Use of sodium dodecyl sulfate-polymyxin B-sucrose medium for isolation of Vibrio vulnificus from shellfish.

The differential and selective sodium dodecyl sulfate-polymyxin B-sucrose medium (SPS) of Kitaura et al. (T. Kitaura, S. Doke, I. Azuma, M. Imaida, K. Miyano, K. Harada, and E. Yabuuchii, FEMS Microbiol. Lett. 17:205-209, 1983), which highlights alkylsulfatase activity, was evaluated for its potential use in the direct isolation and enumeration of Vibrio vulnificus from shellfish. V. vulnificus was detected by this method in six of nine shellfish samples collected from diverse geographic locales during the summer of 1986. Direct enumeration of V. vulnificus at 7.0 X 10(2) to 2.2 X 10(4) CFU/g of shellfish was achieved on SPS agar. All sample results were confirmed in parallel examinations by using conventional glucose-salt-Teepol (Shell Oil Co.) broth and alkaline peptone water enrichment with plating onto thiosulfate-citrate-bile salts-sucrose agar. Additionally, alkylsulfatase activity was evaluated in vitro for 97 strains representing 14 Vibrio spp. V. vulnificus and Vibrio cholerae-01 were the only species consistently found to possess this activity. The range of plating efficiencies for random V. vulnificus strains analyzed on SPS was 11 to 74% (mean, 39%). The use of SPS shows great promise for the study of shellfish and other environmental sources for V. vulnificus.     

Direct plating procedure for enumerating Vibrio vulnificus in oysters (Crassostrea virginica).

A procedure for enumerating and identifying Vibrio vulnificus in oysters was developed and evaluated. This method consists of growth on a direct plating medium (VVE medium) for isolating the organism from shellfish tissues, followed by biochemical tests for differentiating and identifying presumptively positive isolates. Densities of V. vulnificus are reliably obtained in 2 to 4 days, and as few as 10 culturable cells per 100 g can be identified. The procedure was evaluated by using a DNA probe technique specific for the cytotoxin-hemolysin gene of V. vulnificus and gas chromatographic analysis of the fatty acid contents of positive isolates. Only 3.2 and 0.4% of the isolates gave false-positive and false-negative results, respectively. The average level of recovery on VVE medium for 33 strains, including both clinical and environmental isolates, was 92% of the level of recovery obtained with brain heart infusion agar supplemented with 1% NaCl. The densities of V. vulnificus in oyster homogenates and individual oysters harvested from gulf and Atlantic coastal waters revealed that seasonally high levels occurred. The VVE medium procedure facilitated enumeration of this pathogen in molluscan shellfish and had a distinct advantage over the widely used most-probable-number procedure for V. vulnificus enumeration, which requires 5 to 7 days and often gives improbable and imprecise results.     

New selective and differential medium for Vibrio cholerae and Vibrio vulnificus

Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate. Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species. Cellobiose-polymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vibrio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V. vulnificus and V. cholerae were able to grow. Furthermore, the fermentation of cellobiose by V. vulnificus allowed for the easy differentiation of these two species. This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios.     

New selective and differential medium for Vibrio cholerae and Vibrio vulnificus.

Thiosulfate-citrate-bile salts-sucrose agar has been routinely used for the isolation of pathogenic vibrios, although its selectivity for Vibrio cholerae and Vibrio vulnificus is inadequate. Therefore, a new plating medium, cellobiose-polymyxin B-colistin agar, was developed for the isolation of these two species. Cellobiose-polymyxin B-colistin agar demonstrated a significant advantage over other media designed for the isolation or differentiation of vibrios: of both the 136 strains representing 19 Vibrio species and the marine isolates of the genera Pseudomonas, Flavobacterium, and Photobacterium, only V. vulnificus and V. cholerae were able to grow. Furthermore, the fermentation of cellobiose by V. vulnificus allowed for the easy differentiation of these two species. This medium offers significant potential as a selective and differential medium for these two pathogenic vibrios.     

Real-time PCR analysis of Vibrio vulnificus from oysters

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 micro l of PCR mixture(-1) or the equivalent of six cells. Similar sensitivity was observed in DNA extracted from mixtures of V. vulnificus and V. parahaemolyticus cells. Real-time PCR enumeration of artificially inoculated oyster homogenates correlated well with colony hybridization counts (r(2) = 0.97). Numbers of indigenous V. vulnificus cells in oysters by real-time PCR showed no significant differences from numbers from plate counts with probe (t test; P = 0.43). Viable but nonculturable cells were also enumerated by real-time PCR and confirmed by the BacLight viability assay. These data indicate that real-time PCR can provide sensitive species-specific detection and enumeration of V. vulnificus in seafood.     

Methods for isolation and confirmation of Vibrio vulnificus from oysters and environmental sources: a review

The gram-negative bacterium Vibrio vulnificus is a natural inhabitant of estuarine waters and poses a significant health threat to humans who suffer from immune disorders, liver disease, or hemochromatosis (iron overload). V. vulnificus enters human hosts via wound infections or consumption of raw shellfish (primarily oysters), and infections frequently progress to septicemia and death in susceptible individuals. Prevalence in waters and shellfish is not correlated with fecal indicator organisms; therefore, species-specific detection and enumeration of V. vulnificus in the environment has become a priority for agencies that are responsible for shellfish safety. The many selective-differential media developed for isolation of Vibrio spp., and specifically for V. vulnificus detection, are reviewed here; however, none of the media developed to date combines the sensitivity to low numbers with the specificity necessary to inhibit growth of other organisms. Therefore, immunological and molecular protocols are needed for confirmation of the identity of the organism and are discussed in detail. Methods under development that hold promise for rapid, accurate, and sensitive detection and enumeration of the organism include multiplex and real-time PCR. Developing technologies that have proven useful for detection and investigation of other pathogens such as biosensors, spectroscopy and microarrays may provide the next generation of tools for investigation of the prevalence and ecology of V. vulnificus.     

Use of colistin-polymyxin B-cellobiose agar for isolation of Vibrio vulnificus from the environment.

Colistin-polymyxin B-cellobiose agar was employed for the isolation of Vibrio vulnificus from shellfish. Isolates were examined phenotypically and with a gene probe and monoclonal antibody specific for V. vulnificus. Results indicated that colistin-polymyxin B-cellobiose agar is superior to both sodium dodecyl sulfate-polymyxin B-sucrose agar and thiosulfate-citrate-bile salts-sucrose agar in its ability to select and differentiate this species from background vibrios.     

Use of colistin-polymyxin B-cellobiose agar for isolation of Vibrio vulnificus from the environment.

Colistin-polymyxin B-cellobiose agar was employed for the isolation of Vibrio vulnificus from shellfish. Isolates were examined phenotypically and with a gene probe and monoclonal antibody specific for V. vulnificus. Results indicated that colistin-polymyxin B-cellobiose agar is superior to both sodium dodecyl sulfate-polymyxin B-sucrose agar and thiosulfate-citrate-bile salts-sucrose agar in its ability to select and differentiate this species from background vibrios.     

A selective medium and a specific probe for detection of Vibrio vulnificus

A selective medium (VVM) and a specific 16S rRNA gene (rDNA) probe (V3VV) for the detection of Vibrio vulnificus were developed. The medium contains D-(+)-cellobiose as the main carbon source and electrolytes (MgCl(2)-6H(2)O and KCl), which stimulate bacterial growth. Polymyxin B, colistin, and moderate alkalinity and salinity provide selectivity properties. V. vulnificus grows on VVM as flat, bright yellow colonies. Other Vibrio species tested either did not grow or showed green-bluish colonies, with the exception of V. campbelli, V. carchariae, and V. navarrensis. There is a higher colony count on VVM agar than on cellobiose-colistin agar or on modified cellobiose-polymyxin B-colistin agar. The specific probe was evaluated by colony hybridization and dot blot hybridization with PCR-amplified 16S rDNA using collection strains and environmental isolates. No strain studied other than V. vulnificus showed positive hybridization with this oligonucleotide. The combined use of VVM agar and the V3VV probe provided the recovery of V. vulnificus from mixed bacterial suspensions and spiked mussels.     

Evaluation of a simplified semi-quantitative protocol for the estimation of Vibrio vulnificus in bathing water using cellobiose-colistin agar: a collaborative study with 13 municipal food controlling units in Denmark

A simplified semi-quantitative method using pre-enrichment in alkaline peptone water supplemented with polymyxin B and plating onto cellobiose-colistin (CC) agar for the estimation of Vibrio vulnificus in bathing water was evaluated. This protocol was tested in a collaborative study with 13 food controlling laboratories in Denmark during the 1999 bathing season in periods when water temperatures exceeded 20 degrees C. The average percentage of yellow colonies larger than 1 mm in diameter on CC agar that could be identified as V. vulnificus by colony hybridization with a species-specific DNA probe was 79%. This high percentage of specificity demonstrated that by using CC agar in estimating the level of V. vulnificus in bathing water, recognition of yellow colonies larger than 1 mm is sufficient for the identification of V. vulnificus with no further characterization needed. The simplified protocol may be included in the routine control of the microbial quality of bathing water done by the local food controlling laboratories, since it involves simple traditional and low-cost microbiological methods with no use of molecular skills or sophisticated equipment.     

Comparison of selective media for the detection of Vibrio vulnificus in environmental samples

AIMS: To compare two selective agars, cellobiose-colistin (CC) agar and a modification of the Vibrio vulnificus medium (VVMc agar), for the isolation of Vibrio vulnificus from environmental samples. METHODS AND RESULTS: The efficiencies of recovery of V. vulnificus collection strains on CC, VVM, VVMc and on thiosulphate-citrate-bile salts-sucrose (TCBS) agar were compared and similar efficiencies were obtained. A slightly higher recovery was observed on VVMc agar. The detection of V. vulnificus in environmental samples (eels and water) was performed by combining culture-based methods (CC and VVMc agars) with DNA-based methods using species-specific probes based on the cytolysin-haemolysin and the 16S rDNA genes. A lower accompanying microbiota was found on CC agar than on VVMc agar. CONCLUSION: The comparison between CC and VVMc agars confirms that both are useful for the detection of V. vulnificus in environmental samples. However, the use of any of these media should be combined with a species-specific probe. SIGNIFICANCE AND IMPACT OF THE STUDY: The combined use of a selective medium and a specific probe provides a feasible method for the detection of V. vulnificus for epidemiological and ecological studies.     

Comparison of a commercial biochemical kit and an oligonucleotide probe for identification of environmental isolates of Vibrio vulnificus

A. DALSGAARD, I. DALSGAARD, L. HØI AND J.L. LARSEN. 1996. Methods for the identification and isolation of environmental isolates of Vibrio vulnificus were evaluated. Alkaline peptone water supplemented with polymyxin B and colistin-polymyxin B-cellobiose agar were employed for the isolation of suspected V. vulnificus from water, sediment and shellfish samples. When comparing the identification of putative V. vulnificus obtained with the API 20E assay and an oligonucleotide probe, 29 API 20E profiles were obtained with only four profiles (representing 20 isolates) reaching the identification threshold of V. vulnificus among a total of 66 isolates hybridizing with the probe. The results indicated that, compared with colony hybridization, the API 20E assay was not adequate for the identification of environmental isolates of V. vulnificus .     

EVALUATION OF TWO NONRADIOACTIVE GENE PROBES FOR THE ENUMERATION OF VIBRIO PARAHAEMOLYTICUS IN CRABMEAT

This study evaluated two nonradioactive DNA probe procedures for the detection and enumeration of Vibrio parahaemolyticus in crabmeat by comparing counts obtained by direct plating and by most probable number (MPN) procedures. The nonradioactive probes evaluated were an alkaline phosphatase-labeled thermolabile direct hemolysin (AP-tdh) and a digoxigenin-labeled thermostable direct hemolysin (DG-tdh) for detection and enumeration of total and pathogenic V. parahaemolyticus, respectively. Inoculated samples (50 g each) of steamed crabmeat were analyzed by nine analysts in seven laboratories. Samples were inoculated with various Vibrio strains and combinations of strains at different levels of inoculation (0 to 92,000 cells/g). The results indicated that the AP-tlh probe was reliable for identification and enumeration of total V. parahaemolyticus and the DG-tdh probe was specific for the pathogenic strains. Both probes required less effort and expense than the biochemical testing and hemolysin assays that have previously been used. The MPN and direct plating procedures using the nonradioactive probes were both effective for enumeration of total and pathogenic V. parahaemolyticus in the absence of competing microflora, but direct plating was preferable to MPN for enumeration of V. parahaemolyticus, especially pathogenic strains, in the presence of competitors.     

Identification of Vibrio spp. (other than V. vulnificus) recovered on CPC agar from marine natural samples.

Two hundred and eighty four presumptive but not confirmed Vibrio vulnificus isolates grown on cellobiose-polymixin B-colistin agar (CPC) at 40 degrees C, recovered from sea water samples from Valencia, Spain, during a microbiological survey for V. vulnificus, were phenotypically identified. Most of the isolates (91%) corresponded to Vibrio species. V. harveyi (24%) and V. splendidus(19%) were the most abundant species identified, followed by V. navarrensis (13%), V. alginolyticus (8%) and V. parahaemolyticus (5%). The ability to grow on CPC agar and ferment cellobiose of several V. vulnificus strains from different origins and serovars, including reference strains, was tested. Most serovar E isolates and 25% of non-serovar E isolates could not grow on CPC agar.     

Detection of Vibrio vulnificus biotypes 1 and 2 in eels and oysters by PCR amplification.

DNA extraction procedures and PCR conditions to detect Vibrio vulnificus cells naturally occurring in oysters were developed. In addition, PCR amplification of V. vulnificus from oysters seeded with biotype 1 cells was demonstrated. By the methods described, V. vulnificus cells on a medium (colistin-polymyxin B-cellobiose agar) selective for this pathogen were detectable in oysters harvested in January and March, containing no culturable cells (< 67 CFU/g), as well as in oysters harvested in May and June, containing culturable cells. It was possible to complete DNA extraction, PCR, and gel electrophoresis within 10 h by using the protocol described for oysters. V. vulnificus biotype 2 cells were also detected in eel tissues that had been infected with this strain and subsequently preserved in formalin. The protocol used for detection of V. vulnificus cells in eels required less than 5 h to complete. Optimum MgCl2 concentrations for the PCR of V. vulnificus from oysters and eels were different, although the same primer pair was used for both. This is the first report on the detection of cells of V. vulnificus naturally present in shellfish and represents a potentially powerful method for monitoring this important human and eel pathogen.     

Validation of a green fluorescent protein-labeled strain of Vibrio vulnificus for use in the evaluation of postharvest strategies for handling of raw oysters

In this paper we describe a biological indicator which can be used to study the behavior of Vibrio vulnificus, an important molluscan shellfish-associated human pathogen. A V. vulnificus ATCC 27562 derivative that expresses green fluorescent protein (GFP) and kanamycin resistance was constructed using conjugation. Strain validation was performed by comparing the GFP-expressing strain (Vv-GFP) and the wild-type strain (Vv-WT) with respect to growth characteristics, heat tolerance (45 degrees C), freeze-thaw tolerance (-20(o) and -80 degrees C), acid tolerance (pH 5.0, 4.0, and 3.5), cold storage tolerance (5 degrees C), cold adaptation (15 degrees C), and response to starvation. Levels of recovery were evaluated using nonselective medium (tryptic soy agar containing 2% NaCl) with and without sodium pyruvate. The indicator strain was subsequently used to evaluate the survival of V. vulnificus in oysters exposed to organic acids (citric and acetic acids) and various cooling regimens. In most cases, Vv-GFP was comparable to Vv-WT with respect to growth and survival upon exposure to various biological stressors; when differences between the GFP-expressing and parent strains occurred, they usually disappeared when sodium pyruvate was added to media. When V. vulnificus was inoculated into shellstock oysters, the counts dropped 2 log(10) after 11 to 12 days of refrigerated storage, regardless of the way in which the oysters were initially cooled. Steeper population declines after 12 days of refrigerated storage were observed for both iced and refrigerated products than for slowly cooled product and product held under conservative harvest conditions. By the end of the refrigeration storage study (22 days), the counts of Vv-GFP in iced and refrigerated oysters had reached the limit of detection (10(2) CFU/oyster), but slowly cooled oysters and oysters stored under conservative harvest conditions still contained approximately 10(3) and >10(4) CFU V. vulnificus/oyster by day 22, respectively. The Vv-GFP levels in the oyster meat remained stable for up to 24 h when the meat was exposed to acidic conditions at various pH values. Ease of detection and comparability to the wild-type parent make Vv-GFP a good candidate for use in studying the behavior of V. vulnificus upon exposure to sublethal stressors that might be encountered during postharvest handling of molluscan shellfish.     

Virulence factors and pathogenicity of Vibrio vulnificus strains isolated from seafood

The virulence factors of Vibrio vulnificus are not yet well understood. So far, many hydrolytic enzymes have been implicated in the pathogenesis of this micro-organism. The present research was carried out in order to study the presence of some of these enzymes in 133 V. vulnificus strains isolated from 45 seafood samples. The results showed that 100% of these strains were positive for the production of lecithinase and lipase (Tween-80), 99·2% for caseinolytic protease, 96·9% for DNase, 65·4% for mucinase and 46·6% for elastase. None of the strains was positive for the production of collagenase and 96% were haemolytic against sheep blood cells. In relation to colony morphology on brain heart infusion (BHI) agar and nutrient agar, 59·4% of strains showed opaque morphology on BHI agar and 57·9% on nutrient agar, 10·5% presented translucent morphology on both agars and 30·1 and 31·6% of strains showed a mixture of opaque and translucent morphology on BHI agar and nutrient agar, respectively. None of the translucent colonies was virulent to mice. Therefore, opacity was a useful marker for potential virulence. Of 45 food samples contaminated with V. vulnificus , 29 (64·4%) presented strains lethal to adult mice.     

Multiplex PCR detection of clinical and environmental strains of Vibrio vulnificus in shellfish

In this study, we developed a PCR-based rapid detection method for clinically important pathogenic strains of Vibrio vulnificus. Positive amplification of the 504-bp viuB fragment was seen in all 22 clinical isolates tested but only in 8 out of 33 environmental isolates. The combination of the species-specific 205-bp vvh fragment along with viuB in a multiplexed PCR enabled us to confirm the presence of potentially pathogenic strains of V. vulnificus. No amplification of other Vibrio spp. or non-Vibrio bacteria was evidenced, suggesting a high specificity of detection by this method. The sensitivity of detection for both targeted genes was 10 pg of purified DNA, which correlated with 10(3) V. vulnificus CFU in 1 mL of pure culture or 1 g un-enriched seeded oyster tissue homogenate. This sensitivity was improved to 1 CFU per gram of oyster tissue homogenate in overnight-enriched samples. A SYBR Green I based real-time PCR method was also developed that was shown to produce results consistent with the conventional PCR method. Application of the multiplexed real-time PCR to natural oyster tissue homogenates exhibited positive detection of vvh in 51% of the samples collected primarily during the summer months; however, only 15% of vvh positive samples exhibited viuB amplicons. The rapid, sensitive, and specific detection of clinically important pathogenic V. vulnificus in shellfish would be beneficial in reducing illnesses and deaths caused by this pathogen.     

Viability of Vibrio vulnificus in Association with Hemocytes of the American Oyster (Crassostrea virginica)

Certain indigenous estuarine bacteria, such as Vibrio vulnificus, may cause opportunistic human infections after consumption of raw oysters or exposure of tissues to seawater. V. vulnificus is known to be closely associated with oyster (Crassostrea virginica) tissues and is not removed by controlled purification methods, such as UV light-assisted depuration. In fact, when live shellfish are subjected to controlled purification, the number of V. vulnificus cells can markedly increase. A review of previous studies showed that few workers have examined mechanisms in oysters which may influence the persistence of V. vulnificus in shellfish, such as the fate of V. vulnificus following phagocytosis by molluscan hemocytes. The objectives of this study were to define the intracellular viability and extracellular viability of V. vulnificus during the phagocytic process and to study the release of specific lysosomal enzymes. The viability of a virulent estuarine V. vulnificus isolate with opaque morphology was compared with the viability of a translucent, nonvirulent form, the viability of Vibrio cholerae, and the viability of Escherichia coli in phagocytosis experiments. Our results showed that the levels of phagocytosis and bactericidal degradation of the opaque V. vulnificus isolate were less than the levels of phagocytosis and bactericial degradation of the translucent morphotype. These findings indicate that encapsulation may contribute to resistance to ingestion and degradation by hemocytes. The rates of intracellular death of V. cholerae and E. coli exceeded the rate of intracellular death of the opaque V. vulnificus isolate, even though the ingestion or uptake rates did not differ significantly. The levels of lysozyme activity and acid phosphatase activity were not significantly different in hemocyte monolayers inoculated with V. vulnificus.     

Distribution of Vibrio vulnificus phage in oyster tissues and other estuarine habitats.

Phages lytic to Vibrio vulnificus were found in estuarine waters, sediments, plankton, crustacea, molluscan shellfish, and the intestines of finfish of the U.S. Gulf Coast, but no apparent relationship between densities of V. vulnificus and its phages was observed. Phage diversity and abundance in molluscan shellfish were much greater than in other habitats. V. vulnificus phages isolated from oysters did not lyse other mesophilic bacteria also isolated from oysters. Both V. vulnificus and its phages were found in a variety of oyster tissues and fluids with lowest densities in the hemolymph and mantle fluid. These findings suggest a close ecological relationship between V. vulnificus phages and molluscan shellfish.