Excision dynamics of <Emphasis Type="Italic">Vibrio</Emphasis> pathogenicity island-2 from <Emphasis Type="Italic">Vibrio cholerae:</Emphasis> role of a recombination directionality factor VefA

Background  

Vibrio Pathogenicity Island-2 (VPI-2) is a 57 kb region present in choleragenic V. cholerae isolates that is required for growth on sialic acid as a sole carbon source. V. cholerae non-O1/O139 pathogenic strains also contain VPI-2, which in addition to sialic acid catabolism genes also encodes a type 3 secretion system in these strains. VPI-2 integrates into chromosome 1 at a tRNA-serine site and encodes an integrase intV2 (VC1758) that belongs to the tyrosine recombinase family. IntV2 is required for VPI-2 excision from chromosome 1, which occurs at very low levels, and formation of a non-replicative circular intermediate.     

Genomic and Phenotypic Characterization of Vibrio cholerae Non-O1 Isolates from a US Gulf Coast Cholera Outbreak

Between November 2010, and May 2011, eleven cases of cholera, unrelated to a concurrent outbreak on the island of Hispaniola, were recorded, and the causative agent, Vibrio cholerae serogroup O75, was traced to oysters harvested from Apalachicola Bay, Florida. From the 11 diagnosed cases, eight isolates of V. cholerae were isolated and their genomes were sequenced. Genomic analysis demonstrated the presence of a suite of mobile elements previously shown to be involved in the disease process of cholera (ctxAB, VPI-1 and -2, and a VSP-II like variant) and a phylogenomic analysis showed the isolates to be sister taxa to toxigenic V. cholerae V51 serogroup O141, a clinical strain isolated 23 years earlier. Toxigenic V. cholerae O75 has been repeatedly isolated from clinical cases in the southeastern United States and toxigenic V. cholerae O141 isolates have been isolated globally from clinical cases over several decades. Comparative genomics, phenotypic analyses, and a Caenorhabditis elegans model of infection for the isolates were conducted. This analysis coupled with isolation data of V. cholerae O75 and O141 suggests these strains may represent an underappreciated clade of cholera-causing strains responsible for significant disease burden globally.     

Genetic characterization of toxigenic Vibrio cholerae non-O1/non-O139 strains, isolated in the Middle Asia

Here, we report on the characterization of 22 clinical toxigenic V. cholerae non-O1/non-O139 strains isolated in the Middle Asia (Uzbekistan) in 1971–1990. PCR analysis has revealed that these strains contain the main virulence genes such as ctxA, zot, ace (CTXφ); rstC (RS1φ); tcpA, toxT, aldA (pathogenicity island VPI), but they lack both pandemic islands VSP-I and VSP-II specific to epidemic strains of O1 serogroup of El Tor biotype and O139 serogroup. Only two of the twenty two toxigenic strains have tcpA gene of El Tor type, one strain has tcpA gene of classical type, while nineteen other strains carry a new variant of this gene, designated as tcpA ^uzb. Nucleotide sequences analysis of virulence genes in toxigenic V. cholerae non-O1/non-O139 strains from Uzbekistan showed that they differ significantly from the sequences of these genes in epidemic O1 and O139 strain indicating that they belong to a separate line of evolution of virulent V. cholerae strains. For the first time it is shown that V. cholerae non-O1/non-O139 toxigenic strains of different serogroups may belong to the same clone.     

Rapid detection by multiplex PCR of Genomic Islands, prophages and Integrative Conjugative Elements in V. cholerae 7th pandemic variants

Vibrio cholerae poses a threat to human health, and new epidemic variants have been reported so far. Seventh pandemic V. cholerae strains are characterized by highly related genomic sequences but can be discriminated by a large set of Genomic Islands, phages and Integrative Conjugative Elements. Classical serotyping and biotyping methods do not easily discriminate among new variants arising worldwide, therefore the establishment of new methods for their identification is required. We developed a multiplex PCR assay for the rapid detection of the major 7th pandemic variants of V. cholerae O1 and O139. Three specific genomic islands (GI-12, GI-14 and GI-15), two phages (Kappa and TLC), Vibrio Seventh Pandemic Island 2 (VSP-II), and the ICEs of the SXT/R391 family were selected as targets of our multiplex PCR based on a comparative genomic approach. The optimization and specificity of the multiplex PCR was assessed on 5 V. cholerae 7th pandemic reference strains, and other 34 V. cholerae strains from various epidemic events were analyzed to validate the reliability of our method. This assay had sufficient specificity to identify twelve different V. cholerae genetic profiles, and therefore has the potential to be used as a rapid screening method.     

Conversion of viable but nonculturable Vibrio cholerae to the culturable state by co‐culture with eukaryotic cells

VBNC Vibrio cholerae O139 VC‐280 obtained by incubation in 1% solution of artificial sea water IO at 4°C for 74 days converted to the culturable state when co‐cultured with CHO cells. Other eukaryotic cell lines, including HT‐29, Caco‐2, T84, HeLa, and Intestine 407, also supported conversion of VBNC cells to the culturable state. Conversion of VBNC V. cholerae O1 N16961 and V. cholerae O139 VC‐280/pG13 to the culturable state, under the same conditions, was also confirmed. When VBNC V. cholerae O139 VC‐280 was incubated in 1% IO at 4°C for up to 91 days, the number of cells converted by co‐culture with CHO cells declined with each additional day of incubation and after 91 days conversion was not observed.     

Vibrio vulnificus Phage PV94 Is Closely Related to Temperate Phages of V. cholerae and Other Vibrio Species

Background

Vibrio vulnificus is an important pathogen which can cause serious infections in humans. Yet, there is limited knowledge on its virulence factors and the question whether temperate phages might be involved in pathogenicity, as is the case with V. cholerae. Thus far, only two phages (SSP002 and VvAW1) infecting V. vulnificus have been genetically characterized. These phages were isolated from the environment and are not related to Vibrio cholerae phages. The lack of information on temperate V. vulnificus phages prompted us to isolate those phages from lysogenic strains and to compare them with phages of other Vibrio species.

Results

In this study the temperate phage PV94 was isolated from a V. vulnificus biotype 1 strain by mitomycin C induction. PV94 is a myovirus whose genome is a linear double-stranded DNA of 33,828 bp with 5′-protruding ends. Sequence analysis of PV94 revealed a modular organization of the genome. The left half of the genome comprising the immunity region and genes for the integrase, terminase and replication proteins shows similarites to V. cholerae kappa phages whereas the right half containing genes for structural proteins is closely related to a prophage residing in V. furnissii NCTC 11218.

Conclusion

We present the first genomic sequence of a temperate phage isolated from a human V. vulnificus isolate. The sequence analysis of the PV94 genome demonstrates the wide distribution of closely related prophages in various Vibrio species. Moreover, the mosaicism of the PV94 genome indicates a high degree of horizontal genetic exchange within the genus Vibrio, by which V. vulnificus might acquire virulence-associated genes from other species.     

Activation of Cholera Toxin Production by Anaerobic Respiration of Trimethylamine N-oxide in Vibrio cholerae

Vibrio cholerae is a Gram-negative bacterium that causes cholera. Although the pathogenesis caused by this deadly pathogen takes place in the intestine, commonly thought to be anaerobic, anaerobiosis-induced virulence regulations are not fully elucidated. Anerobic growth of the V. cholerae strain, N16961, was promoted when trimethylamine N-oxide (TMAO) was used as an alternative electron acceptor. Strikingly, cholera toxin (CT) production was markedly induced during anaerobic TMAO respiration. N16961 mutants unable to metabolize TMAO were incapable of producing CT, suggesting a mechanistic link between anaerobic TMAO respiration and CT production. TMAO reductase is transported to the periplasm via the twin arginine transport (TAT) system. A similar defect in both anaerobic TMAO respiration and CT production was also observed in a N16961 TAT mutant. In contrast, the abilities to grow on TMAO and to produce CT were not affected in a mutant of the general secretion pathway. This suggests that V. cholerae may utilize the TAT system to secrete CT during TMAO respiration. During anaerobic growth with TMAO, N16961 cells exhibit green fluorescence when stained with 2′,7′-dichlorofluorescein diacetate, a specific dye for reactive oxygen species (ROS). Furthermore, CT production was decreased in the presence of an ROS scavenger suggesting a positive role of ROS in regulating CT production. When TMAO was co-administered to infant mice infected with N16961, the mice exhibited more severe pathogenic symptoms. Together, our results reveal a novel anaerobic growth condition that stimulates V. cholerae to produce its major virulence factor.     

Distribution of Virulence-Associated Genes and Genetic Relationships in Non-O1/O139 Vibrio cholerae Aquatic Isolates from China

Non-O1/O139 Vibrio cholerae is naturally present in aquatic ecosystems and has been linked with cholera-like diarrhea and local outbreaks. The distribution of virulence-associated genes and genetic relationships among aquatic isolates from China are largely unknown. In this study, 295 aquatic isolates of V. cholerae non-O1/O139 serogroups from different regions in China were investigated. Only one isolate was positive for ctxB and harbored a rare genotype; 10 (3.4%) isolates carried several types of rstR sequences, eight of which carried rare types of toxin-coregulated pili (tcpA). Furthermore, 16 (5.4%) isolates carried incomplete (with partial open reading frames [ORFs]) vibrio seventh pandemic island I (VSP-I) or VSP-II clusters, which were further classified as 11 novel types. PCR-based analyses revealed remarkable variations in the distribution of putative virulence genes, including mshA (95.6%), hlyA (95.3%), rtxC (89.8%), rtxA (82.7%), IS1004 (52.9%), chxA (30.2%), SXT (15.3%), type III secretion system (18.0%), and NAG-ST (3.7%) genes. There was no correlation between the prevalence of putative virulence genes and that of CTX prophage or TCP genes, whereas there were correlations among the putative virulence genes. Further multilocus sequence typing (MLST) placed selected isolates (n = 70) into 69 unique sequence types (STs), which were different from those of the toxigenic O1 and O139 counterparts, and each isolate occupied a different position in the MLST tree. The V. cholerae non-O1/O139 aquatic isolates predominant in China have high genotypic diversity; these strains constitute a reservoir of potential virulence genes, which may contribute to evolution of pathogenic isolates.     

Isolation ofVibrio parahaemolyticus andVibrio cholerae (Non-O1 and O139) from moribund shrimp (Penaeus monodon) and experimental challenge study against post larvae and juveniles

Outbreak ofVibrio infection was reported from a shrimp farm near Chennai, south India. Both green and yellowVibrio were isolated from disease outbreak ofPenaeus monodon (Milne Edwards) culture farm and biochemically confirmed asVibrio parahaemolyticus andVibrio cholerae. Randomly selectedV. parahaemolyticus (VP1) andV. cholerae (VC1) were reconfirmed by partial 16S rRNA gene analysis. Both were tested against post larvae and juveniles ofP. monodon by bath challenge test and intramuscular injection respectively. The study revealed that VP1 is more virulent than VC1 isolated from same source againstP. monodon post larvae and juveniles. Phylogenetic tree based on comparison of partial 16S rRNA gene analysis revealed close relationship of VP1 with other shrimp pathogens likeVibrio harveyi andVibrio alginolyticus. There might be some close relationship for disease development among all these strains.     

Antimicrobial activity of some sulfonamide derivatives on clinical isolates of Staphylococus aureus

Background

Human infections with non-O1, non-O139 V. cholerae have been described from Laos. Elsewhere, non cholera-toxin producing, non-O1, non-O139 V. cholerae have been described from blood cultures and ascitic fluid, although they are exceedingly rare isolates.

Case presentation

We describe a farmer who died with Vibrio cholerae O21 bacteremia and peritonitis in Vientiane, Laos, after eating partially cooked apple snails (Pomacea canaliculata) and mussels (Ligumia species). The cultured V. cholerae were non-motile. PCR detected ompW and toxR gene regions but not the ctxA, ompU, omp K and TCP gene regions. Although the organisms lacked flagellae on scanning electron microscopy, they possessed the Vibrio flagellin flaA gene.

Conclusion

Severe bacteremic non-O1, non-O139 V. cholerae is reported from Laos. The organisms were unusual in being non-motile. They possessed the Vibrio flagellin flaA gene. Further research to determine the reasons for the non-motility and virulence is required.     

Alkaline protease from a non-toxigenic mangrove isolate of Vibrio sp. V26 with potential application in animal cell culture

Vibrio sp. V26 isolated from mangrove sediment showed 98 % similarity to 16S rRNA gene of Vibrio cholerae, V. mimicus, V. albensis and uncultured clones of Vibrio. Phenotypically also it resembled both V. cholerae and V. mimicus. Serogrouping, virulence associated gene profiling, hydrophobicity, and adherence pattern clearly pointed towards the non—toxigenic nature of Vibrio sp. V26. Purification and characterization of the enzyme revealed that it was moderately thermoactive, nonhemagglutinating alkaline metalloprotease with a molecular mass of 32 kDa. The application of alkaline protease from Vibrio sp. V26 (APV26) in sub culturing cell lines (HEp-2, HeLa and RTG-2) and dissociation of animal tissue (chick embryo) for primary cell culture were investigated. The time required for dissociation of cells as well as the viable cell yield obtained by while administering APV26 and trypsin were compared. Investigations revealed that the alkaline protease of Vibrio sp. V26 has the potential to be used in animal cell culture for subculturing cell lines and dissociation of animal tissue for the development of primary cell cultures, which has not been reported earlier among metalloproteases of Vibrios.     

Genotypic and PFGE/MLVA analyses of Vibrio cholerae O1: geographical spread and temporal changes during the 2007-2010 cholera outbreaks in Thailand

Background

Vibrio cholerae O1 El Tor dominated the seventh cholera pandemic which occurred in the 1960s. For two decades, variants of V. cholerae O1 El Tor that produce classical cholera toxin have emerged and spread globally, replacing the prototypic El Tor biotype. This study aims to characterize V. cholerae O1 isolates from outbreaks in Thailand with special reference to genotypic variations over time.

Methods/Findings

A total of 343 isolates of V. cholerae O1 from cholera outbreaks from 2007 to 2010 were investigated, and 99.4% were found to carry the classical cholera toxin B subunit (ctxB) and El Tor rstR genes. Pulsed-field gel electrophoresis (PFGE) differentiated the isolates into 10 distinct pulsotypes, clustered into two major groups, A and B, with an overall similarity of 88%. Ribotyping, multiple-locus variable-number tandem-repeat analysis (MLVA), and PCR to detect Vibrio seventh pandemic island II (VSP-II) related genes of randomly selected isolates from each pulsotype corresponded to the results obtained by PFGE. Epidemiological investigations revealed that MLVA type 2 was strongly associated with a cholera outbreak in northeastern Thailand in 2007, while MLVA type 7 dominated the outbreaks of the southern Gulf areas in 2009 and MLVA type 4 dominated the outbreaks of the central Gulf areas during 2009–2010. Only MLVA type 16 isolates were found in a Thai-Myanmar border area in 2010, whereas those of MLVA types 26, 39, and 41 predominated this border area in 2008. Type 39 then disappeared 1–2 years later as MLVA type 41 became prevalent. Type 41 was also found to infect an outbreak area.

Conclusions

MLVA provided a high-throughput genetic typing tool for understanding the in-depth epidemiology of cholera outbreaks. Our epidemiological surveys suggest that some clones of V. cholerae O1 with similar but distinctive genetic traits circulate in outbreak sites, while others disappear over time.     

Transcriptomic profiling of the oyster pathogen Vibrio splendidus opens a window on the evolutionary dynamics of the small RNA repertoire in the Vibrio genus

Work in recent years has led to the recognition of the importance of small regulatory RNAs (sRNAs) in bacterial regulation networks. New high-throughput sequencing technologies are paving the way to the exploration of an expanding sRNA world in nonmodel bacteria. In the Vibrio genus, compared to the enterobacteriaceae, still a limited number of sRNAs have been characterized, mostly in Vibrio cholerae, where they have been shown to be important for virulence, as well as in Vibrio harveyi. In addition, genome-wide approaches in V. cholerae have led to the discovery of hundreds of potential new sRNAs. Vibrio splendidus is an oyster pathogen that has been recently associated with massive mortality episodes in the French oyster growing industry. Here, we report the first RNA-seq study in a Vibrio outside of the V. cholerae species. We have uncovered hundreds of candidate regulatory RNAs, be it cis-regulatory elements, antisense RNAs, and trans-encoded sRNAs. Conservation studies showed the majority of them to be specific to V. splendidus. However, several novel sRNAs, previously unidentified, are also present in V. cholerae. Finally, we identified 28 trans sRNAs that are conserved in all the Vibrio genus species for which a complete genome sequence is available, possibly forming a Vibrio “sRNA core.”     

Isolation of Non-O1 Vibrio cholerae Serovars from Oregon Coastal Environments

Water, sediment, and shellfish from three Oregon estuaries were cultured for pathogenic Vibrio species. Non-O1 serovars of V. cholerae were the most common pathogenic Vibrio species recovered. Non-O1 V. cholerae were isolated from all three estuaries sampled, covering an area of about 170 miles along the Oregon coast. Non-O1 V. cholerae were isolated from water and sediment, but not shellfish, at temperatures ranging from 11 to 19°C and salinities of 2.3 to 26‰. Sixteen isolates representing 12 different non-O1 serovars were identified, while four non-O1 V. cholerae isolates failed to react with any of the 54 antisera tested. These results indicate that non-O1 V. cholerae serovars can be found over a large geographic area and under a variety of environmental conditions. These organisms are apparently an autochthonous component of these estuarine microbial communities.     

Clinical Isolates of Vibrio cholerae O1 El Tor Ogawa of 2009 from Kolkata,India: Preponderance of SXT Element and Presence of Haitian ctxB Variant

Background

Increase in the number of multidrug resistant pathogens and the accompanied rise in case fatality rates has hampered the treatment of many infectious diseases including cholera. Unraveling the mechanisms responsible for multidrug resistance in the clinical isolates of Vibrio cholerae would help in understanding evolution of these pathogenic bacteria and their epidemic potential. This study was carried out to identify genetic factors responsible for multiple drug resistance in clinical isolates of Vibrio cholerae O1, serotype Ogawa, biotype El Tor isolated from the patients admitted to the Infectious Diseases Hospital, Kolkata, India, in 2009.

Methodology/Principal Findings

One hundred and nineteen clinical isolates of V. cholerae were analysed for their antibiotic resistance phenotypes. Antibiogram analysis revealed that majority of the isolates showed resistance to co-trimoxazole, nalidixic acid, polymixin B and streptomycin. In PCR, SXT integrase was detected in 117 isolates and its sequence showed 99% identity notably to ICEVchInd5 from Sevagram, India, ICEVchBan5 from Bangladesh and VC1786ICE sequence from Haiti outbreak among others. Antibiotic resistance traits corresponding to SXT element were transferred from the parent Vibrio isolate to the recipient E. coli XL-1 Blue cells during conjugation. Double-mismatch-amplification mutation assay (DMAMA) revealed the presence of Haitian type ctxB allele of genotype 7 in 55 isolates and the classical ctxB allele of genotype 1 in 59 isolates. Analysis of topoisomerase sequences revealed the presence of mutation Ser83 → Ile in gyrA and Ser85→ Leu in parC. This clearly showed the circulation of SXT-containing V. cholerae as causative agent for cholera in Kolkata.

Conclusions

There was predominance of SXT element in these clinical isolates from Kolkata region which also accounted for their antibiotic resistance phenotype typical of this element. DMAMA PCR showed them to be a mixture of isolates with different ctxB alleles like classical, El Tor and Haitian variants.     

Dynamics of Vibrio with Virulence Genes Detected in Pacific Harbor Seals (Phoca vitulina richardii) Off California: Implications for Marine Mammal Health

Given their coastal site fidelity and opportunistic foraging behavior, harbor seals (Phoca vitulina) may serve as sentinels for coastal ecosystem health. Seals using urbanized coastal habitat can acquire enteric bacteria, including Vibrio that may affect their health. To understand Vibrio dynamics in seals, demographic and environmental factors were tested for predicting potentially virulent Vibrio in free-ranging and stranded Pacific harbor seals (Phoca vitulina richardii) off California. Vibrio prevalence did not vary with season and was greater in free-ranging seals (29 %, n?=?319) compared with stranded seals (17 %, n?=?189). Of the factors tested, location, turbidity, and/or salinity best predicted Vibrio prevalence in free-ranging seals. The relationship of environmental factors with Vibrio prevalence differed by location and may be related to oceanographic or terrestrial contributions to water quality. Vibrio parahaemolyticus, Vibrio alginolyticus, and Vibrio cholerae were observed in seals, with V. cholerae found almost exclusively in stranded pups and yearlings. Additionally, virulence genes (trh and tdh) were detected in V. parahaemolyticus isolates. Vibrio cholerae isolates lacked targeted virulence genes, but were hemolytic. Three out of four stranded pups with V. parahaemolyticus (trh+ and/or tdh+) died in rehabilitation, but the role of Vibrio in causing mortality is unclear, and Vibrio expression of virulence genes should be investigated. Considering that humans share the environment and food resources with seals, potentially virulent Vibrio observed in seals also may be of concern to human health.     

Isolation and Characterization of the New Mosaic Filamentous Phage VFJ Φ of Vibrio cholerae

Filamentous phages have distinguished roles in conferring many pathogenicity and survival related features to Gram-negative bacteria including the medically important Vibrio cholerae, which carries factors such as cholera toxin on phages. A novel filamentous phage, designated VFJΦ, was isolated in this study from an ampicillin and kanamycin-resistant O139 serogroup V. cholerae strain ICDC-4470. The genome of VFJΦ is 8555 nucleotides long, including 12 predicted open reading frames (ORFs), which are organized in a modular structure. VFJΦ was found to be a mosaic of two groups of V. cholerae phages. A large part of the genome is highly similar to that of the fs2 phage, and the remaining 700 bp is homologous to VEJ and VCYΦ. This 700 bp region gave VFJΦ several characteristics that are not found in fs2 and other filamentous phages. In its native host ICDC-4470 and newly-infected strain N16961, VFJΦ was found to exist as a plasmid but did not integrate into the host chromosome. It showed a relatively wide host range but did not infect the classical biotype O1 V. cholerae strains. After infection, the host strains exhibited obvious inhibition of both growth and flagellum formation and had acquired a low level of ampicillin resistance and a high level of kanamycin resistance. The antibiotic resistances were not directly conferred to the hosts by phage-encoded genes and were not related to penicillinase. The discovery of VFJΦ updates our understanding of filamentous phages as well as the evolution and classification of V. cholerae filamentous phage, and the study provides new information on the interaction between phages and their host bacteria.     

An experimental study of the population and evolutionary dynamics of Vibrio cholerae O1 and the bacteriophage JSF4

Studies of Vibrio cholerae in the environment and infected patients suggest that the waning of cholera outbreaks is associated with rise in the density of lytic bacteriophage. In accordance with mathematical models, there are seemingly realistic conditions where phage predation could be responsible for declines in the incidence of cholera. Here, we present the results of experiments with the El Tor strain of V. cholerae (N16961) and a naturally occurring lytic phage (JSF4), exploring the validity of the main premise of this model: that phage predation limits the density of V. cholerae populations. At one level, the results of our experiments are inconsistent with this hypothesis. JSF4-resistant V. cholerae evolve within a short time following their confrontation with these viruses and their populations become limited by resources rather than phage predation. At a larger scale, however, the results of our experiments are not inconsistent with the hypothesis that bacteriophage modulate outbreaks of cholera. We postulate that the resistant bacteria that evolved play an insignificant role in the ecology or pathogenicity of V. cholerae. Relative to the phage-sensitive cells from whence they are derived, the evolved JSF4-resistant V. cholerae have fitness costs and other characters that are likely to impair their ability to compete with the sensitive cells in their natural habitat and may be avirulent in human hosts. The results of this in vitro study make predictions that can be tested in natural populations of V. cholerae and cholera-infected patients.     

Application of Chitosan Microparticles for Reduction of Vibrio Species in Seawater and Live Oysters (Crassostrea virginica)

Human Vibrio infections associated with consumption of raw shellfish greatly impact the seafood industry. Vibrio cholerae-related disease is occasionally attributed to seafood, but V. vulnificus and V. parahaemolyticus are the primary targets of postharvest processing (PHP) efforts in the United States, as they pose the greatest threat to the industry. Most successful PHP treatments for Vibrio reduction also kill the molluscs and are not suitable for the lucrative half-shell market, while nonlethal practices are generally less effective. Therefore, novel intervention strategies for Vibrio reduction are needed for live oyster products. Chitosan is a bioactive derivative of chitin that is generally recognized as safe as a food additive by the FDA, and chitosan microparticles (CMs) were investigated in the present study as a potential PHP treatment for live oyster applications. Treatment of broth cultures with 0.5% (wt/vol) CMs resulted in growth cessation of V. cholerae, V. vulnificus, and V. parahaemolyticus, reducing culturable levels to nondetectable amounts after 3 h in three independent experiments. Furthermore, a similar treatment in artificial seawater at 4, 25, and 37°C reduced V. vulnificus levels by ca. 7 log CFU/ml after 24 h of exposure, but 48 h of exposure and elevated temperature were required to achieve similar results for V. parahaemolyticus and V. cholerae. Live oysters that either were artificially inoculated or contained natural populations of V. vulnificus and V. parahaemolyticus showed significant and consistent reductions following CM treatment (5%) compared to the amounts in the untreated controls. Thus, the results strongly support the promising potential for the application of CMs as a PHP treatment to reduce Vibrio spp. in intact live oysters.