Vibrio vulnificus in Denmark

Summary

Vibrio vulnificus, a halophilic marine vibrio, is an opportunistic human pathogen that can cause severe wound infections and septicemias with mortalities as high as 60 % (Oliver, 1989; Hlady & Klontz, 1996).

V. vulnificus can be isolated from a wide variety of aquatic ecosystems, but the occurrence of the organism is favored by high temperatures (>20°C) and intermediate salinities (15-25l) (Motes et al., 1998). In temperate areas, V. vulnificus is less abundant than in subtropical waters, but V. vulnificus has been isolated from coastal waters or implicated in human infections during the summer months in Denmark, Sweden, Germany, Holland, and Belgium (Mertens et al., 1979; Veenstra et al., 1994; Hoyer et al., 1995; Melhus et al., 1995; Dalsgaard et al., 1996b). V. vulnificus has also caused disease in eel farms in Japan, Spain, Norway, Sweden, and Denmark (Muroga et al., 1976, Biosca et al., 1991; Høi et al., 1998b; Dalsgaard et al., in press).

During the unusually warm summer in Denmark in 1994, coastal water temperatures exceeded 20°C for about four weeks (Dalsgaard et al., 1996b). At the end of August 1994, 11 patients were admitted to hospitals with V. vulnificus infections; this was presented in the first report of a series of human V. vulnificus infections from a temperate zone (Dalsgaard et al., 1996b). All patients contracted their disease after exposure to coastal water. In 1995 and 1997, a total of 3 and 4 V. vulnificus infections, respectively, were reported in Denmark. In 1996 and 1998, water temperatures were low and no cases were reported (Bruun, 1997). The clinical cases in 1994 prompted the Royal

Veterinary and Agricultural University, The Danish Institute for Fisheries Research, The Environmental Protection Agency and The Ministry of Food, Agriculture, and Fisheries to fund studies on the occurrence of V. vulnificus in Danish costal waters.

Overall conclusions

The overall conclusions of the studies were that V. vulnificus occur in Danish marine environments including coastal water, sediment, wild fish, and shellfish. Low concentrations of V. vulnificus were recorded and growth was favored when water temperatures exceeded 20°C for several weeks during warm summers. In this period health authorities should be aware of wound infections associated with V. vulnificus and other Vibrio spp. V. vulnificus was also associated with disease outbreaks in Danish eel farms (Høi et al., 1998b, Dalsgaard et al., in press). V. vulnificus was occasionally isolated from raw, frozen seafood imported from South East Asia and in Danish blue mussels but do not constitute a hazard to public health in Denmark providing correct handling and cooking.

Identification

The name V. vulnificus was given official taxonomic status in 1980 (Farmer, 1980). It is believed that V. vulnificus formerly often was misidentified as V. parahaemolyticus (Hollis et al., 1976). The species V. vulnificus comprises two biotypes which in the original definition differed phenotypically, serologically, and in host range (Tison et al., 1982). V. vulnificus biotype 1 is ubiquitous to estuarine environments and is an opportunistic human pathogen (Oliver, 1989). Biotype 2 is typically recovered from diseased eels but is also reported to cause illness in humans after handling eels (e.g. Mertens et al., 1979; Høi et al., 1997).

Biochemical identification of V. vulnificus requires a number of biochemical assays which are costly and time-consuming (Colwell, 1984; Murray, 1995). The API 20E index, where information on V. vulnificus is based on 31 V. vulnificus strains only, differs from the reactions shown in several manuals for the lysine decarboxylase and Voges-Proskauer reactions (Barrow & Feltham, 1993;. Holt et al., 1994; Murray, 1995; Sinding, 1998). In general, characters listed in reference manuals are based on a limited number of clinical strains and do not include the more heterogenous reaction patterns of environmental strains.

Molecular techniques

Molecular techniques, particularly specific oligonucleotide probes, constitute a very sensitive and specific tool for detecting V. vulnificus. An alkaline phosphatase-labeled oligonucleotide probe directed towards the cytolysin gene of V. vulnificus was constructed by Wright et al. (1985,1993). This probe, termed VVAP, demonstrated 100% specificity and sensitivity for clinical and environmental isolates of V. vulnificus and numerous investigators have shown that cytolysin is produced by all V. vulnificus strains, including both biotypes, and is species-specific (Kaysner et al., 1987; Morris et al., 1987a; Parker & Lewis, 1995; Biosca et al., 1996c). The sequence of the cytolysin gene has also been used for constructing primers for PCR identification (Brauns et al., 1991; Coleman et al., 1996).

DNA probe detection

In the present project, suspected V. vulnificus isolates from environmental samples were more frequently identified as V. vulnificus by the VVAP probe than by the API 20E system (Dalsgaard et al., 1996a). Sixty-six isolates identified as V. vulnificus with the specific DNA probe were tested with the API 20E strip according to the manufacturers instructions. A total of 29 API 20E profiles were obtained. Only four of these profiles, representing 20 isolates, reached the identification threshold of V. vulnificus. The reasons for these difficulties could be discrepancies between the species-specific reaction pattern in the API 20E database and the reactions given in standard identification tables, the heterogeneity of environmental strains, and the limited number of V. vulnificus strains (31 strains) included in the API 20E database (Dalsgaard et al., 1996a; Sinding, 1998).

Biotype 2 isolates are not included in the API 20E system and can not be identified by this system (Biosca et al., 1993a). Furthermore, commonly used reference manuals do not recognize the existence of V. vulnificus biotype 2 strains since they do not include indole negative strains.

Conclusions

In conclusion, identification of V. vulnificus with the commercial biochemical testing kit API 20E is not reliable because V. vulnificus isolates are very heterogenous in their biochemical characters. Serological identification requires preparation of antibodies since no commercial V. vulnificus-specific antibodies are available. Colony hybridization with the V. vulnificus-specific oligonucleotide probe is specific, fast and cost-effective. Identification of V. vulnificus with oligonucleotide probes or PCR primers directed against DNA or rRNA sequences is recommended.

The choice of including a pre-enrichment step in isolation of V. vulnificus depends on four factors: (i) the expected concentration of V. vulnificus in the samples, (ii) if a quantitative or qualitative result is needed, (iii) the conditions of the cells, and (iv) the level and composition of background flora. The pre-enrichment step should improve the ratio of target to background flora before a selective plating step.

Pre-enrichment broths and selective agars

The isolation of pathogenic Vibrio spp. is usually accomplished by culture methods that start with pre-enrichment in alkaline peptone water (APW; 1% peptone, pH 8.6) with 1% NaCl to recover sublethal injured organisms, followed by plating onto thiosulfate-citrate-bile salts-sucrose (TCBS) agar (Colwell, 1984).

Alkaline peptone water with polymyxin B

Overnight pre-enrichment in APW with polymyxin B (20 U/ml; APWP) gave higher recovery rate than pre-enrichment in regular APW in combination with modified cellobiose polymyxin B colistin (mCPC) agar when analyzing samples of coastal water and sediment in Denmark (Dalsgaard et al., 1996a). APWP and mCPC agar was subsequently used with success for the isolation of V. vulnificus from fresh and frozen seafood (Dalsgaard & Høi, 1997; Høi et al., 1998c). However, when analyzing gills, mucus, and intestinal content from cultured diseased eels and wild fish from Danish coastal waters pre-

enrichment in APW for 6-8 h proved more favorable than overnight pre-enrichment in APWP (Høi et al., 1998c; unpublished results). Studies with heavily infected eels showed that direct plating of tissue samples homogenized in phosphate-buffered-saline (PBS) gave the same or sometimes even better recovery of V. vulnificus than by pre-enrichment (unpublished results). Other studies have also reported that different sample types requires different isolation strategies for V. vulnificus (Biosca et al., 1997b; Kaysner et al., 1989).

Studies of 50 V. vulnificus strains from various sources and countries showed that no strains had a minimal inhibitory concentration (MIC) lower than 779 U colistin/ml (Høi et al., 1998a); thus recovery should not be reduced by adding as high a concentration as 20 U colistin/ml to the enrichment broth (Høi et al., 1998a). Colistin, which also is termed polymyxin E, shows a similar mode of action on bacteria as polymyxin B.

The use of a selective and indicative medium for isolation of V. vulnificus serves two purposes: (i) to allow growth of V. vulnificus while inhibiting growth of more abundant marine species, (ii) to allow differentiation of V. vulnificus from other bacterial species so suspect colonies can be further identified.

mCPC agar

Tamplin et al. (1991,1992) described a less selective modification of the CPC agar termed mCPC with a reduced concentration of colistin. This medium was reported to be effective in isolating V. vulnificus from environmental sources (Tamplin et al., 1991; Tamplin & Capers, 1992; Dalsgaard & Høi, 1997; Høi et al., 1998c). In Denmark, more than 95% of presumptive colonies on mCPC agar could be identified as V. vulnificus when taking into consideration the typical colony morphology of V. vulnificus on this medium (flat, yellow colonies of approximately 2 mm in diameter) (Høi et al., 1998a; Høi et al., 1998c). It was very important for the identification-success rate to include the criterion Aflat@ in the evaluation of suspect colonies (Høi et al., 1998a; Høi et al., 1998c). Høi et al. (1998a) recommended a new medium termed cellobiose colistin (CC) agar which gave a better recovery of V. vulnificus than TCBS, CPC and mCPC agar in laboratory studies using pure cultures and Danish water and sediment samples. TCBS agar gave a very low plating efficiency (1%) of both clinical and environmental V. vulnificus strains and should not be recommended for the isolation of V. vulnificus (Høi et al., 1998a). This is in agreement with other reports of low recovery of V. vulnificus on TCBS (Brayton et al., 1983; Beazley & Palmer, 1992).

Conclusions

In conclusion, the isolation strategy for recovery and enumeration of V. vulnificus from environmental samples depends on the sample type, the level of background flora, and the expected concentration of V. vulnificus. In Denmark, where V. vulnificus levels are generally low (less than 10 CFU per ml or gram), CC agar significantly increased the isolation rate of V. vulnificus from coastal water and sediment samples compared to mCPC agar when used in combination with pre-enrichment in APWP. More than 95% of the presumptive colonies on CC agar were identified as V. vulnificus with the VVAP probe indicating that identification and enumeration of V. vulnificus using CC agar only may be sufficient in laboratories where colony hybridization can not be done. TCBS gave very low plating efficiencies and can not be recommended for the isolation of V. vulnificus.

V. vulnificus causes primary septicemias and wound infections in humans (Blake et al., 1979). Most primary septicemias are associated with raw seafood consumption, especially raw oysters and in almost every case the patient has a chronic underlying disease. V. vulnificus differs from other food borne pathogens as it is seldom reported to cause diarrhea and vomiting (Hollis et al., 1976; Blake et al., 1979; Hlady & Klontz, 1996). V. vulnificus causes only sporadic disease and outbreaks have never been reported (Whitman, 1995).

The fatality rate is high with almost 60% of the patients with primary septicemia dying within a few days (Oliver, 1989). A high prevalence of liver disease and alcoholism among patients with septicaemia has supported a requirement of V. vulnificus for free iron via saturated transferrin or excess of iron (Hlady & Klontz, 1996). Other risk factors include the use of immunosuppressive agents, gastric diseases, and blood disorders (Oliver, 1989).

V. vulnificus causes wound infections by entering a pre-existing skin lesion during exposure to saline waters. Patients are often employed as fishermen or in other jobs with close contact to the marine environment (Dalsgaard et al., 1996b; Hlady & Klontz, 1996). The fatality rate of reported cases is approximately 20% but amputation or surgical debridement is often necessary (Oliver, 1989).

V. vulnificus in Denmark

In Denmark, four of 11 patients in 1994 developed septicemia, of which one subsequently died. Nine patients exhibited skin manifestations and six underwent surgical debridement. Four patients contracted their disease during fishing and at least one patient had been handling eels (Dalsgaard et al., 1996b). In 1995 and 1997 three and four patients were likely to have contracted their disease during fishing. None of the patients had consumed any seafood prior to infection.

V. vulnificus is sensitive to most antibiotics and infections have been treated with antibiotics, e.g. ampicillin, tetracycline, chloramphenicol or third-generation cephalosporins (Klontz et al., 1988; Chuang et al., 1992; Fang, 1992; Dalsgaard et al., 1996b). Antibiotic treatment is often ineffective unless initiated as soon as the first clinical symptoms appear (Oliver, 1989). However, in cases of serious wound infections, the primary treatment is a proper surgical debridement with antibiotics playing a secondary role (Dalsgaard et al., 1996b).

V. vulnificus biotype 2 causes serious economic losses in aquaculture in Denmark and other countries where eels are kept in brackish water around 20-24°C. The Danish production of eels in aquaculture is expanding with a production in 1999 of 3,000 metric tonnes. The majority of eel farms in Denmark use fresh water to culture eels. Disease outbreaks caused by V. vulnificus and other pathogenic Vibrio spp. have not been reported in Denmark with recirculating systems that use freshwater continuously. One exception is V. anguillarium that has been reported to casue infections in eels after salt-treatment (Mellergaard & Dalsgaard, 1987). It is preferable to culture eels in brackish water instead of fresh water since it leads to better growth rates, feed conversion and taste. However, brackish water can be a reservoir or vehicle of V. vulnificus biotype 2 and might facilitate the spread to cultured eels (Høi et al., 1998c). Further, water temperatures in eel farms favor V. vulnificus growth (Høi et al.,1998c). V. vulnificus biotype 2 was isolated from wound infections in humans and coastal water in Denmark in 1994 but was not isolated from diseased eels in Danish farms using brackish water until 1995 (Dalsgaard et al., 1996b; Høi et al., 1997; Høi et al., 1998b; Høi et al., 1998c; Dalsgaard et al, in press). Since 1995 recurrent outbreaks of V. vulnificus have occurred in two Danish eel farms both using brackish water. The disease has caused economic losses (Høi et al.,1998b; Dalsgaard et al.,in press).

Although the disease is a septicemic infection and bacteria are easily isolated from blood samples from moribund eels (Amaro et al., 1997), antibiotic treatment of V. vulnificus infections in eels has a limited effect and outbreaks are recurrent. No antibiotic resistance has been demonstrated so far (Dalsgaard et al., in press). Changes to production in freshwater usually reduce the eel mortality, but in one Danish eel farm the infection with V. vulnificus is persisting at the present time. Research on survival and spread of V. vulnificus in eel farms and the efficiency of vaccination is needed to make culture of eels in brackish water profitable. V. vulnificus has recently been isolated from the gills and intestinal contents of eels cultured in freshwater but these eels had been kept in brackish water in the past (unpublished results). These findings suggested that once V.vulnificus enter the eel farm and colonize the eels, then Na⁺ or other cations present in the eel may be sufficient for growth of this halophilic bacteria. The concentration of Na⁺ in blood an extracellular fluids in eels is approximately 150 mmol (8.8 g/L) which theoretically is sufficient for growth and persistence of V. vulnificus (Scholz & Zerbst-Boroffka, 1994).

Virulence factors

The high virulence of V. vulnificus can not be assigned to a single factor but is likely to be influenced by capsule production, ability to acquire iron in human serum, lipopolysaccharide (LPS) type, production of exoenzymes and exotoxins, and a susceptible host.

The two biotypes share many of the same virulence factors, including (i) the capsule, a protective surface antigen that allows cells to resist phagocytosis and lysis by human serum but not by eel complement (Biosca et al., 1993b; Amaro et al.,1994); (ii) various iron uptake systems, including siderophore production and the ability to utilize hemoglobin and hemin as iron sources (Amaro et al., 1994; Biosca et al., 1996b); and (iii) a cytolysin, with hemolytic activity together with potent proteases, which are active involved in the lesions produced in different organs (Amaro et al., 1992).

Plasmids

A relationship between high molecular weight plasmids and eel virulence was first suggested by Biosca et al. (1997a), who found that a plasmid-free biotype 2 strain had a significantly higher LD₅₀ in eels (the lethal dose of V. vulnificus that kills 50% of eels tested) than biotype 2 strains harboring high molecular weight plasmids. These findings are corroborated by Høi et al. (1998b) who found that 93 of 97 biotype 2 strains isolated from diseased eels contained one to three high molecular weight plasmids of varying sizes. Restriction digests of plasmids from a number of biotype 2 strains from Denmark revealed a high degree of homology (Lewin, 1998). The role of plasmids requires further studies.

Risk for eel farmers

Danish farmers when handling eels are exposed to V. vulnificus during outbreaks but at the present time no infections of eel farmers has been reported.

V. vulnificus in Danish coastal waters

In conclusion, human infection with V. vulnificus following exposure to coastal water in Denmark occur when water temperatures exceed 20°C and fishermen, especially eel fishermen, appear to be at the greatest risk. Consumption of raw shellfish has not been associated with V. vulnificus infections in Denmark, despite that V. vulnificus occasionally was isolated in low numbers. V. vulnificus presents a serious economic problem to the Danish eel farmers wanting to use brackish water and warrant for both therapeutic and prophylactic measures. Research is needed to understand the ecology of V. vulnificus in eel farms and to develop a vaccine against eel-pathogenic V. vulnificus strains.

A comprehensive environmental survey of V. vulnificus in Danish marine environments was done during 1996 (Høi et al., 1998c). The aims of this survey were to investigate the occurrence of V. vulnificus in Danish coastal waters, shellfish, and wild fish (Høi et al., 1998c).

From May to October 1996, water was sampled weekly at seven sites and sediment samples were collected weekly from two sites. Blue mussels (Mytilus edulis) and oysters (Oestra edulis and Crassostrea gigas) were sampled from July until December 1996 from a total of 13 sites. From July until October 1996, a total of 136 wild fish were analyzed, including 29 flounders (Platichthys flesus), 14 eel pouts (Zoarches viviparus), and 93 eels (A. anguilla) that were caught at various locations in Køge Bay (Høi et al., 1998c).

In Denmark, biotype 2 strains were isolated from sediment and coastal water samples although the frequency of isolating such strains was very low (3 of 706 strains were indole negative and designated biotype 2). The low incidence of V. vulnificus biotype 2 strains in environmental samples may explain why its occurrence in coastal water has not been reported earlier. The isolation procedure may also influence which biotype is detected. Our study suggests differences in the ecology of the two biotypes: biotype 1 was isolated from various environmental sources whereas biotype 2 was rarely isolated. Thus, Danish marine environments are potential reservoirs of V. vulnificus biotype 2. The distribution of eel-pathogenic V. vulnificus strains in Danish coastal waters may have been underestimated because not all eel-pathogenic V. vulnificus strains are indole negative. This knowledge is particular important for fish farmers since the use of brackish water for culturing eels may introduce pathogenic V. vulnificus biotype 2 strains in the farms.

Levels of V. vulnificus correlated with water temperatures

Low densities of V. vulnificus were detected at the seven costal sites in water (0.8 to 19 cell forming units (CFU)/liter) from June until mid-September and in sediment (0.04 to >11 CFU/g) (Høi et al., 1998c). The occurrence of V. vulnificus was strongly correlated to water temperatures, as reported by other researchers (Wright et al., 1996; O'Neill et al., 1992; Kelly, 1982). V. vulnificus was rarely isolated when water temperatures were below 15°C. However, V. vulnificus was detected in coastal waters at one mussel farm at 7°C which is lower than previously reported (Wright et al., 1996).

The control of Danish coastal bathing water is based on presumptive Escherichia coli and coliforms as indicators of water quality. As reported elsewhere, analysis of the data collected in Denmark in 1996 did not reveal any correlation between presumptive E. coli and V. vulnificus (Høi et al., 1998c).

V. vulnificus was mainly found in wild fish when water temperatures were high. The highest incidence of V. vulnificus was found in the gills from eels, but 4 of 75 samples from eelpouts and flounders did also contain V. vulnificus.

Low levels in mussels

Danish oysters and mussels and their surrounding waters were analyzed for V. vulnificus. V. vulnificus was isolated from both water and blue mussels from one cultivation site of a total of 13 areas examined. Concentrations of V. vulnificus in blue mussels were very low (# 10 CFU per gram of mussel tissue) and the low probability of detecting a viable V. vulnificus cell may explain why it was not isolated from other areas. Location of mussels in surface waters provides more favorable temperatures for growth of V. vulnificus during summer time, and the high level of nutrients may support growth and persistence of V. vulnificus, even at low temperatures. V. vulnificus infections have not been associated with consumption of raw shellfish in Denmark or reported elsewhere in Europe. These findings suggest minimal risk associated with consumption of raw shellfish containing V. vulnificus in low numbers.

Conclusions

In conclusions, our findings of V. vulnificus in Danish wild fish during the summer suggest that fishermen, especially those with abrasions on their hands may be at risk for V. vulnificus wound infections. In 1994 and 1995, seven people contracted V. vulnificus wound infections while fishing or handling eels (Dalsgaard et al.,1996b; Bruun, 1997). V. vulnificus infections were not reported during the summer of 1996 when low concentrations (<2 CFU/100 ml) were observed in coastal waters. V. vulnificus levels were probably too low to cause infection, in susceptible individuals, and colder temperatures discouraged bathers from contact with coastal waters. Epidemiological data from 1994 and 1995 suggest that the risk of contracting a V. vulnificus infection following exposure to coastal water was correlated with high water temperature (>20°C). Thus, surveillance and monitoring efforts surveillance and monitoring efforts should be initiated when water temperatures exceed 20°C.

V. vulnificus is a naturally occurring bacterium in warm estuarine environments and should therefore be expected in shrimp produced in brackish-water aquaculture in tropical countries. The European Union imported approximately 75 metric tonnes of these warm-water shrimp through Denmark in 1995 (Dalsgaard & Høi, 1997). The prevalence of V. vulnificus in a total of 107 samples representing 37 consignments of frozen shrimp imported from South East Asia was determined. V. vulnificus was detected in three of 46 (7 %) frozen raw shrimp samples but was not recovered from any of the 61 frozen cooked products. Absence of V. vulnificus in frozen cooked shrimp products indicated proper processing such as adequate heat treatment and sanitation (Dalsgaard & Høi, 1997). The low prevalence of V. vulnificus in frozen raw shrimp products was likely due to its poor resistance to cold (Oliver, 1981; Boutin et al., 1985; Parker et al., 1994). Frozen shrimp products are usually kept at temperatures at -20EC before and after shipping, often for substantial periods, and a significant decrease in the number of V. vulnificus would be anticipated. In conclusion, the absence of V. vulnificus in frozen cooked shrimp products and the low prevalence of V. vulnificus in frozen raw shrimp suggested that V. vulnificus does not constitute a hazard to public health in Denmark (Dalsgaard & Høi, 1997).