Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V. vulnificus CPS locus, which included an upstream ops element, a wza gene (wza Vv ), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli. Polar transposon mutations in wza Vv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza Vv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wza Vv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V. vulnificus.Vibrio vulnificus is indigenous to the estuarine environment and can produce rapidly fatal human infections associated with consumption of raw oysters. Pathogenesis of this gram-negative species involves a combination of host-pathogen interactions that are not completely understood. Predisposing host factors include iron overload, hepatic disease, and immune system dysfunction (3,22,45). This organism asymptomatically colonizes both fish (8) and shellfish (51) at relatively high levels (10 3 to 10 6 CFU/g of body weight) during warmer months, but mouse models, using exogenous iron, suggest that the infectious dose may be Ͻ10 CFU (48). Mortalities exceed 50% for septicemic patients, and V. vulnificus disease remains the leading cause of fatal infections associated with seafood consumption (37). Although symptoms of V. vulnificus septicemia resemble endotoxic shock, the lipopolysaccharide (LPS) of this organism is relatively inert, and the contribution of LPS to virulence remains unclear (24, 31). Conversely, expression of capsular polysaccharide (CPS) is clearly a prerequisite for virulence and correlates with lethality in mice (41, 54), resistance to phagocytosis (46) and complement-mediated lysis (40), cytokine induction (31), and opaque colonies. Phase variation to a phenotype with reduced CPS expression occurs at a frequency of about 10 Ϫ4 and correlates with translucent colonies, increased serum sensitivity, and reduced virulence. CPS is a protective antigen in mice (9, 18), and its relationship to V. vulnificus disease was confirmed by the loss of virulence in acapsular transposon mutants (49,52). CPS-independent virulence factors indicate that pathogenesis is multifac...

Vibrio vulnificus is a human pathogen that produces lethal septicemia in susceptible persons, and the primary virulence factor for this organism is capsular polysaccharide (CPS). The role of the capsule in V. vulnificus biofilms was examined under a variety of conditions, by using either defined CPS mutants or spontaneous CPS expression phase variants derived from multiple strains. CPS expression was shown to inhibit attachment and biofilm formation, which contrasted with other studies describing polysaccharides as integral to biofilms in related species.Vibrio vulnificus is indigenous to estuarine environments (9,18,24,36,43) and causes human infections associated with raw oyster consumption (3). Pathogenesis was recently reviewed, and virulence is primarily attributed to capsular polysaccharide (CPS) expression (34). Opaque (O) colony morphology, indicative of a virulent, encapsulated phenotype, exhibits reversible phase variation to translucent (T) colony types with reduced CPS expression and decreased virulence (30,47). Defined mutations in the CPS operon confirmed the relationship of CPS and virulence (28,42,44,45). Vibrio spp. attach to algae and zooplankton (5,15,16,17,19,22), and V. vulnificus may be more concentrated in oysters and fish which feed on these organisms (9,29,36,43). Microbial communities attached to nutrient-rich surfaces are generally referred to as biofilms and are thought to engage in complex signaling for expression of CPS and other factors (7,25,26). For example, V. cholerae biofilms require production of polysaccharide, pili, and flagella (5,21,23,37,38,39,46). Biofilms for V. vulnificus biotype 2 eel pathogens were recently described (20); however, this group differs from human pathogens of biotype 1 in that biotype 2 lipopolysaccharide (LPS) is homogeneous (serovar E) and CPS may not always be required for virulence (2). The role of CPS in biofilms of either biotype has not been addressed; therefore, our studies examined V. vulnificus biofilms in O versus T phase variants and CPS mutants that differed in their abilities to produce capsular polysaccharide.CPS expression inhibits V. vulnificus biofilm formation. Surface CPS displays a continuum of expression among V. vulnificus strains (44). Strains for this study are detailed in Table 1 and were stored at Ϫ70°C in 50% glycerol to ensure stability of phase variants. O strains are completely encapsulated, while T strains either are acapsular or have reduced, patchy capsules. Mutant strains are acapsular but differ in CPS biosynthesis: CVD752 contains a polar transposon mutation in the CPS operon that eliminates biosynthesis, while MO6-24/31T contains a nonpolar mutation, specifically targeting the CPS transport function of the wza gene, and can synthesize CPS but is unable to transport it to the cell surface (44, 45).Biofilm formation on abiotic surfaces was examined by crystal violet absorption assays, and the relative biofilm content was estimated from the concentration of dye eluted from destained cells and matrix (32). Examination...

Translucent, avirulent spontaneous phase variants of Vibrio vulnificus M06-24 reverted back to the original opaque, encapsulated phenotype under both in vivo and in vitro conditions. Two translucent, acapsular mutants, which did not show phase variation, were constructed by using the transposon Tn5 ISSOL: :phoA (TnphoA). Loss of capsule was accompanied by decreases in virulence, hydrophilicity, and serum resistance. The ability to utilize transferrin-bound iron for growth was lost in only one of the two unencapsulated mutants.

Vibrio vulnificus produces human disease associated with raw-oyster consumption or wound infections, but fatalities are limited to persons with chronic underlying illness. Capsular polysaccharide (CPS) is required for virulence, and CPS expression correlates with opaque (Op) colonies that show "phase variation" to avirulent translucent (Tr) phenotypes with reduced CPS. The results discussed here confirmed homology of a V. vulnificus CPS locus to the group 1 CPS operon in Escherichia coli. However, two distinct V. vulnificus genotypes or alleles were associated with the operon, and they diverged at sequences encoding hypothetical proteins and also at unique, intergenic repetitive DNA elements. Phase variation was examined under conditions that promoted high-frequency transition of Op to Tr forms. Recovery of Tr isolates in these experiments showed multiple genotypes, which were designated TR1, TR2, and TR3: CPS operons of TR1 isolates were identical to the Op parent, and cells remained phase variable but expressed reduced CPS. TR2 and TR3 showed deletion mutations in one (wzb) or multiple genes, respectively, and deletion mutants were acapsular and locked in the Tr phase. Complementation in trans restored the Op phenotype in strains with the wzb deletion mutation. Allelic variation in repetitive elements determined the locations, rates, and extents of deletion mutations. Thus, different mechanisms are responsible for reversible phase variation in CPS expression versus genetic deletions in the CPS operon of V. vulnificus. Repetitive-element-mediated deletion mutations were highly conserved within the species and are likely to promote survival in estuarine environments.Vibrio vulnificus causes systemic human infections with high mortality (Ͼ50%), and death can occur within hours or days following exposure (3). Illness is generally a consequence of raw oyster consumption or exposure of wounds to seawater. Underlying liver disease, diabetes, hemochromatosis (iron overload), and immune system dysfunction are prerequisites for life-threatening infections. The pathogenesis of V. vulnificus has been reviewed (14, 54), and disease symptoms resemble endotoxic shock. Although the contribution of lipopolysaccharide (LPS) to virulence remains unclear (31, 38), lethality in animal models is clearly related to capsular polysaccharide (CPS) expression (38,49,62,63,64,69). Both CPS expression and virulence are associated with opaque (Op) colony morphology, but colonies can spontaneously revert to the translucent (Tr) phenotype in a process termed phase variation. Tr colonies have reduced CPS expression and diminished virulence in mouse models (64). These phenotypes are reversible for both Op3Tr and Tr3Op transitions at rates of about 10 Ϫ3 to 10 Ϫ4 , and colony morphology is currently the most reliable virulence marker for V. vulnificus (51).Bacterial phase variation typically is related to reversible mutations that alter the expression of cell surface structures (CPS, LPS, pili, flagella, or outer membrane proteins) and increase...

Vibrio vulnificus is an opportunistic human pathogen commonly found in estuarine environments. Infections are associated with raw oyster consumption and can produce rapidly fatal septicemia in susceptible individuals. Standard enumeration of this organism in shellfish or seawater is laborious and inaccurate; therefore, more efficient assays are needed. An oligonucleotide probe derived from the cytolysin gene, vvhA, was previously used for colony hybridizations to enumerate V. vulnificus. However, this method requires overnight growth, and vibrios may lack culturability under certain conditions. In the present study, we targeted the same locus for development of a TaqMan real-time PCR assay. Probe specificity was confirmed by amplification of 28 V. vulnificus templates and by the lack of a PCR product with 22 non-V. vulnificus strains. Detection of V. vulnificus in pure cultures was observed over a 6-log-unit linear range of concentration (10 2 to 10 8 CFU ml ؊1 ), with a lower limit of 72 fg of genomic DNA l of PCR mixture Vibrio vulnificus produces a rapidly fatal septicemia, which is primarily associated with the ingestion of raw oysters (3, 16). Contact of wounds with seawater or shellfish can also lead to serious infections that can progress to septicemia or require limb amputation. Methods for examination of seafood safety currently rely on fecal coliform analysis; however, V. vulnificus is not associated with fecal contamination (30). Although current standards do not regulate the number of V. vulnificus organisms in shellfish, the Food and Drug Administration supports postharvest treatments that will greatly reduce or eliminate the numbers of this organism. Therefore, quantitative methods are needed to accurately and efficiently validate application of these treatments to the seafood industry.V. vulnificus is indigenous to estuaries worldwide and can be readily isolated from the environment (24, 30, 37); however, standard detection methods require enrichment and selective plating media to reduce the growth of background organisms (10). Species identification requires additional time-consuming assays that are frequently inaccurate and laborious. Standard plate count based on DNA probe hybridization for colony identification can also be used for enumeration in oyster tissues (19,36,37), but this assay still requires overnight growth of bacteria and usually involves several days of processing time for large numbers of samples. Numerous studies have indicated that, under conditions of reduced temperatures (4 to 5°C) and nutrient availability, vibrios become nonculturable on standard media while retaining viability (8,13,26,32,33). Although the contribution of a viable but nonculturable (VBNC) population to V. vulnificus disease is unknown, human infections have been reported with VBNC V. cholerae (8). Therefore, enumeration assays that do not require cultivation may be useful for risk assessment. Several PCR assays (5, 6, 23), as well as reverse transcription-PCR (RT-PCR) (9) for detection of vibrios without ...

f Irrigation water has been implicated as a likely source of produce contamination by Salmonella enterica. Therefore, the distribution of S. enterica was surveyed monthly in irrigation ponds (n ‫؍‬ 10) located within a prime agricultural region in southern Georgia and northern Florida. All ponds and 28.2% of all samples (n ‫؍‬ 635) were positive for Salmonella, with an overall geometric mean concentration (0.26 most probable number [MPN]/liter) that was relatively low compared to prior reports for rivers in this region. Salmonella peaks were seasonal; the levels correlated with increased temperature and rainfall (P < 0.05). The numbers and occurrence were significantly higher in water (0.32 MPN/liter and 37% of samples) than in sediment (0.22 MPN/ liter and 17% of samples) but did not vary with depth. Representative isolates (n ‫؍‬ 185) from different ponds, sample types, and seasons were examined for resistance to 15 different antibiotics; most strains were resistant to streptomycin (98.9%), while 20% were multidrug resistant (MDR) for 2 to 6 antibiotics. DiversiLab repetitive extragenic palindromic-element sequence-based PCR (rep-PCR) revealed genetic diversity and showed 43 genotypes among 191 isolates, as defined by >95% similarity. The genotypes did not partition by pond, season, or sample type. Genetic similarity to known serotypes indicated Hadar, Montevideo, and Newport as the most prevalent. All ponds achieved the current safety standards for generic Escherichia coli in agricultural water, and regression modeling showed that the E. coli level was a significant predictor for the probability of Salmonella occurrence. However, persistent populations of Salmonella were widely distributed in irrigation ponds, and the associated risks for produce contamination and subsequent human exposure are unknown, supporting continued surveillance of this pathogen in agricultural settings.

A study of prevalence, diversity, and antimicrobial resistance of Salmonella enterica in surface water in the southeastern United States was conducted. A new scheme was developed for recovery of Salmonella from irrigation pond water and compared with the FDA's Bacteriological Analytical Manual (8th ed., 2014) (BAM) method. Fifty-one isolates were recovered from 10 irrigation ponds in produce farms over a 2-year period; nine Salmonella serovars were identified by pulsed-field gel electrophoresis analysis, and the major serovar was Salmonella enterica serovar Newport (S. Newport, n ‫؍‬ 29), followed by S. enterica serovar Enteritidis (n ‫؍‬ 6), S. enterica serovar Muenchen (n ‫؍‬ 4), S. enterica serovar Javiana (n ‫؍‬ 3), S. enterica serovar Thompson (n ‫؍‬ 2), and other serovars. It is noteworthy that the PulseNet patterns of some of the isolates were identical to those of the strains that were associated with the S. Thompson outbreaks in 2010, 2012, and 2013, S. Enteritidis outbreaks in 2011 and 2013, and an S. Javiana outbreak in 2012. Antimicrobial susceptibility testing confirmed 16 S. Newport isolates of the multidrug resistant-AmpC (MDR-AmpC) phenotype, which exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT), and to the 1st, 2nd, and 3rd generations of cephalosporins (cephalothin, amoxicillin-clavulanic acid, and ceftriaxone). Moreover, the S. Newport MDR-AmpC isolates had a PFGE pattern indistinguishable from the patterns of the isolates from clinical settings. These findings suggest that the irrigation water may be a potential source of contamination of Salmonella in fresh produce. The new Salmonella isolation scheme significantly increased recovery efficiency from 21.2 (36/170) to 29.4% (50/170) (P ‫؍‬ 0.0002) and streamlined the turnaround time from 5 to 9 days with the BAM method to 4 days and thus may facilitate microbiological analysis of environmental water.

Vibrio vulnificus causes rare but frequently fatal septicemia associated with raw oyster consumption by persons with underlying hepatic or immune system dysfunction. The virulence potential of environmental reservoirs appears widely distributed, because most strains are virulent in animal models; however, several investigations recently demonstrated genetic divergence among strains from clinical versus environmental origin at independent genetic loci. The present study used PCR to screen DNA polymorphisms in strains from environmental (n ‫؍‬ 35) or clinical (n ‫؍‬ 33) sources, and genomic relationships were determined by repetitive extragenic palindromic DNA PCR (rep-PCR) typing. Significant (P < 0.01) association was observed for typical "clinical" or "environmental" polymorphism profiles based on strain origin. Most oyster isolates (88%), including all of those with the "environmental" profile, also formed a single rep-PCR genogroup. Clinical isolates within this group did not have the typical "clinical" profile. On the other hand, clinical isolates with the typical polymorphism profile were distributed among multiple rep-PCR genogroups, demonstrating greater genetic diversity than was evident by profiling genetic polymorphisms. Wound isolates were genetically distinct from typical blood isolates by all assays. Strains from an outbreak of wound infections in Israel (biotype 3) were closely related to several U.S. strains by rep-PCR, indicating potential reservoirs of emerging disease. Strains genetically related to blood isolates appeared to be relatively rare in oysters, as only one had the "clinical" polymorphism profile or clustered by rep-PCR. However, this study was not an extensive survey, and more sampling using rep-PCR for sensitive genetic discrimination is needed to determine the virulence potential of environmental reservoirs.Vibrio vulnificus is associated with serious wound infections or frequently fatal (mortality rates are generally Ͼ50%) septicemia related to consumption of raw shellfish, particularly oysters (5, 37). The bacterium is indigenous to temperate estuaries, and prevalence in oysters and seawater approaches 100% during warmer months (25,45). Most strains isolated from environmental reservoirs appear to be as virulent as clinical strains in animal models (13,35,36,39). Also, multiple virulence factors have been proposed for V. vulnificus but are generally present in most strains and do not provide predictive value (16,36,39,50). Virulent strains are distinguished by opaque colony morphology (34, 49), which reflects expression of a protective capsular polysaccharide (CPS); however, both clinical and environmental strains are generally encapsulated (45). Thus, appropriate markers to screen the virulence potential of V. vulnificus in environmental reservoirs are not available.Recently, DNA sequence polymorphisms at individual loci discriminated isolates from clinical versus oyster origin in several independent studies. Polymorphic variants generally included two genotypes, such as types ...