A novel multiplex PCR for the identification of Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus

AIM: To establish a simple multiplex polymerase chain reaction (PCR) that will identify Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus. METHODS AND RESULTS: A total of 429 Vibrio spp. from various origins were tested with the novel primers targeting toxR. The reverse primers were all designed to be species specific, while the forward primer was universal. The primers correctly identified all the V. parahaemolyticus, V. cholerae and V. vulnificus isolates tested. CONCLUSIONS: The toxR multiplex PCR works well when the initial colony morphology is known. If not, Vibrio alginolyticus might represent a diagnostic obstacle. SIGNIFICANCE AND IMPACT OF THE STUDY: The method provides a fast and reliable way of identifying the main Vibrio spp. involved in food-borne disease. The method could prove very useful for laboratories working with identification of these Vibrio spp.     

Temperature effects on the viable but non-culturable state of Vibrio vulnificus

Abstract The non-culturable state of Vibrio vulnificus , strain C7184, was studied in artificial seawater microcosms held at 5, 10, 15, 20, and 30°C. Plate counts were made on a non-selective medium, total cell counts were monitored by acridine orange epifluorescence, and direct viable counts (DVSs) by the method of Kogure et al. (Can J. Microbiol. 25, 415–420; 1986) and by the INT method. From an initial inoculum of 10⁷ cells/ml, V. vulnificus became non-culturable within 40 days at 5°C, although both indicators of viability revealed a viable population exceeding 10⁶ cells/ml. Cells at all higher temperatures remained culturable (at least 10⁴/ml) throughout the study. The non-culturable states of the opaque and translucent colony variants of V. vulnificus , as well as those of six other clinical and environmental strains of V. vulnificus , were examined at 5°C; all but one strain and both colony variants also became non-culturable within 40 days. In contrast, six other Vibrio spp. ( V. cholerae, V. mimicus, V. parahaemolyticus, V. natriegens, V. proteolyticus , and V. campbelli ) remained culturable at 5°C. Thus, entrance of V. vulnificus into the non-culturable state appears to be highly temperature dependent and, among the vibrios, this species may be especially sensitive to low temperature. The public health aspects of these findings are discussed.     

Isolation and preliminary characterization of bacteriocins produced by Vibrio vulnificus

AIMS: The aim of this work was to isolate bacteriocins from the environment that would be effective in neutralizing Vibrio vulnificus in seafood. METHODS AND RESULTS: Water samples from Wilmington (NC, USA) were plated to determine total viable counts and to isolate presumptive Vibrio spp. Isolates containing plasmids were checked for antimicrobial activity which was not due to lytic bacteriophage or small, non-specific molecules. Three bacteriocin producers were detected and their inhibitory spectra determined: IW1 inhibited few strains of V. vulnificus; BC1 inhibited several strains of V. vulnificus, V. cholerae and V. parahaemolyticus and BC2 inhibited all tested Vibrio spp., Plesiomonas shigelloides and Escherichia coli. Loss of inhibitory activity coincided with loss of the bacteriocinogenic plasmid. The bacteriocins were found to be between 1.3 and 9.0 kDa. IW1 was heat labile, while BC1 was moderately stable except at extreme temperatures. BC2 was very stable and maintained its activity when frozen, autoclaved or exposed to extreme pH values. CONCLUSIONS: Bacteriocins have been isolated from environmental isolates of V. vulnificus and V. cholerae. BC2, with its broad spectrum and stability, may be useful in neutralizing V. vulnificus. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have significance in relation to reducing the occurrence of food poisoning caused by V. vulnificus.     

The ability of two different Vibrio spp. bacteriophages to infect Vibrio harveyi, Vibrio cholerae and Vibrio mimicus

AIMS: To determine the host range of the Vibrio harveyi myovirus-like bacteriophage (VHML) and the cholera toxin conversion bacteriophage (CTX Phi) within a range of Vibrio cholerae and V. mimicus and V. harveyi, V. cholerae and V. mimicus isolates respectively. METHODS AND RESULTS: Three V. harveyi, eight V. cholerae and five V. mimicus isolates were incubated with VHML and CTX Phi. Polymerase chain reaction (PCR) was used to determine the presence of VHML and CTX Phi in infected isolates. We demonstrated that it was possible to infect one isolate of V. cholerae (isolate ACM #2773/ATCC #14035) with VHML. This isolate successfully incorporated VHML into its genome as evident by positive PCR amplification of the sequence coding part of the tail sheath of VHML. Attempts to infect all other V. cholerae and V. mimicus isolates with VHML were unsuccessful. Attempts to infect V. cholerae non-01, V. harveyi and V. mimicus isolates with CTX Phi were unsuccessful. CONCLUSIONS: Bacteriophage infection is limited by bacteriophage-exclusion systems operating within bacterial strains and these systems appear to be highly selective. One system may allow the co-existence of one bacteriophage while excluding another. VHML appears to have a narrow host range which may be related to a common receptor protein in such strains. The lack of the vibrio pathogenicity island bacteriophage (VPI Phi) in the isolates used in this study may explain why infections with CTX Phi were unsuccessful. SIGNIFICANCE AND IMPACT OF THE STUDY: The current study has demonstrated that Vibrio spp. bacteriophages may infect other Vibrio spp.     

Cyclo(Phe-Pro) modulates the expression of ompU in Vibrio spp

Vibrio vulnificus was found to produce a chemical that induced the expression of Vibrio fischeri lux genes. Electron spray ionization-mass spectrometry and 1H nuclear magnetic resonance analyses indicated that the compound was cyclo(L-Phe-L-Pro) (cFP). The compound was produced at a maximal level when cell cultures reached the onset of stationary phase. Sodium dodecyl sulfate-polyacrylamide gel analysis of the total proteins of V. vulnificus indicated that expression of OmpU was enhanced by exogenously added synthetic or purified cFP. A toxR-null mutant failed to express ompU despite the addition of cFP. The related Vibrio spp. V. cholerae, V. parahaemolyticus, and V. harveyi also produced cFP, which induced the expression of their own ompU genes. cFP also enhanced the expression in V. cholerae of the ctx genes, which are known to be regulated by ToxR. Our results suggest that cFP is a signal molecule controlling the expression of genes important for the pathogenicity of Vibrio spp.     

Occurrence and potential pathogenesis of Vibrio cholerae, Vibrio parahaemolyticus and Vibrio vulnificus on the South Coast of Sweden

During the summer of 2006, several wound infections - of which three were fatal - caused by Vibrio cholerae were reported from patients who had been exposed to water from the Baltic Sea. Before these reports, we initiated a sampling project investigating the occurrence of potential human pathogenic V. cholerae, Vibrio vulnificus and Vibrio parahaemolyticus in The Sound between Sweden and Denmark. The Blue mussel (Mytilus edulis) was used as an indicator to follow the occurrence of vibrios over time. Molecular analyses showed high frequencies of the most potent human pathogenic Vibrio spp.; 53% of mussel samples were positive for V. cholerae (although none were positive for the cholera toxin gene), 63% for V. vulnificus and 79% for V. parahaemolyticus (of which 47% were tdh(+) and/or trh(+)). Viable vibrios were also isolated from the mussel meat and screened for virulence by PCR. The mortality of eukaryotic cells when exposed to bacteria was tested in vivo, with results showing that the Vibrio strains, independent of species and origin, were harmful to the cells. Despite severe infections and several deaths, no report on potential human pathogenic vibrios in this area had been published before this study.     

Occurrence, seasonality and genetic diversity of Vibrio vulnificus in coastal seaweeds and water along the Kii Channel, Japan

Vibrio vulnificus is a ubiquitous toxigenic bacterium found in a coastal environment but little is known about its occurrence and seasonality among seaweeds, which are widely consumed as seafood in Japan. Therefore, we have observed the bacterium's abundance in seawater and seaweed samples from three areas of the Kii Channel, Japan, during June 2003 to May 2004. A total of 192 samples were collected: 24 from each source in summer, autumn, winter and spring. The samples were selectively cultivated following the most probable number (MPN) technique. Vibrio vulnificus population ranged from 0 to 10(3) MPN 100 mL(-1) seawater or 10 g seaweeds; higher counts were observed during summer. The optimum temperature, salinity and pH for the bacterium were 20-24 degrees C, 24-28 p.p.t. and 7.95-8.15, respectively. However, seaweeds always contained higher V. vulnificus than seawater. Among 280 V. vulnificus strains, detected by species-specific colony hybridization and PCR, 78, 74, 11 and 16 were from seaweeds and 46, 42, 2 and 11 were from seawater during summer, autumn, winter and spring, respectively. Ribotyping of 160 selected strains revealed a higher genotypic diversity (18 patterns) among strains from seaweeds than from seawater (10 patterns). Seaweeds can thus act as a potential habitat for V. vulnificus and are more unsafe for consumption during summer.     

Catecholamine-induced stimulation of growth in Vibrio species

AIMS: To evaluate the effect of norepinephrine (NE) and related compounds on the growth of bacteria, we have examined the effect of the neuroendocrine hormone NE and related compounds on the growth of Vibrio parahaemolyticus and other human-pathogenic Vibrio species (Vibrio cholerae, Vibrio vulnificus, and Vibrio mimicus). METHODS AND RESULTS: The effects on bacterial growth were examined using the serum-based medium and viable cells were counted using agar plates. We have shown that NE and its related compounds stimulate growth of V. parahaemolyticus in serum-based medium. This NE-induced growth stimulation was dependent upon the presence of transferrin. NE also stimulated growth of V. mimicus, but not V. cholerae and V. vulnificus. CONCLUSIONS: These results suggest that the Vibrio species differ in their ability to respond to NE. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible that NE and related compounds modulate the pathogenicity of V. parahaemolyticus and V. mimicus.     

A comparison of thiosulphate-citrate-bile salts-sucrose (TCBS) agar and thiosulphate-chloride-iodide (TCI) agar for the isolation of Vibrio species from estuarine environments

AIMS: The purpose of this study was to compare a recently described medium, thiosulphate-chloride-iodide (TCI), for the isolation of estuarine vibrios with thiosulphate-citrate-bile salts-sucrose (TCBS). METHODS: A total of 492 colonies which developed on these media from estuarine water samples taken monthly over a 10-month period were examined. RESULTS: A much larger number of colonies developed on TCBS than TCI, and minimal taxonomic criteria indicated that a higher percentage (61%) of TCBS colonies could be identified as Vibrio spp. when compared with TCI (46%). SIGNIFICANCE: This study suggests that TCBS is a superior medium when compared with TCI for the isolation of Vibrio spp. from estuarine waters. Because of the public health risk presented by V. vulnificus, V. parahaemolyticus, V. cholerae and other vibrios, the selection of the most appropriate medium for their isolation is extremely important.     

Environmental influences on Vibrio populations in northern temperate and boreal coastal waters (Baltic and Skagerrak Seas)

Even if many Vibrio spp. are endemic to coastal waters, their distribution in northern temperate and boreal waters is poorly studied. To identify environmental factors regulating Vibrio populations in a salinity gradient along the Swedish coastline, we combined Vibrio-specific quantitative competitive PCR with denaturant gradient gel electrophoresis-based genotyping. The total Vibrio abundance ranged from 4 x 10(3) to 9.6 x 10(4) cells liter(-1), with the highest abundances in the more saline waters of the Skagerrak Sea. Several Vibrio populations were present throughout the salinity gradient, with abundances of single populations ranging from 5 x 10(2) to 7 x 10(4) cells liter(-1). Clear differences were observed along the salinity gradient, where three populations dominated the more saline waters of the Skagerrak Sea and two populations containing mainly representatives of V. anguillarum and V. aestuarianus genotypes were abundant in the brackish waters of the Baltic Sea. Our results suggest that this apparent niche separation within the genus Vibrio may also be influenced by alternate factors such as nutrient levels and high abundances of dinoflagellates. A V. cholerae/V. mimicus population was detected in more than 50% of the samples, with abundances exceeding 10(3) cells liter(-1), even in the cold (annual average water temperature of around 5 degrees C) and low-salinity (2 to 4 per thousand) samples from the Bothnian Bay (latitude, 65 degrees N). The unsuspected and widespread occurrence of this population in temperate and boreal coastal waters suggests that potential Vibrio pathogens may also be endemic to cold and brackish waters and hence may represent a previously overlooked health hazard.     

Construction of cholera toxin B subunit-producing Vibrio cholerae strains using the Mariner-FRT transposon delivery system

The most widely used oral whole-cell-recombinant B subunit cholera vaccine contains the nontoxic cholera toxin B subunit (CTXB) and either heat- or formalin-killed Vibrio cholerae O1 strains. Vibrio cholerae O1 strains in the vaccine provide antibacterial immunity, and CTXB contributes to the vaccine's efficacy by stimulating production of anti-CTXB antibody. Various attempts have been made to increase CTXB production. In this study, the mariner-FRT transposon delivery system developed by Chiang and Mekalanos was used to place the ctxB gene under the control of a strong chromosomal promoter in a nontoxigenic V. cholerae El Tor strain, M7922. The expression level of CTXB in transposon insertion mutant clones was screened by ganglioside-dependent enzyme-linked immunosorbent assay. Among CTXB-producing V. cholerae clones that were isolated, M7922-C1 produced the highest amount of CTXB (3.17+/-1.69 microg mL(-1)). M7922-C1 harbors a single insertion of ctxB into VC0972, which encodes a putative porin protein. Although the level of CTXB expression in this strain was not exceptionally high, this study indicates the possibility of using this delivery system to construct vaccine strains that overexpress specific antigens.     

Sequence Analyses of Type IV Pili from Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus

Bacterial surface structures called pili have been studied extensively for their role as possible colonization factors. Most sequenced Vibrio genomes predict a variety of pili genes in these organisms, including several types of type IV pili. In particular, the mannose-sensitive hemagglutinin (MSHA) and the PilA pili, also known as the chitin-regulated pilus (ChiRP), are type IVa pili commonly found in Vibrio genomes and have been shown to play a role in the colonization of Vibrio species in the environment and/or host tissue. Here, we report sequence comparisons of two type IVa pilin subunit genes, mshA and pilA, and their corresponding amino acid sequences, for several strains from the three main human pathogenic Vibrio species, V. cholerae, V. parahaemolyticus, and V. vulnificus. We identified specific groupings of these two genes in V. cholerae, whereas V. parahaemolyticus and V. vulnificus strains had no apparent allelic clusters, and these genes were strikingly divergent. These results were compared with other genes from the MSHA and PilA operons as well as another Vibrio pili from the type IVb group, the toxin co-regulated pilus (TCP) from V. cholerae. Our data suggest that a selective pressure exists to cause these strains to vary their MSHA and PilA pilin subunits. Interestingly, V. cholerae strains possessing TCP have the same allele for both mshA and pilA. In contrast, V. cholerae isolates without TCP have polymorphisms in their mshA and pilA sequences similar to what was observed for both V. parahaemolyticus and V. vulnificus. This data suggests a possible linkage between host interactions and maintaining a highly conserved type IV pili sequence in V. cholerae. Although the mechanism underlying this intriguing diversity has yet to be elucidated, our analyses are an important first step towards gaining insights into the various aspects of Vibrio ecology.     

Rapid serological identification of Vibrio vulnificus by anti-H coagglutination

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.     

Detection and identification of Vibrio species using whole-cell protein pattern analysis

Outbreaks of foodborne diseases associated with Vibrio species such as V. parahaemolyticus, V. vulnificus, and V. cholerae frequently occur in countries having a dietary habit of raw seafood consumption. For rapid identification of different Vibrio species involved in foodborne diseases, whole-cell protein pattern analysis for 13 type strains of 12 Vibrio species was performed using SDS-PAGE analysis. Pathogenic Vibrio species such as V. parahaemolyticus, V. vulnificus, V. cholerae, V. alginolyticus, V. fluvialis, and V. mimicus were included in the 12 Vibrio species used in this study. Each of the 12 Vibrio species showed clearly specific band patterns of its own. Two different strains of V. parahaemolyticus showed two different SDS-PAGE wholecell protein patterns, giving the possibility of categorizing isolated strains in the same V. parahaemolyticus species into two subgroups. The 36 Vibrio isolates collected from sushi restaurants in Busan were all identified as V. parahaemolyticus by comparing their protein patterns with those of Vibrio type strains. The identified isolates were categorized into two different subgroups of V. parahaemolyticus. The whole-cell protein pattern analysis by SDS-PAGE can be used as a specific, rapid, and simple identification method for Vibrio spp. involved in foodborne diseases at the subspecies level.     

Temporal and spatial variability in culturable pathogenic Vibrio spp. in Lake Pontchartrain, Louisiana, following hurricanes Katrina and Rita

We investigated the abundance, distribution, and virulence gene content of Vibrio cholerae, V. parahaemolyticus, and V. vulnificus in the waters of southern Lake Pontchartrain in Louisiana on four occasions from October 2005 to September 2006, using selective cultivation and molecular assays. The three targeted pathogenic vibrios were generally below the detection level in January 2006, when the water was cold (13°C), and most abundant in September 2006, when the lake water was warmest (30°C). The maximum values for these species were higher than reported previously for the lake by severalfold to orders of magnitude. The only variable consistently correlated with total vibrio abundance within a single sampling was distance from shore (P = 0.000). Multiple linear regression of the entire data set revealed that distance from shore, temperature, and turbidity together explained 82.1% of the variability in total vibrio CFU. The log-transformed mean abundance of V. vulnificus CFU in the lake was significantly correlated with temperature (P = 0.014), but not salinity (P = 0.625). Virulence-associated genes of V. cholerae (ctx) and V. parahaemolyticus (trh and tdh) were not detected in any isolates of these species (n = 128 and n = 20, respectively). In contrast, 16S rRNA typing of V. vulnificus (n = 298) revealed the presence of both environmental (type A) and clinical (type B) strains. The percentage of the B-type V. vulnificus was significantly higher in the lake in October 2005 (35.8% of the total) than at other sampling times (P ≤ 0.004), consistent with the view that these strains represent distinct ecotypes.     

溶藻弧菌HY9901 AcrA基因克隆与序列分析

Touchdown PCR扩增溶藻弧菌HY9901 AcrA基因部分序列,得一460bp片段,再以反向PCR和巢式PCR联合扩增其侧翼序列,拼接得一由1101 nt组成,共编码366 aa的完整基因.该基因演绎的氨基酸序列与几种弧菌的同源性都比较高,与创伤弧菌YJ016、副溶血弧菌RIMD 2210633、灿烂弧菌12B01、霍乱弧菌O1 N16961同源性分别为76%、73%、71%和70%.     

Rapid serological identification of Vibrio vulnificus by anti-H coagglutination.

Staphylococcus aureus Cowan 1 cells were armed with anti-flagellar (anti-H) antibody produced in rabbits immunized with flagellar core protein prepared from Vibrio vulnificus. This reagent was assessed by coagglutination for its capacity to agglutinate and identify V. vulnificus. A species-specific H antigen is expressed in the core proteins of the polar flagella of V. vulnificus. Of 435 V. vulnificus isolates identified bacteriologically, 432 (99.3%) were agglutinated in the slide test within 2 min after the addition of the anti-V. vulnificus H coagglutination reagent. Other than Vibrio pelagius, the reagent did not agglutinate 19 heterologous Vibrio spp. tested, including 290 V. cholerae, 22 V. mimicus, 395 V. parahaemolyticus, and 16 V. fluvialis isolates recovered from seafood and the marine environment. The serological resolution of the coagglutination reaction was enhanced if the organism under test was suspended in 0.1 M Tris buffer-0.1 mM EDTA-1.0% Triton X-100 (TET) for 24 h before serological examination. The TET buffer also increased the sensitivity of the coagglutination reaction 100-fold over that for isolates suspended in 0.3% formalinized phosphate-buffered saline before testing. The anti-H coagglutination test is a rapid, serologically specific, and inexpensive procedure for identifying V. vulnificus one step beyond primary isolation.     

Abundance and antibiotic resistance of non-O1 Vibrio cholerae strains in domestic wastewater before and after treatment in stabilization ponds in an arid region (Marrakesh, Morocco)

Abstract: The abundance and antibiotic resistance of non-O1 Vibrio cholerae strains were studied in wastewater before and after treatment in stabilization ponds in an arid Mediterranean climate. The seasonal abundance of non-O1 Vibrio cholerae was the inverse of those of fecal coliforms, with high densities in hot periods and low densities in cold periods. Although the stabilization pond presents a good efficiency in removing fecal coliforms (97.97%), this treatment system did not produce any significant reduction in non-O1 V. cholerae abundances between the inflow and outflow stations. Among the 240 non-O1 V. cholerae strains isolated before and after treatment in the stabilization ponds, 89 (37.1%) isolates were resistant to at least one of 14 tested antibiotics. The levels of antibiotic resistance at the inflow and outflow points of the system were respectively 40 and 34%. High ampicillin, amoxicillin and mezlocillin resistance was observed at all sampling points, followed by resistance to cefalexin, cefoperazone and amikacin. Antibiotic resistance can be transferred from non-O1 V. cholerae to other members of the Enterobacteriaceae family such as Escherichia coli K12. Transfer frequencies in nutrient broth and filtered wastewater were 3 × 10⁻⁵ and 2 × 10⁻⁸, respectively.     

Characterization of the antiseptic-resistance gene qacEΔ1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.