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.     

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 .     

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.     

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.     

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.     

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.     

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.     

Low incidence of Vibrio vulnificus among Vibrio isolates from sea water and shellfish of the western Mediterranean coast

A specific search for Vibrio vulnificus in natural marine samples from the Spanish Mediterranean Sea was carried out by nested PCR and cultural approaches using thiosulphate-citrate-bile salts-sucrose agar (TCBS) and cellobiose-polymixin B-colistin agar (CPC), incubated at 40 degrees C, as selective media. Presumptive colonies were identified by PCR using specific primers against 23S rRNA sequences. This species was isolated from sea water and edible bivalves, mainly after preenrichment in alkaline peptone water (APW) at 40 degrees C followed by CPC agar. None of the V. vulnificus isolates identified corresponded to serovar E. Dominant Vibrio species on directly inoculated TCBS plates incubated at 25 degrees C were V. splendidus below 20 degrees C and V. harveyi and V. mediterranei above that temperature. Low percentages of several pathogenic vibrios were recorded but V. vulnificus was never recovered at this incubation temperature. The incidence of this species in the samples studied was lower than that described for other geographical areas, probably due to the high salinity values of the Mediterranean Sea.     

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.     

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.     

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.     

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.     

Sandwich enzyme-linked immunosorbent assay for Vibrio vulnificus hemolysin to detect V. vulnificus in environmental specimens.

Vibrio vulnificus hemolysin, purified by quantitative isoelectric focusing, was used to prepare rabbit and goat anti-hemolysin. The resulting antibodies were used as capture and detector antibody reagents in a sandwich enzyme-linked immunosorbent assay (ELISA) to detect V. vulnificus in environmental samples. By this technique, 4 laboratory-maintained V. vulnificus strains and 33 environmental V. vulnificus isolates were detected. Also, the technique distinguished five other Vibrio species from V. vulnificus, and when it was used in combination with colistin-polymyxin-cellobiose agar, 31 non-V. vulnificus isolated were excluded. This sandwich ELISA compared favorably with the current Food and Drug Administration standard immunoassay in confirming presumptive V. vulnificus colonies from environmental specimens: oysters, sediment, and seawater. Among 340 presumptive V. vulnificus colonies, the sandwich ELISA detected 95% of the confirmed V. vulnificus colonies. Equally important, the technique correctly distinguished 99% of the non-V. vulnificus colonies. The sandwich ELISA offers time-saving and labor-saving advantages over the currently accepted immunoassay.     

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.     

Protocol for specific isolation of virulent strains of Vibrio vulnificus serovar E (biotype 2) from environmental samples

The eel pathogen Vibrio vulnificus biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections. The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources. The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples. The key element of the new protocol is the broth used for the first step (saline eel serum broth [SEB]), which contains eel serum as a nutritive and selective component. This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors. The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species). The selective and differential agar Vibrio vulnificus medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other V. vulnificus groups, including biotype 3. The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control. V. vulnificus was isolated by both protocols, but serovar E was only recovered by the new method described here. All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera. In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E.     

Regulation of fatty acid metabolism by FadR is essential for Vibrio vulnificus to cause infection of mice

The opportunistic bacterial pathogen Vibrio vulnificus causes severe wound infection and fatal septicemia. We used alkaline phosphatase insertion mutagenesis in a clinical isolate of V. vulnificus to find genes necessary for virulence, and we identified fadR, which encodes a regulator of fatty acid metabolism. The fadR::mini-Tn5Km2phoA mutant was highly attenuated in a subcutaneously inoculated iron dextran-treated mouse model of V. vulnificus disease, was hypersensitive to the fatty acid synthase inhibitor cerulenin, showed aberrant expression of fatty acid biosynthetic (fab) genes and fatty acid oxidative (fad) genes, produced smaller colonies on agar media, and grew slower in rich broth than did the wild-type parent. Deletion of fadR essentially recapitulated the phenotypes of the insertion mutant, and the DeltafadR mutation was complemented in trans with the wild-type gene. Further characterization of the DeltafadR mutant showed that it was not generally hypersensitive to envelope stresses but had decreased motility and showed an altered membrane lipid profile compared to that of the wild type. Supplementation of broth with the unsaturated fatty acid oleate restored wild-type growth in vitro, and infection with oleate in the inoculum increased the ability of the DeltafadR mutant to infect mice. We conclude that fadR and regulation of fatty acid metabolism are essential for V. vulnificus to be able to cause disease in mammalian hosts.     

Effect of low temperature on starvation-survival of the eel pathogen Vibrio vulnificus biotype 2.

At present, no reports exist on the isolation of the eel pathogen Vibrio vulnificus biotype 2 from water samples. Nevertheless, it has recently been demonstrated that this biotype can use water as a route of infection. In the present study, the survival of this pathogen in artificial seawater (ASW) microcosms at different temperatures (25 and 5 degrees C) was investigated during a 50-day period, with biotype 1 as a control, V. vulnificus biotype 2 was able to survive in the culturable state in ASW at 25 degrees C in the free-living form, at least for 50 days, entering into the nonculturable state when exposed to low temperature. In this state, this microorganism survived with reduced rates of activity, showing marked changes in size and morphology. The rate at which cells became nonculturable was dependent on their physiological age. The capsule seems not to be necessary for the survival of biotype 2 in aquatic environments as a free-living organism. Culturability remained the highest on modified salt water yeast extract agar, which is closer in salt and nutrient composition to ASW than heart infusion agar. Biotype 2 cells recovered culturability on solid media after an increase of incubation temperature from 5 to 25 degrees C. Culturable cells of this bacterium maintained infectivity for either eel or mice, while dormant cells seemed to lose their virulence. The former finding suggests that the aquatic environment is a reservoir and vehicle of transmission of this pathogen.     

Isolation of medically significant Vibrio species from riverine sources in south east Queensland

Vibrio and Vibrio-like bacteria were isolated from water, sediments, plants and faeces from eight riverine sites in South East Queensland, Australia, using filtrations, enrichments and selective growth on thiosulphate-citrate-bile salts-sucrose (TCBS) and Simidu agars. Isolates (402 in toto) were classified by numerical analysis of phenotypic characteristics and comparison with fifteen reference cultures. Of the isolates 41 (10.2%) were identified as Vibrio cholerae, 33 (8.2%) as V. fluvialis and 118 (29.4%) as motile Aeromonas species. Other isolates resembled V. parahaemolyticus, V. alginolyticus, V. vulnificus and V. hollisae. No isolates were positively identified as V. damsela or Plesiomonas shigelloides. Vibrio species tolerant of low salt levels and aeromonads were widely distributed in riverine locations but more halophilic species were restricted to more saline areas. Simidu agar failed to select for vibrios as effectively as TCBS agar. Enrichment for 18 h produced more isolates than for 6 h.     

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.