. I will emphasise some of the novel findings that make studying the VBNC state now more exciting than ever and its significance in the epidemiology of these pathogens and critical role in food safety.

The increased prevalence of Vibrio species and the first reporting of Vibrio jasicida and Vibrio rotiferianus at UK shellfish sites.

Warming sea-surface temperature has led to an increase in the prevalence of Vibrio species in marine environments. This can be observed particularly in temperate regions where conditions for their growth has become more favourable. The increased prevalence of pathogenic Vibrio species has resulted in a worldwide surge of Vibriosis infections in human and aquatic animals. This study uses sea-surface temperature data around the English and Welsh coastlines to identify locations where conditions for the presence and growth of Vibrio species is favourable. Shellfish samples collected from three locations that were experiencing an increase in sea-surface temperature were found to be positive for the presence of Vibrio species. We identified important aquaculture pathogens Vibrio rotiferianus and Vibrio jasicida from these sites that have not been reported in UK waters. We also isolated human pathogenic Vibrio species including V. parahaemolyticus from these sites. This paper reports the first isolation of V. rotiferianus and V. jasicida from UK shellfish and highlights a growing diversity of Vibrio species inhabiting British waters.

The Application of Imaging Flow Cytometry for Characterisation and Quantification of Bacterial Phenotypes.

Bacteria modify their morphology in response to various factors including growth stage, nutrient availability, predation, motility and long-term survival strategies. Morphological changes may also be associated with specific physiological phenotypes such as the formation of dormant or persister cells in a "viable but non-culturable" (VBNC) state which frequently display different shapes and size compared to their active counterparts. Such dormancy phenotypes can display various degrees of tolerance to antibiotics and therefore a detailed understanding of these phenotypes is crucial for combatting chronic infections and associated diseases. Cell shape and size are therefore more than simple phenotypic characteristics; they are important physiological properties for understanding bacterial life-strategies and pathologies. However, quantitative studies on the changes to cell morphologies during bacterial growth, persister cell formation and the VBNC state are few and severely constrained by current limitations in the most used investigative techniques of flow cytometry (FC) and light or electron microscopy. In this study, we applied high-throughput Imaging Flow Cytometry (IFC) to characterise and quantify, at single-cell level and over time, the phenotypic heterogeneity and morphological changes in cultured populations of four bacterial species, Bacillus subtilis, Lactiplantibacillus plantarum, Pediococcus acidilactici and Escherichia coli. Morphologies in relation to growth stage and stress responses, cell integrity and metabolic activity were analysed. Additionally, we were able to identify and morphologically classify dormant cell phenotypes such as VBNC cells and monitor the resuscitation of persister cells in Escherichia coli following antibiotic treatment. We therefore demonstrate that IFC, with its high-throughput data collection and image capture capabilities, provides a platform by which a detailed understanding of changes in bacterial phenotypes and their physiological implications may be accurately monitored and quantified, leading to a better understanding of the role of phenotypic heterogeneity in the dynamic microbiome.

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

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:ΔlldD) 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:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.

Campylobacter jejuni 11168H Exposed to Penicillin Forms Persister Cells and Cells With Altered Redox Protein Activity.

The formation of persister cells is one mechanism by which bacteria can survive exposure to environmental stresses. We show that Campylobacter jejuni 11168H forms persister cells at a frequency of 10-3 after exposure to 100 × MIC of penicillin G for 24 h. Staining the cell population with a redox sensitive fluorescent dye revealed that penicillin G treatment resulted in the appearance of a population of cells with increased fluorescence. We present evidence, to show this could be a consequence of increased redox protein activity in, or associated with, the electron transport chain. These data suggest that a population of penicillin G treated C. jejuni cells could undergo a remodeling of the electron transport chain in order to moderate membrane hyperpolarization and intracellular alkalization; thus reducing the antibiotic efficacy and potentially assisting in persister cell formation.

Genome Sequences of Four Vibrio parahaemolyticus Strains Isolated from the English Channel and the River Thames.

Vibrio parahaemolyticus is the lead causative agent for seafood-borne human gastroenteritis. While its occurrence has traditionally been uncommon in Europe and the United Kingdom, rising sea surface temperatures have resulted in an increased prevalence. Here, we present the complete genome sequences of four novel V. parahaemolyticus strains isolated in the United Kingdom.

Genome Resequencing of Laboratory Stocks of Burkholderia pseudomallei K96243.

We have resequenced the genomes of four Burkholderia pseudomallei K96243 laboratory cultures and compared them to the reported genome sequence that was published in 2004. Compared with the reference genome, these laboratory cultures harbored up to 42 single-nucleotide variants and up to 11 indels, including a 31.7-kb deletion in one culture.

Evidence of Clostridium perfringens epsilon toxin associated with multiple sclerosis.

BACKGROUND: It was recently reported that, using Western blotting, some multiple sclerosis (MS) patients in the United States had antibodies against epsilon toxin (Etx) from Clostridium perfringens, suggesting that the toxin may play a role in the disease. OBJECTIVE: We investigated for serum antibodies against Etx in UK patients with clinically definite multiple sclerosis (CDMS) or presenting with clinically isolated syndrome (CIS) or optic neuritis (ON) and in age- and gender-matched controls. METHODS: We tested sera from CDMS, CIS or ON patients or controls by Western blotting. We also tested CDMS sera for reactivity with linear overlapping peptides spanning the amino acid sequence (Pepscan) of Etx. RESULTS: Using Western blotting, 24% of sera in the combined CDMS, CIS and ON groups ( n = 125) reacted with Etx. In the control group ( n = 125), 10% of the samples reacted. Using Pepscan, 33% of sera tested reacted with at least one peptide, whereas in the control group only 16% of sera reacted. Out of 61 samples, 21 (43%) were positive to one or other testing methodology. Three samples were positive by Western blotting and Pepscan. CONCLUSION: Our results broadly support the previous findings and the role of Etx in the aetiology of MS warrants further investigation.

Galleria mellonella as an infection model to investigate virulence of Vibrio parahaemolyticus.

Non-toxigenic V. parahaemolyticus isolates (tdh-/trh-/T3SS2-) have recently been isolated from patients with gastroenteritis. In this study we report that the larvae of the wax moth (Galleria mellonella) are susceptible to infection by toxigenic or non-toxigenic clinical isolates of V. parahaemolyticus. In comparison larvae inoculated with environmental isolates of V. parahaemolyticus did not succumb to disease. Whole genome sequencing of clinical non-toxigenic isolates revealed the presence of a gene encoding a nudix hydrolase, identified as mutT. A V. parahaemolyticus mutT mutant was unable to kill G. mellonella at 24 h post inoculation, indicating a role of this gene in virulence. Our findings show that G. mellonella is a valuable model for investigating screening of possible virulence genes of V. parahaemolyticus and can provide new insights into mechanisms of virulence of atypical non-toxigenic V. parahaemolyticus. These findings will allow improved genetic tests for the identification of pathogenic V. parahaemolyticus to be developed and will have a significant impact for the scientific community.

A proteasome inhibitor produced by Burkholderia pseudomallei modulates intracellular growth.

The NRPS/PKS cluster encodes the enzymes necessary for glidobactin synthesis it is partially conserved in various members of the Burkholderia genus including B. pseudomallei. In this study we have shown that the insertional inactivation or deletion of glbC in this cluster in B. pseudomallei could reduce the ability of the bacterium to survive or grow in murine macrophages or in human neutrophils. Exogenously added proteasome inhibitors were able to chemically complement the mutation. The insertional inactivation or deletion of glbC increased virulence in an acute model of infection in Balb/c or C57BL/6 mice but virulence in a chronic model of infection was similar to that of the wild type. Our findings contrast with the previous finding that inactivation of the glb gene cluster in B. pseudomallei strain 1026b resulted in marked attenuation, and provides evidence of differential roles for some genes in virulence of different strains of B. pseudomallei.

Mapping epigenetic changes to the host cell genome induced by Burkholderia pseudomallei reveals pathogen-specific and pathogen-generic signatures of infection.

The potential for epigenetic changes in host cells following microbial infection has been widely suggested, but few examples have been reported. We assessed genome-wide patterns of DNA methylation in human macrophage-like U937 cells following infection with Burkholderia pseudomallei, an intracellular bacterial pathogen and the causative agent of human melioidosis. Our analyses revealed significant changes in host cell DNA methylation, at multiple CpG sites in the host cell genome, following infection. Infection induced differentially methylated probes (iDMPs) showing the greatest changes in DNA methylation were found to be in the vicinity of genes involved in inflammatory responses, intracellular signalling, apoptosis and pathogen-induced signalling. A comparison of our data with reported methylome changes in cells infected with M. tuberculosis revealed commonality of differentially methylated genes, including genes involved in T cell responses (BCL11B, FOXO1, KIF13B, PAWR, SOX4, SYK), actin cytoskeleton organisation (ACTR3, CDC42BPA, DTNBP1, FERMT2, PRKCZ, RAC1), and cytokine production (FOXP1, IRF8, MR1). Overall our findings show that pathogenic-specific and pathogen-common changes in the methylome occur following infection.

Galleria mellonella as a model host for microbiological and toxin research.

Mammals are widely used by microbiologists as a model host species to study infectious diseases of humans and domesticated livestock. These studies have been pivotal for our understanding of mechanisms of virulence and have allowed the development of diagnostics, pre-treatments and therapies for disease. However, over the past decade we have seen efforts to identify organisms which can be used as alternatives to mammals for these studies. The drivers for this are complex and multifactorial and include cost, ethical and scientific considerations. Galleria mellonella have been used as an alternative infection model since the 1980s and its utility for the study of bacterial disease and antimicrobial discovery was recently comprehensively reviewed. The wider applications of G. mellonella as a model host, including its susceptibility to 29 species of fungi, 7 viruses, 1 species of parasite and 16 biological toxins, are described in this perspective. In addition, the latest developments in the standardisation of G. mellonella larvae for research purposes has been reviewed.

Differences in carbon source utilisation distinguish Campylobacter jejuni from Campylobacter coli.

BACKGROUND: Campylobacter jejuni and C. coli are human intestinal pathogens that are the most frequent causes of bacterial foodborne gastroenteritis in humans in the UK. In this study, we aimed to characterise the metabolic diversity of both C. jejuni and C. coli using a diverse panel of clinical strains isolated from the UK, Pakistan and Thailand, thereby representing both the developed and developing world. Our aim was to apply multi genome analysis and Biolog phenotyping to determine differences in carbon source utilisation by C. jejuni and C. coli strains. RESULTS: We have identified a core set of carbon sources (utilised by all strains tested) and a set that are differentially utilised for a diverse panel of thirteen C. jejuni and two C. coli strains. This study used multi genome analysis to show that propionic acid is utilised only by C. coli strains tested. A broader PCR screen of 16 C. coli strains and 42 C. jejuni confirmed the absence of the genes needed for propanoate metabolism. CONCLUSIONS: From our analysis we have identified a phenotypic method and two genotypic methods based on propionic utilisation that might be applicable for distinguishing between C. jejuni and C. coli.

The twin arginine translocation system is essential for aerobic growth and full virulence of Burkholderia thailandensis.

The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some ß-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated.

Isolation of pandemic Vibrio parahaemolyticus from UK water and shellfish produce.

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium found commonly in temperate and warm estuarine waters worldwide. V. parahaemolyticus is considered an emerging bacterial pathogen in Europe and has been responsible for several recent seafood-associated outbreaks. During ad hoc testing of raw shellfish produce in May 2012, pandemic group (O3:K6) V. parahaemolyticus was isolated from Pacific oysters (Crassostrea gigas), harvested in Southern England. Follow-on testing of water and shellfish, encompassing a small number geographically diverse sites, also retrieved pandemic group isolates. These strains are amongst the most northerly pandemic strains described to date and represent the first instance of pandemic V. parahaemolyticus isolated in the UK, highlighting the expanding geographical distribution of these foodborne pathogens in the environment.

Detection and identification of tdh- and trh-positive Vibrio parahaemolyticus strains from four species of cultured bivalve molluscs on the Spanish Mediterranean Coast.

Presented here is the first report describing the detection of potentially diarrheal Vibrio parahaemolyticus strains isolated from cultured bivalves on the Mediterranean coast, providing data on the presence of both tdh- and trh-positive isolates. Potentially diarrheal V. parahaemolyticus strains were isolated from four species of bivalves collected from both bays of the Ebro delta, Spain.

Yersinia ruckeri biotype 2 isolates from mainland Europe and the UK likely represent different clonal groups.

There have been increased reports of outbreaks of enteric redmouth disease (ERM) caused by Yersinia ruckeri in previously vaccinated salmonids in Europe, with some of these outbreaks being attributed to emergent non-motile, Tween 80-negative, biotype 2 isolates. To gain information about their likely origins and relationships, a geographically and temporally diverse collection of isolates were characterised by serotyping, biotyping, pulsed-field gel electrophoresis (PFGE) and outer membrane protein (OMP) profiling. A total of 44 pulsotypes were identified from 160 isolates by PFGE, using the restriction enzyme NotI. Serotype O1 isolates responsible for ERM in rainbow trout in both the US and Europe, and including biotype 2 isolates, represented a distinct subgroup of similar pulsotypes. Biotype 2 isolates, responsible for outbreaks of the disease in rainbow trout in the UK, Denmark and Spain, had different pulsotypes, suggesting that they represented different clones that may have emerged separately. Danish biotype 2 isolates recovered since 1995 were indistinguishable by PFGE from the dominant biotype 1 clone responsible for the majority of outbreaks in Denmark and the rest of mainland Europe. In contrast, US biotype 2 isolate YRNC10 had an identical pulsotype and OMP profile to UK biotype 2 isolates, suggesting that there had been exchange of these isolates between the UK and the US in the past. UK Atlantic salmon isolates were genetically and serologically diverse, with 12 distinct pulsotypes identified among 32 isolates.

Prevalence and potential pathogenicity of Vibrio parahaemolyticus in Chinese mitten crabs (Eriocheir sinensis) harvested from the River Thames estuary, England.

Chinese mitten crabs (Eriocheir sinensis) have been described as an alien invasive species in the River Thames, United Kingdom, and elsewhere in Europe. The crabs can cause considerable physical damage to the riverbeds and threaten native ecosystems. Trapping has been considered an option, but such attempts to control mitten crab populations in Germany in the 1930s failed. In the United Kingdom, it has been suggested that commercial exploitation of the species could be employed as a control option. This study was conducted as part of a larger program to assess the suitability of a commercial Chinese mitten crab fishery in the River Thames. Crabs and water samples from the River Thames between 2003 and 2006 were examined for the human pathogenic bacterium Vibrio parahaemolyticus. All samples throughout this testing period were positive for V. parahaemolyticus. The putative pathogenicity markers, thermostable direct hemolysin and thermostable direct-related hemolysin, were detected in one sample, indicating that the crabs possessed the potential to cause V. parahaemolyticus-associated illness if consumed without further processing. Levels of V. parahaemolyticus were higher during the summer than in the winter. This is the first study of V. parahaemolyticus prevalence in European-adapted Chinese mitten crabs.

Comparison of V. parahaemolyticus isolated from seafoods and cases of gastrointestinal disease in the UK.

In this study the prevalence of Vibrio parahaemolyticus in shellfish and estuarine waters from the UK was examined using cultural and nucleic acid hybridisation approaches. Forty-nine isolates derived from environmental sources were characterised using serotyping, PCR, nucleic acid hybridisation and pulsed field gel electrophoresis (PFGE). The serotypic and molecular profiles of these isolates were compared to 20 clinical isolates, including representatives of the pandemic O3:K6 clone. Thirty percent of environmental samples were positive for V. parahaemolyticus. The tdh gene was identified in 12% of samples tested. Environmentally derived tdh+ strains were highly heterogeneous with neither association between isolates from similar origins nor seafood type. Previously uncharacterised clinical strains from UK patients with travel related V. parahaemolyticus associated gastroenteritis, were unrelated to tdh+ or tdh- environmental isolates but 2 were clonally indistinguishable from the pandemic O3:K6 strain responsible for outbreaks in Spain, Korea, Japan and Laos.

Evaluation of an alkaline phosphatase-labeled oligonucleotide probe for the detection and enumeration of the thermostable-related hemolysin (trh) gene of Vibrio parahaemolyticus.

Reliable methods are needed to detect total and pathogenic Vibrio parahaemolyticus. One marker of V. parahaemolyticus virulence is the thermostable-related hemolysin. We developed an alkaline phosphatase-labeled DNA probe method for the specific detection and enumeration of trh-positive V. parahaemolyticus by colony hybridization. The probe was tested against a panel of 200 bacterial strains and determined to be specific for trh-positive V. parahaemolyticus. Additionally, the trh alkaline phosphatase probe colony hybridization was successfully used to detect and enumerate trh-positive V. parahaemolyticus in seafood and water samples collected from the United States and the United Kingdom.