Tracking Vibrio: population dynamics and ecology of Vibrio parahaemolyticus and V. vulnificus in an Alabama estuary.

Vibrio is a genus of halophilic, gram-negative bacteria found in estuaries around the globe. Integral parts of coastal cultures often involve contact with vectors of pathogenic Vibrio spp. (e.g., consuming raw shellfish). High rates of mortality from certain Vibrio spp. infections demonstrate the need for an improved understanding of Vibrio spp. dynamics in estuarine regions. Our study assessed meteorological, hydrographic, and biological correlates of Vibrio parahaemolyticus and V. vulnificus at 10 sites in the Eastern Mississippi Sound System (EMSS) from April to October 2019. During the sampling period, median abundances of V. parahaemolyticus and V. vulnificus were 2.31 log MPN/L and 2.90 log MPN/L, respectively. Vibrio spp. dynamics were largely driven by site-based variation, with sites closest to freshwater inputs having the highest abundances. The E-W wind scalar, which affects Ekman transport, was a novel Vibrio spp. correlate observed. A potential salinity effect on bacterial-particle associations was identified, where V. vulnificus was associated with larger particles in conditions outside of their optimal salinity. Additionally, V. vulnificus abundances were correlated to those of harmful algal species that did not dominate community chlorophyll. Correlates from this study may be used to inform the next iteration of regionally predictive Vibrio models and may lend additional insight to Vibrio spp. ecology in similar systems. IMPORTANCE: Vibrio spp. are bacteria found in estuaries worldwide; some species can cause illness and infections in humans. Relationships between Vibrio spp. abundance, salinity, and temperature are well documented, but correlations to other environmental parameters are less understood. This study identifies unique correlates (e.g., E-W wind scalar and harmful algal species) that could potentially inform the next iteration of predictive Vibrio models for the EMSS region. Additionally, these correlates may allow existing environmental monitoring efforts to be leveraged in providing data inputs for future Vibrio risk models. An observed correlation between salinity and V. vulnificus/particle-size associations suggests that predicted environmental changes may affect the abundance of Vibrio spp. in certain reservoirs, which may alter which vectors present the greatest vibrio risk.

Sewage Promotes Vibrio vulnificus Growth and Alters Gene Transcription in Vibrio vulnificus CMCP6.

Vibrio vulnificus is a naturally occurring, potentially lethal pathogen found in coastal waters, fish, and shellfish. Sewage spills in coastal waters occur when infrastructure fails due to severe storms or age, and may affect bacterial populations by altering nutrient levels. This study investigated effects of sewage on clonal and natural V. vulnificus populations in microcosms. Addition of 1% sewage to estuarine water caused the density of a pure culture of V. vulnificus CMCP6 and a natural V. vulnificus population to increase significantly, by two to three orders of magnitude, whether measured by quantitative PCR (qPCR) or culture and in batch and continuous cultures. Changes in the transcription of six virulence- and survival-associated genes in response to sewage were assessed using continuous culture. Exposure to sewage affected transcription of genes that may be associated with virulence, i.e., it modulated the oxidative stress response by altering superoxide dismutase transcription, significantly increasing sodB transcription while repressing sodA. Sewage also repressed transcription of nptA, which encodes a sodium-phosphate cotransporter. Sewage had no effect on sodC transcription or the putative virulence-associated genes hupA or wza. The effects of environmentally relevant levels of sewage on V. vulnificus populations and gene transcription suggest that sewage spills that impact warm coastal waters could lead to an increased risk of V. vulnificus infections. IMPORTANCE Vibrio vulnificus infections have profound impacts such as limb amputation and death for individuals with predisposing conditions. The warming climate is contributing to rising V. vulnificus prevalence in waters that were previously too cold to support high levels of the pathogen. Climate change is also expected to increase precipitation in many regions, which puts more pressure on wastewater infrastructure and will result in more frequent sewage spills. The finding that 1% wastewater in estuarine water leads to 100 to over 1,000-fold greater V. vulnificus concentrations suggests that human exposure to oysters and estuarine water could have greater health impacts in the future. Further, wastewater had a significant effect on gene transcription and has the potential to affect virulence during the initial environment-to-host transition.

Regulator of ribonuclease activity modulates the pathogenicity of Vibrio vulnificus.

RraA, a protein regulator of RNase E activity, plays a unique role in modulating the mRNA abundance in Escherichia coli. The marine pathogenic bacterium Vibrio vulnificus also possesses homologs of RNase E (VvRNase E) and RraA (VvRraA1 and VvRraA2). However, their physiological roles have not yet been investigated. In this study, we demonstrated that VvRraA1 expression levels affect the pathogenicity of V. vulnificus. Compared to the wild-type strain, the VvrraA1-deleted strain (ΔVvrraA1) showed decreased motility, invasiveness, biofilm formation ability as well as virulence in mice; these phenotypic changes of ΔVvrraA1 were restored by the exogenous expression of VvrraA1. Transcriptomic analysis indicated that VvRraA1 expression levels affect the abundance of a large number of mRNA species. Among them, the half-lives of mRNA species encoding virulence factors (e.g., smcR and htpG) that have been previously shown to affect VvrraA1 expression-dependent phenotypes were positively correlated with VvrraA1 expression levels. These findings suggest that VvRraA1 modulates the pathogenicity of V. vulnificus by regulating the abundance of a subset of mRNA species.

Analysis of Gene Expression Profiles, Cytokines, and Bacterial Loads Relevant to Alcoholic Liver Disease Mice Infected With V. vulnificus.

Patients with liver disease are susceptible to infection with Vibrio vulnificus (V. vulnificus), but the specific reasons remain elusive. Through RNA-seq, we found that when mice with alcoholic liver disease (ALD) were infected with V. vulnificus by gavage, compared with the Pair group, the small intestinal genes affecting intestinal permeability were upregulated; and the number of differentially expressed genes related to immune functions (e.g., such as cell chemotaxis, leukocyte differentiation, and neutrophil degranulation) decreased in the liver, spleen, and blood. Further analysis showed that the number of white blood cells decreased in the Pair group, whereas those in the ALD mice did not change significantly. Interestingly, the blood bacterial load in the ALD mice was about 100 times higher than that of the Pair group. After the ALD mice were infected with V. vulnificus, the concentrations of T cell proliferation-promoting cytokines (IL-2, IL-23) decreased. Therefore, unlike the Pair group, ALD mice had weaker immune responses, lower T cell proliferation-promoting cytokines, and higher bacterial loads post-infection, possibly increasing their susceptibility to V. vulnificus infection. These new findings we presented here may help to advance the current understanding of the reasons why patients with liver disease are susceptible to V. vulnificus infection and provides potential targets for further investigation in the context of treatment options for V. vulnificus sepsis in liver disease patient.

Characterization of bacteriophage VVP001 and its application for the inhibition of Vibrio vulnificus causing seafood-borne diseases.

Vibrio vulnificus is a major food-borne pathogen that causes septicemia and cellulitis with a mortality rate of >50%. However, there are no efficient natural food preservatives or biocontrol agents to control V. vulnificus in seafood. In this study, we isolated and characterized a novel bacteriophage VVP001. Host range and transmission electron microscopy morphology observations revealed that VVP001 belongs to the family Siphoviridae and specifically infects V. vulnificus. Phage stability tests showed that VVP001 is stable at a broad temperature range of -20 °C to 65 °C and a pH range from 3 to 11, which are conditions for food applications (processing, distribution, and storage). In vitro challenge assays revealed that VVP001 inhibited V. vulnificus MO6-24/O (a clinical isolate) growth up to a 3.87 log reduction. In addition, complete genome analysis revealed that the 76 kb VVP001 contains 102 open reading frames with 49.64% G + C content and no gene encoding toxins or other virulence factors, which is essential for food applications. Application of VVP001 to fresh abalone samples contaminated with V. vulnificus demonstrated its ability to inhibit V. vulnificus growth, and an in vivo mouse survival test showed that VVP001 protects mice against high mortality (survival rate >70% at a multiplicity of infection of 1000 for up to 7 days). Therefore, the bacteriophage VVP001 can be used as a good natural food preservative and biocontrol agent for food applications.

rpoB mutations conferring rifampicin-resistance affect growth, stress response and motility in Vibrio vulnificus.

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the ß subunit of RNA polymerase, rpoB. Different rpoB mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. rpoB mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen Vibrio vulnificus, even though rifampicin-resistant strains of V. vulnificus have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of V. vulnificus have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of rpoB mutations. In this work, effects on growth, stress response and virulence of V. vulnificus were investigated using a set of nine spontaneous rifampicin-resistant derivatives of V. vulnificus CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml-1], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of rpoB mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Effects of tumbling, refrigeration and subsequent resubmersion on the abundance of Vibrio vulnificus and Vibrio parahaemolyticus in cultured oysters (Crassostrea virginica).

Routine handling of oysters is a common industry practice for off-bottom oyster aquaculture, which aims to produce a high-quality oyster. These practices expose oysters to elevated temperatures and interrupt filter feeding, which can increase Vibrio vulnificus and V. parahaemolyticus levels within the oyster. The resubmersion of oysters after exposure to conditions where the time-temperature controls are exceeded is as an effective mitigation strategy to allow elevated levels of Vibrio spp. to "recover", or return to ambient levels, prior to harvest. Previous work examined the effect of desiccation on recovery times; the objective of this study was to evaluate the effect of additional handling treatments [tumbled and refrigerated (TR), tumbled and not refrigerated (TNR), not tumbled and refrigerated (NTR), and not tumbled and not refrigerated (NTNR)] on the time needed for V. vulnificus, total V. parahaemolyticus, and pathogenic V. parahaemolyticus (tdh+/trh+) to recover in oysters. A set of non-treated (control) oysters remained submerged throughout the study to determine the ambient Vibrio spp. (inclusive of genotypes) levels within oysters. Vibrio spp. levels were measured immediately before (pre) and after (post) the treatments, and 1, 2, 4, 7, 10, and 14 days after resubmersion using a three-tube MPN real-time PCR method. The non-refrigerated oysters (TNR, NTNR) had Vibrio spp. levels 1.54 to 2.10 log MPN/g higher than the pre-treatment levels, while the Vibrio spp. levels in refrigerated oysters were not significantly higher than pre-treatment levels. After resubmersion, Vibrio spp. levels increased by 0.84 to 1.78 log MPN/g in the refrigerated oysters (TR, NTR). Vibrio spp. levels in oysters returned to ambient after 1-7 days of resubmersion, depending on the handling treatment and the Vibrio spp. These results provide data on handling treatments not previously reported and further support the seven-day resubmersion requirement for farmers in Alabama using the adjustable longline system.

Interaction of Escherichia coli and its culture supernatant with Vibrio vulnificus during biofilm formation.

Vibrio vulnificus is a foodborne pathogen causing septicemia with high mortality rate. In this study, we explored how Escherichia coli, one of the commensal bacteria in the human gastrointestinal tract, can interact with V. vulnificus. Our study results show that the amount of biofilm produced by V. vulnificus was reduced in the presence of E. coli ATCC 35218, although the growth of V. vulnificus L-180 remained unaffected. We also detected an antibiofilm effect of E. coli culture supernatant against V. vulnificus, which could not be reduced even after heat treatment. These findings indicate that E. coli and its culture supernatant may be suitable to prevent biofilm formation by V. vulnificus. By contrast, live cells of V. vulnificus could reduce the amount of preformed E. coli biofilm, but its culture supernatant could not. This suggests that the cell-associated factors contribute toward reduction in E. coli biofilm. Therefore, we speculate that ingestion of an infectious dose of V. vulnificus might induce dislodging of the commensal bacteria from the intestinal epithelia and thus can colonize to initiate the infection.

Differential expression of vvhA and CPS operon allele 1 genes in Vibrio vulnificus under biofilm and planktonic conditions.

Examination of genes encoding for the virulence factors, hemolysin/cytolysin (vvhA) and capsular polysaccharide (CPS allele 1), during biofilm formation revealed that their expression was influenced by the maturity of the biofilm as well as by temperature. At 24 °C, expression of vvhA during biofilm formation was low between 4 and 12 h but increased 10-fold by 24 h to (5.1 × 104 ± 6.3 × 103mRNA copies/ml) as the biofilm matured. Compared to planktonic cells, expression of vvhA during biofilm formation at 24 °C was initially up-regulated at 4 h (1.07 ± 0.00-fold) but then was down-regulated almost four-fold during the intermediate and mature stages of biofilm formation. In contrast, vvhA expression at 37 °C was up-regulated almost four-fold in the early stages (4 and 6 h) of biofilm formation and remained two-fold up-regulated by 24 h even as the biofilm was deteriorating. CPS allele 1 expression at 24 °C during biofilm formation was up-regulated (1.50 ± 0.18-fold) during the initial attachment phase of the cells but was strongly down-regulated during the intermediate phases at 8 and 10 h (74.42 ± 42.16-fold and 453.76 ± 193.32-fold, respectively), indicating that capsular polysaccharide (CPS) is not important to intermediate biofilm architecture. Interestingly, as the biofilm matured by 24 h, expression of CPS allele 1 was again up-regulated (1.88 ± 1.07), showing that CPS plays a role in mature biofilm. At 37 °C, CPS allele 1 expression was significantly up-regulated (up to 105) during biofilm formation, indicating that the biofilm form of V. vulnificus may be preferred over the planktonic form in the human host.

Phylogeny and life cycle of the zoonotic pathogen Vibrio vulnificus.

Vibrio vulnificus is a zoonotic pathogen able to cause diseases in humans and fish that occasionally result in sepsis and death. Most reviews about this pathogen (including those related to its ecology) are clearly biased towards its role as a human pathogen, emphasizing its relationship with oysters as its main reservoir, the role of the known virulence factors as well as the clinic and the epidemiology of the human disease. This review tries to give to the reader a wider vision of the biology of this pathogen covering aspects related to its phylogeny and evolution and filling the gaps in our understanding of the general strategies that V. vulnificus uses to survive outside and inside its two main hosts, the human and the eel, and how its response to specific environmental parameters determines its survival, its death, or the triggering of an infectious process.

Influence of farming methods and seawater depth on Vibrio species in New Zealand Pacific oysters.

Studies conducted in seawaters around New Zealand have shown the numbers of human pathogenic Vibrio spp. are usually low, but high numbers sometimes occur during warmer summer/autumn months (January - April). In this study, Pacific oysters (Crassostrea gigas) were grown at Kaipara Harbour and Mahurangi Harbour in New Zealand at different heights from the seafloor in different ways: fixed positons intertidally and subtidally, and as floating long lines over the 2013 and 2014 summer periods. Two geographically distinct commercial harvest areas: Coromandel Harbour (North Island) and Croisilles Harbour (South Island) in New Zealand were also compared in 2015 where oysters are grown under different methods. Detection and enumeration of Vibrio spp. was performed according to the Bacteriological Analytical Manual using the Most Probable Number approach and real-time polymerase chain reaction technique. The only significant growing method effect was observed in Mahurangi Harbour, where intertidal oysters at 1.5 m from the seafloor had higher numbers of trh + Vibrio parahaemolyticus than other intertidal samples from Kaipara Harbour and Coromandel Harbour. All other samples showed a relationship with surface seawater temperature, but not with distance from seafloor or farming method. Overall, there is no clear evidence that different oyster farming methods (floating, subtidal or intertidal at different depths) affect Vibrio spp. population sizes, which were dominated by seasonal changes and environmental parameters.

Isolation and characterization of potentially pathogenic Vibrio species in a temperate, higher latitude hotspot.

The recent emergence of Vibrio infections at high latitudes represents a clear human health risk attributable to climate change. Here, we investigate the population dynamics of three Vibrio species: Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae within a British coastal estuarine site, with contrasting salinity and temperature regimes during an intense heatwave event. Water samples were collected weekly through the summer of 2018 and 2019 and filtered using membrane filtration and subsequently grown on selective media. Suspected vibrios were confirmed using a conventional species-specific PCR assay and further analysed for potential pathogenic markers. Results showed that Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio cholerae were present at high concentrations throughout both years, with their populations at substantially greater abundances corresponding to conditions of higher water temperatures during the heatwave of 2018 and at lower salinity sites, which is comparable to the results of previous studies. A subset of strains isolated during the extreme heatwave event in 2018 (46 Vibrio parahaemolyticus, 11 Vibrio cholerae and 4 Vibrio vulnificus) were genomically sequenced. Analysis of these 63 sequenced strains revealed a broad phenotypic and genomic diversity of strains circulating in the environment. An analysis of pathogenicity attributes identified a broad array of virulence genes across all three species, including a variety of genes associated with human disease. This study highlights the importance of the need for an increased Vibrio spp. surveillance system in temperate regions and the potential impact warming events such as heatwaves may have on the abundance of potentially pathogenic bacteria in the environment.

The cytochrome d oxidase complex regulated by fexA is an Achilles' heel in the in vivo survival of vibrio vulnificus.

Vibrio vulnificus is a halophilic estuarine bacterium causing severe opportunistic infections. To successfully establish an infection, V. vulnificus must adapt to redox fluctuations in vivo. In the present study, we show that deletion of V. vulnificus fexA gene caused hypersensitivity to acid and reactive oxygen species. The ΔfexA mutant exhibited severe in vivo survival defects. For deeper understanding the role of fexA gene on the successful V. vulnificus infection, we analyzed differentially expressed genes in ΔfexA mutant in comparison with wild type under aerobic, anaerobic or in vivo culture conditions by genome-scale DNA microarray analyses. Twenty-two genes were downregulated in the ΔfexA mutant under all three culture conditions. Among them, cydAB appeared to dominantly contribute to the defective phenotypes of the ΔfexA mutant. The fexA deletion induced compensatory point mutations in the cydAB promoter region over subcultures, suggesting essentiality. Those point mutations (PcydSMs) restored bacterial growth, motility, cytotoxicity ATP production and mouse lethality in the ΔfexA mutant. These results indicate that the cydAB operon, being regulated by FexA, plays a crucial role in V. vulnificus survival under redox-fluctuating in vivo conditions. The FexA-CydAB axis should serve an Achilles heel in the development of therapeutic regimens against V. vulnificus infection.

A stealth adhesion factor contributes to Vibrio vulnificus pathogenicity: Flp pili play roles in host invasion, survival in the blood stream and resistance to complement activation.

The tad operons encode the machinery required for adhesive Flp (fimbrial low-molecular-weight protein) pili biogenesis. Vibrio vulnificus, an opportunistic pathogen, harbors three distinct tad loci. Among them, only tad1 locus was highly upregulated in in vivo growing bacteria compared to in vitro culture condition. To understand the pathogenic roles of the three tad loci during infection, we constructed single, double and triple tad loci deletion mutants. Interestingly, only the Δtad123 triple mutant cells exhibited significantly decreased lethality in mice. Ultrastructural observations revealed short, thin filamentous projections disappeared on the Δtad123 mutant cells. Since the pilin was paradoxically non-immunogenic, a V5 tag was fused to Flp to visualize the pilin protein by using immunogold EM and immunofluorescence microscopy. The Δtad123 mutant cells showed attenuated host cell adhesion, decreased biofilm formation, delayed RtxA1 exotoxin secretion and subsequently impaired translocation across the intestinal epithelium compared to wild type, which could be partially complemented with each wild type operon. The Δtad123 mutant was susceptible to complement-mediated bacteriolysis, predominantly via the alternative pathway, suggesting stealth hiding role of the Tad pili. Complement depletion by treating with anti-C5 antibody rescued the viable count of Δtad123 in infected mouse bloodstream to the level comparable to wild type strain. Taken together, all three tad loci cooperate to confer successful invasion of V. vulnificus into deeper tissue and evasion from host defense mechanisms, ultimately resulting in septicemia.

In Vitro Synergy and In Vivo Activity of Tigecycline-Ciprofloxacin Combination Therapy against Vibrio vulnificus Sepsis.

The mortality rate associated with Vibrio vulnificus sepsis remains high. An in vitro time-kill assay revealed synergism between tigecycline and ciprofloxacin. The survival rate was significantly higher in mice treated with tigecycline plus ciprofloxacin than in mice treated with cefotaxime plus minocycline. Thus, combination treatment with tigecycline-ciprofloxacin may be an effective novel antibiotic regimen for V. vulnificus sepsis.

Characterization of temperature-dependent hemin uptake receptors HupA and HvtA in Vibrio vulnificus.

The Gram-negative pathogen Vibrio vulnificus produces several iron-sequestration systems including a hemin uptake system in response to iron limitation as a means to acquire this essential element. Strains of this organism are capable of causing serious septicemia in humans and eels, where hemin is abundant and an advantageous source of iron. Vibrio vulnificus hemin uptake systems consist of HupA, a well studied outer membrane protein, and a recently identified HvtA protein receptor. In this study, we confirmed that the expression of the hvtA gene is iron-regulated in a fur-dependent manner. When analyzed for virulence in a hemin-overloaded murine model system, the hupA gene was more important for establishing infection than the hvtA gene. Transcriptional profiling of these genes using strains of two different biotypes, biotype 1 (human pathogen) and biotype 2 (eel pathogen), showed that the expression of the two receptors was also regulated in response to temperature. The expression of hupA was highly induced in elevated temperatures in the human pathogenic strain when tested in iron-depleted conditions. Conversely, hvtA expression was induced significantly in the eel pathogenic strain at a lower temperature, a condition where the hupA locus was relatively repressed. Our results indicate that although both hupA and hvtA are involved for optimal hemin uptake in V. vulnificus, their expression is dually regulated by the environmental cues of iron concentration and temperature. Together, these data suggest that the virulence genes hupA and hvtA are tightly regulated and strictly induced during iron limitation combined with the physiological temperature of the host organism.

Structure and assembly of pilotin-dependent and -independent secretins of the type II secretion system.

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.

Unveiling the acid stress response of clinical genotype Vibrio vulnificus isolated from the marine environments of Mangaluru coast, India.

Gastric acidity is one of the earliest host defences faced by ingested organisms, and successful pathogens need to overcome this hurdle. The objective of this study was the systematic assessment of acid-stress response of Vibrio vulnificus isolated from coastal regions of Mangaluru. Acid-shock experiments were carried out at pH 4.0 and pH 4.5, with different experimental conditions expected to produce a varied acid response. Exposure to mild acid before the acid shock was favourable to the bacteria but was dependent on cell population and pH of the media and was independent of the strains tested. Lysine-dependent acid response was demonstrated with reference to the previously identified lysine decarboxylase system. Additionally, the results showed that inoculation into oysters provided some level of protection against acid stress. Increased expression of lysine/cadaverine genes was observed upon the addition of ground oyster and was confirmed by quantitative real-time PCR. The potential role of ornithine was analyzed with regard to acid stress, but no change in the survival pattern was observed. These findings highlight the physiology of bacteria in acid stress.

Small-molecule inhibitor of HlyU attenuates virulence of Vibrio species.

Increasing antibiotic resistance has led to the development of new strategies to combat bacterial infection. Anti-virulence strategies that impair virulence of bacterial pathogens are one of the novel approaches with less selective pressure for developing resistance than traditional strategies that impede viability. In this study, a small molecule CM14 [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] that inhibits the activity of HlyU, a transcriptional regulator essential for the virulence of the fulminating human pathogen Vibrio vulnificus, has been identified. Without affecting bacterial growth or triggering the host cell death, CM14 reduces HlyU-dependent expression of virulence genes in V. vulnificus. In addition to the decreased hemolysis of human erythrocytes, CM14 impedes host cell rounding and lysis caused by V. vulnificus. Notably, CM14 significantly enhances survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. Biochemical, mass spectrometric, and mutational analyses revealed that CM14 inhibits HlyU from binding to target DNA by covalently modifying Cys30. Remarkably, CM14 decreases the expression of various virulence genes of other Vibrio species and thus attenuates their virulence phenotypes. Together, this molecule could be an anti-virulence agent against HlyU-harboring Vibrio species with a low selective pressure for the emergence of resistance.

Vibrio vulnificus meningoencephalitis in a patient with thalassemia and a splenectomy.

Vibrio vulnificus usually causes wound infection, gastroenteritis, and septicemia. However, it is a rare conditional pathogen causing meningoencephalitis. We report a case of a young, immunocompromised man presenting with severe sepsis after exposure to sea water and consumption of seafood. The patient subsequently developed meningoencephalitis, and Vibrio vulnificus was isolated from his blood culture. The sequence was confirmed by Next-generation sequencing of a sample of cerebrospinal fluid, as well as from a bacteria culture. After the pathogen was detected, the patient was treated with ceftriaxone, doxycycline, and moxifloxacin for 6 weeks, which controlled his infection. In this case, we acquired his clinical and dynamic MRI presentations, which were never reported. Physicians should consider Vibrio vulnificus infections when they see a similar clinical course, brain CT and MRI findings, susceptibility factors and recent seafood ingestion or exposure to seawater. Due to high mortality, the early diagnosis and treatment of Vibrio vulnificus infections are crucial. Next-generation sequencing was found to be useful for diagnosis.