Year round patchiness of Vibrio vulnificus within a temperate Texas bay

Aims: To investigate with high geographical resolution the small‐scale spatial and temporal distribution of the pathogen Vibrio vulnificus throughout the water column in a temperate Texas bay where numerous V. vulnificus infections had been reported by the regional media the previous summer. Methods and Results: Surface and bottom water samples were collected from 19 sites between April 2005 and October 2006 from Matagorda Bay, TX. Physicochemical parameters were measured and V. vulnificus were analysed using quantitative polymerase chain reaction (Q‐PCR) as a means of overcoming constraints of traditional culturing techniques. V. vulnificus was detected through out the year, although it’s temporal and spatial distribution was patchy. V. vulnificus abundances at individual sites ranged from <10 to >1·1 × 10³ cells ml^?1. No statistically reliable predictive model related to the physicochemical parameters could be developed for this pathogen. Conclusions: his study demonstrates that year round detection of V. vulnificus while likely in the viable but nonculturable (VBNC) state during the winter months and emphasizes why physicochemical factors are insufficient metrics for robust regression modelling of this pathogen. Significance and Impact of the Study: This study provides an effective new tool, Q‐PCR, to study environmental distribution of V. vulnificus and that in the light of the patchy distribution observed, new reliable approaches and a mechanistic understanding of pathogen ecology need to be considered to effectively model the aquatic distribution of V. vulnificus.     

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.     

Incidence of Vibrio vulnificus in estuarine waters of the south Texas Coastal Bend region

Aims: To determine the occurrence of the human pathogen, Vibrio vulnificus, in south Texas coastal waters. Methods and Results: Coastal waters were sampled monthly between August 2006 and July 2007. Water temperature, dissolved oxygen, pH, salinity, conductivity and turbidity were measured during each sampling event. Culture‐based techniques utilizing Vibrio vulnificus agar (VVA) and membrane‐Enterococcus indoxyl‐β‐d ‐glucoside agar (mEI) were used to assess the occurrence and levels of V. vulnificus and the faecal contamination indicator group, enterococci, respectively. Vibrio vulnificus isolates were confirmed using colony‐blot hybridization with the species‐specific VVAP probe. Vibrio vulnificus was isolated at all sites throughout the year even when the water temperature dropped to 9·71°C. Significant correlations were found between concentrations of V. vulnificus and the abiotic factors, water temperature (P = 0·002) and dissolved oxygen (P = 0·028), as well as between concentrations of V. vulnificus and enterococci (P < 0·001). Conclusions: This study demonstrated the year‐round presence of V. vulnificus in coastal waters of south Texas. Significance and Impact of the Study: These findings indicate that the potential for human exposure to the pathogen, V. vulnificus, exists throughout the year. It also suggests that routinely monitored data might be used to predict the occurrence of the pathogen.     

Occurrence of Vibrio vulnificus in mussel farms from the Varano lagoon environment

Aims: Monitoring the occurrence of the human pathogen Vibrio vulnificus in a mussel farm located in the lagoon of Varano (Italy). Methods and Results: A total of 72 samples of mussel, water and sediment, collected from two locations of Varano lagoon in the Gargano peninsula, during a 7‐ month survey, were analysed. Isolation and PCR characterization of six V. vulnificus environmental genotype strains revealed that this pathogen was isolated when with T was above 22°C and salinity ranged between 22·7 and 26·4‰. No significant correlation of the occurrence of V. vulnificus with water pH or salinity was observed. Moreover, 8% of mussel samples were found to be contaminated by V. vulnificus. All of that positive mussel samples originated from the same sampling station. Conclusion: It is suggested that warmer season are risky to eat raw or undercooked bivalve molluscs in the local area. Significance and Impact of the Study: To increase knowledge about environmental conditions that may affect the occurrence of waterborne pathogen Vibrio vulnificus in seafood.     

创伤弧菌毒力相关因子的全基因组关联分析研究

【目的】创伤弧菌是致死率最高的弧菌物种,但目前尚无在全基因组层面挖掘毒力相关因子的研究。本研究以创伤弧菌分离来源(临床和环境)作为不同表型,通过与260株基因组序列进行关联分析,挖掘毒力相关因子,从而进一步了解创伤弧菌致病因素。【方法】对139株创伤弧菌分离株进行高通量测序,获取其全基因组序列;与公共数据库已公开发表的121株基因组整合,使用pyseer软件进行全基因组关联分析,对与不同分离来源显著相关的基因进行注释和解读。【结果】共发现11个基因与临床分离株显著相关,其中9个是本研究新发现的创伤弧菌潜在毒力相关因子。【结论】本研究使用群体基因组学和统计遗传学方法,在全基因组范围扫描挖掘了创伤弧菌毒力相关因子,为深入揭示该物种致病机制、设计新的疫苗和治疗靶点提供了重要依据。     

Regulation systems of protease and hemolysin production in Vibrio vulnificus

Vibrio vulnificus, a gram‐negative halophilic estuarine bacterium, is an opportunistic human pathogen that causes rapidly progressive fatal septicemia and necrotizing wound infection. This species also causes hemorrhagic septicemia called vibriosis in cultured eels. It has been proposed that a range of virulence factors play roles in pathogenesis during human and/or eel infection. Among these factors, a metalloprotease (V. vulnificus protease [VVP]) and a cytolytic toxin (V. vulnificus hemolysin [VVH]) are of significant importance. VVP elicits the characteristic edematous and hemorrhagic skin damage, whereas VVH exhibits powerful hemolytic and cytolytic activities and contributes to bacterial invasion from the intestine to the blood stream. In addition, a few V. vulnificus strains isolated from diseased eels have recently been found to produce a serine protease designated as V. vulnificus serine protease (VvsA) instead of VVP. Similarly to VVP, VvsA may possess various toxic activities such as collagenolytic, cytotoxic and edema‐forming activity. In this review, regulation of V. vulnificus VVP, VVH and VvsA is clarified in terms of expression at the mRNA and protein levels. The explanation is given on the basis of the quorum sensing system, which is dependent on bacterial cell density. In addition, the roles of environmental factors and global regulators, such as histone‐like nucleoid structuring protein, cyclic adeno monophosphate receptor protein, RpoS, HlyU, Fur, ToxRS, AphB and LeuO, in this regulation are outlined. The cumulative impact of these regulatory systems on the pathogenicity of V. vulnificus is here delineated.     

Genome-Wide SNP-Genotyping Array to Study the Evolution of the Human Pathogen Vibrio vulnificus Biotype 3

Vibrio vulnificus is an aquatic bacterium and an important human pathogen. Strains of V. vulnificus are classified into three different biotypes. The newly emerged biotype 3 has been found to be clonal and restricted to Israel. In the family Vibrionaceae, horizontal gene transfer is the main mechanism responsible for the emergence of new pathogen groups. To better understand the evolution of the bacterium, and in particular to trace the evolution of biotype 3, we performed genome-wide SNP genotyping of 254 clinical and environmental V. vulnificus isolates with worldwide distribution recovered over a 30-year period, representing all phylogeny groups. A custom single-nucleotide polymorphism (SNP) array implemented on the Illumina GoldenGate platform was developed based on 570 SNPs randomly distributed throughout the genome. In general, the genotyping results divided the V. vulnificus species into three main phylogenetic lineages and an additional subgroup, clade B, consisting of environmental and clinical isolates from Israel. Data analysis suggested that 69% of biotype 3 SNPs are similar to SNPs from clade B, indicating that biotype 3 and clade B have a common ancestor. The rest of the biotype 3 SNPs were scattered along the biotype 3 genome, probably representing multiple chromosomal segments that may have been horizontally inserted into the clade B recipient core genome from other phylogroups or bacterial species sharing the same ecological niche. Results emphasize the continuous evolution of V. vulnificus and support the emergence of new pathogenic groups within this species as a recurrent phenomenon. Our findings contribute to a broader understanding of the evolution of this human pathogen.     

Occurrence and distribution of the human pathogen Vibrio vulnificus in a subtropical Gulf of Mexico estuary

Water and sediment samples from Charlotte Harbor, Florida were examined for the autochthonous human pathogen, Vibrio vulnificus, for 1 year (March 1997–February 1998). Within the estuary, mean water column levels of V. vulnificus ranged between 58 CFU/100 ml and 1.21×10³ CFU/100 ml while sediment levels were up to 2 orders of magnitude greater.Vibrio vulnificus was detected throughout the year in Charlotte Harbor. The highest concentrations (5.14×10³ CFU/100 ml) of the year were found at warm temperatures and moderate salinities in September. The lowest mean concentration occurred in March at 26 CFU/100 ml. Although concentrations of Vibrio vulnificus were positively correlated with temperature, salinity was a more important factor influencing variability of this organism. In Charlotte Harbor, an optimal salinity of 15 psu (practical salinity units) was found for recovery of high concentrations of the pathogen. There were significant positive and negative correlations above and below 15 psu, respectively. Results from this study suggest that unlike temperate estuaries, in regions of moderate year round temperatures, such as the tropics or subtropics, salinity strongly controls the geographical and seasonal distribution of V. vulnificus between sediment and water column.     

Histidine phosphocarrier protein regulates pyruvate kinase A activity in response to glucose in Vibrio vulnificus

The bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) consists of two general energy‐coupling proteins [enzyme I and histidine phosphocarrier protein (HPr)] and several sugar‐specific enzyme IIs. Although, in addition to the phosphorylation‐coupled transport of sugars, various regulatory roles of PTS components have been identified in Escherichia coli, much less is known about the PTS in the opportunistic human pathogen Vibrio vulnificus. In this study, we have identified pyruvate kinase A (PykA) as a binding partner of HPr in V. vulnificus. The interaction between HPr and PykA was strictly dependent on the presence of inorganic phosphate, and only dephosphorylated HPr interacted with PykA. Experiments involving domain swapping between the PykAs of V. vulnificus and E. coli revealed the requirement for the C‐terminal domain of V. vulnificus PykA for a specific interaction with V. vulnificus HPr. Dephosphorylated HPr decreased the K_m of PykA for phosphoenolpyruvate by approximately fourfold without affecting V_max. Taken together, these findings indicate that the V. vulnificus PTS catalyzing the first step of glycolysis stimulates the final step of glycolysis in the presence of glucose through the direct interaction of dephospho‐HPr with the C‐terminal domain of PykA.     

Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification

Vibrio vulnificus is a serious bacterial pathogen for humans and aquatic animals. We developed a rapid, sensitive and specific identification method for V. vulnificus using loop-mediated isothermal amplification (LAMP) technique. A set of primers, composed of two outer primers and two inner primers, was designed based on the cytolysin gene sequence of V. vulnificus. The LAMP reaction was processed in a heat block at 65 °C for 60 min. The amplification products were detected by visual inspection using SYBR Green I, as well as by electrophoresis on agarose gels. Our results showed that the LAMP reaction was highly specific to V. vulnificus. This method was 10-fold more sensitive than conventional PCR. In conclusion, the LAMP assay was extremely rapid, simple, cost-effective, sensitive and specific for the rapid identification of V. vulnificus.     

Protective effect of polygoni cuspidati radix and emodin on <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> cytotoxicity and infection

Vibrio vulnificus, a good model organism of bacterial septicemia, causes fatal septicemia manifesting a fulminating course and a high mortality rate within days. In order to identify new natural substances preventing V. vulnificus infection, a plant library was screened for inhibiting cytotoxicity to host cells by using Trypan blue staining and LDH assay. We found that Polygoni Cuspidati Radix potently suppressed the acute death of HeLa and RAW264.7 cells in a dose dependent manner. Further studies revealed that Polygoni Cuspidati Radix inhibited V. vulnificus growth and survival in HI broth and seawater, respectively. We confirmed that Polygoni Cuspidati Radix contained high level of emodin by thin layer chromatography (TLC). Emodin showed direct antibacterial activity against V. vulnificus. In addition, emodin prevented the morphologic damages and acute death of HeLa cells caused from V. vulnificus. The safety of Polygoni Cuspidati Radix and emodin to host cells was confirmed by MTT assay. Polygoni Cuspidati Radix and emodin protected mice from V. vulnificus infection.     

Ecology ofVibrio vulnificus in Galveston Bay oysters,suspended particulate matter,sediment and seawater: Detection by monoclonal antibody — immunoassay — most probable number procedures

Summary Oysters, suspended particulate matter (SPM), sediment and seawater samples were collected from West Galveston Bay, Texas over a 16-month period and analyzed for the presence ofVibrio vulnificus, a naturally-occurring human marine pathogen. Detection and enumeration ofV. vulnificus was performed using a species-specific monoclonal antibody (mAb FRBT37) in an enzyme immunoassay (EIA)-most probable number (MPN) procedure capable of detecting as few as 2000 target organisms.V. vulnificus was not detected in seawater, oyster or SPM samples during the cold weather months, but was detected at low levels in several sediment samples during this time period. Increased levels of the organism were first observed in early spring in the sediment, and then in SPM and oysters. The major increase inV. vulnificus occurred only after the seawater temperature had increased above 20°C and the winter-spring rainfall had lowered the salinity below 16. The highestV. vulnificus levels at each site were associated with suspended particulate matter. These results are consistent with the hypothesis that (1)V. vulnificus over-winters in a floc zone present at the sediment-water interface, (2) is resuspended into the water column in early spring following changes in climatic conditions, (3) colonizes the surfaces of zooplankton which are also blooming during early spring and (4) are ingested by oysters during their normal feeding process.     

Molecular and Physical Factors That Influence Attachment of Vibrio vulnificus to Chitin

The human pathogen Vibrio vulnificus is the leading cause of seafood-related deaths in the United States. Strains are genotyped on the basis of alleles that correlate with isolation source, with clinical (C)-genotype strains being more often implicated in disease and environmental (E)-genotype strains being more frequently isolated from oysters and estuarine waters. Previously, we have shown that the ecologically distinct C- and E-genotype strains of V. vulnificus display different degrees of chitin attachment, with C-genotype strains exhibiting reduced attachment relative to their E-genotype strain counterparts. We identified type IV pili to be part of the molecular basis for this observed genotypic variance, as E-genotype strains exhibit higher levels of expression of these genes than C-genotype strains. Here, we used a C-genotype quorum-sensing (QS) mutant to demonstrate that quorum sensing is a negative regulator of type IV pilus expression, which results in decreased chitin attachment. Furthermore, calcium depletion reduced E-genotype strain attachment to chitin, which suggests that calcium is necessary for proper functioning of the type IV pili in E-genotype strains. We also found that starvation or dormancy can alter the efficiency of chitin attachment, which has significant implications for the environmental persistence of V. vulnificus. With the increasing incidence of wound infections caused by V. vulnificus, we investigated a subset of E-genotype strains isolated from human wound infections and discovered that they attached to chitin in a manner more similar to that of C-genotype strains. This study enhances our understanding of the molecular and physical factors that mediate chitin attachment in V. vulnificus, providing insight into the mechanisms that facilitate the persistence of this pathogen in its native environment.     

Actions of Vibrio vulnificus Metalloprotease on Human Plasma Proteinase-Proteinase Inhibitor Systems: A Comparative Study of Native Protease with Its Derivative Modified by Polyethylene Glycol

Vibrio vulnificus, an opportunistic human pathogen causing wound infection and septicemia, produces a metalloprotease (VVP) which is suspected to be a virulent determinant. The interactions of VVP, as well as its derivative (PEG₁-VVP) modified with polyethylene glycol, with a variety of human plasma proteins were investigated. We found that native VVP and its derivative were able to act directly on many biologically important human plasma proteins even in the presence of α-macroglobulin, the sole plasma inhibitor of native VVP. The activities of both classical and alternative pathways of the complement cascade system were drastically abolished by incubation with either VVP. Furthermore, these proteases rapidly digested the Aα-chain of human fibrinogen into fragment(s) with no clotting ability. Therefore both VVPs are thought to function as a fibrinogenolytic enzyme, causing delay of the coagulation reaction. VVP and PEG₁-VVP were also shown to destroy plasma proteinase inhibitors including α₁-proteinase inhibitor, a major inhibitor in human plasma. Because endogenous proteolytic enzymes and their inhibitors are indispensable in maintaining physiological homeostasis, these findings suggest that VVP (and PEG₁-VVP) may cause an imbalance of human plasma proteinase-proteinase inhibitor systems, thus eliciting an immunocompromised state in the host and facilitating the development of a systemic V. vulnificus infection such as septicemia.     

Multi-site Analysis Reveals Widespread Antibiotic Resistance in the Marine Pathogen <Emphasis Type="Italic">Vibrio vulnificus</Emphasis>

Vibrio vulnificus is a serious opportunistic human pathogen commonly found in subtropical coastal waters, and is the leading cause of seafood-borne mortality in the USA. This taxon does not sustain prolonged presence in clinical or agricultural settings, where it would undergo human-induced selection for antibiotic resistance. Therefore, few studies have verified the effectiveness of commonly prescribed antibiotics in V. vulnificus treatment. Here we screened 151 coastal isolates and 10 primary septicaemia isolates against 26 antimicrobial agents representing diverse modes of action. The frequency of multiple resistances to antibiotics from all sources was unexpectedly high, particularly during summer months, and a substantial proportion of isolates (17.3%) were resistant to eight or more antimicrobial agents. Numerous isolates demonstrated resistance to antibiotics routinely prescribed for V. vulnificus infections, such as doxycycline, tetracycline, aminoglycosides and cephalosporins. These resistances were detected at similar frequencies in virulent and non-virulent strains (PCR-based virulence typing) and were present in septicaemia isolates, underlying the public health implications of our findings. Among environmental isolates, there were no consistent differences in the frequency of resistance between pristine and anthropogenically impacted estuaries, suggesting natural rather than human-derived sources of resistance traits. This report is the first to demonstrate prevalent antibiotic resistance in a human pathogen with no clinical reservoirs, implying the importance of environmental studies in understanding the spread, evolution and public health relevance of antibiotic resistance factors. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Polar landmark protein HubP recruits flagella assembly protein FapA under glucose limitation in Vibrio vulnificus

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum‐driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIA^Glc of the PEP‐dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA‐mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIA^Glc and HubP, in the human pathogen V. vulnificus.     

Evidence that <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> ahpC2 is essential for survival under high salinity by modulating intracellular level of ROS

Expression of ahpC2 encoding an alkyl hydroperoxide reductase of Vibrio vulnificus, a foodborne pathogen, was incrementally induced depending on NaCl concentrations in the culture. Growth of the ahpC2 mutant was significantly impaired with longer lag phase and lower growth rate when cultured under high salinity. ROS was accumulated in V. vulnificus cells when stressed by exposure to high salinity, and the ahpC2 mutant accumulated higher level of ROS as compared with the parental wild type. Consequently, the combined results suggest that AhpC2 contributes to the growth of V. vulnificus under high salinity by scavenging ROS in cells.     

The Interactions of Vibrio vulnificus and the Oyster Crassostrea virginica

The human bacterial pathogen, Vibrio vulnificus, is found in brackish waters and is concentrated by filter-feeding molluscan shellfish, especially oysters, which inhabit those waters. Ingestion of raw or undercooked oysters containing virulent strains of V. vulnificus can result in rapid septicemia and death in 50 % of victims. This review summarizes the current knowledge of the environmental interactions between these two organisms, including the effects of salinity and temperature on colonization, uptake, and depuration rates of various phenotypes and genotypes of the bacterium, and host–microbe immunological interactions.     

Vibrio vulnificus Secretes an Insulin-degrading Enzyme That Promotes Bacterial Proliferation in Vivo

We describe a novel insulin-degrading enzyme, SidC, that contributes to the proliferation of the human bacterial pathogen Vibrio vulnificus in a mouse model. SidC is phylogenetically distinct from other known insulin-degrading enzymes and is expressed and secreted specifically during host infection. Purified SidC causes a significant decrease in serum insulin levels and an increase in blood glucose levels in mice. A comparison of mice infected with wild type V. vulnificus or an isogenic sidC-deletion strain showed that wild type bacteria proliferated to higher levels. Additionally, hyperglycemia leads to increased proliferation of V. vulnificus in diabetic mice. Consistent with these observations, the sid operon was up-regulated in response to low glucose levels through binding of the cAMP-receptor protein (CRP) complex to a region upstream of the operon. We conclude that glucose levels are important for the survival of V. vulnificus in the host, and that this pathogen uses SidC to actively manipulate host endocrine signals, making the host environment more favorable for bacterial survival and growth.