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

Abstract

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؇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؇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؇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. Vibrio vulnificus is an estuarine bacterium which comprises two biotypes. Biotype 1 is an opportunistic human pathogen, ubiquitous in the marine environment, capable of causing either primary septicemia after ingestion of raw shellfish or water-borne infections after exposure to seawater (22). Biotype 2 is classically considered an obligate eel pathogen since until now it has been isolated only from diseased eels and no reports exist on its occurrence in water samples. This biotype seemed to be restricted to Japan until it was recently recovered from diseased eels in Taiwan (31) and Spain (8). From November 1989 to July 1990, biotype 2 produced several epizootic outbreaks with variable mortality rates in a brackish-water Spanish eel farm, until the water salinity decreased to 3‰ (7, 8, 11). During this almost 1-year period this microorganism was never recovered from the water of the culture tanks, while biotype 1 was isolated from either the surface of healthy eels or tank water (7, 11). Despite a decrease in water salinity, three outbreaks were recorded in widely separated periods (1990, 1992, and 1994), coinciding with higher temperatures (2, 7, 11). V. vulnificus biotype 2 grows rapidly in the rich medium represented by the eel (10) and may adopt a starvation-survival state when released into the nutrient-diluted environment of culture tanks. We have recently demonstrated, for the first time, that biotype 2 is able to infect eels by the water-borne route, with transmission dependent on the presence of the capsule, as well as temperature and water salinity (4). Th...

Resuscitation of viable but nonculturable cells of Vibrio parahaemolyticus induced at low temperature under starvation

“…After 10 days in the nonculturable state (22 days after commencement of the experiment), no cells were recovered after resuscitation on the amended plates (data not shown). A similar phenomenon has been observed when V. cholerae and V. vulni¢cus were induced into the VBNC state at a low temperature and subsequent resuscitation was performed by temperature upshift [7,16]. The ability to regain the culturability of nonculturable cells might have decreased during periods after entry into the VBNC state.…”

An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies

“…Since culture methods are unable to recover nonculturable cells, we also evaluated the ability of the immunoassay to detect dormant biotype 2 cells. The dormancy was induced in saline-water microcosms maintained at 4ЊC as previously described (9).…”

“…In fact, the two biotypes share several virulence determinants, such as exotoxin production and expression of capsule and siderophore-dependent iron acquisition mechanisms (3,5,12,14), and the exotoxins produced by the two biotypes are equally lethal for eels and mice (7). Encapsulated cells of biotype 2, like biotype 1 cells, can be transmitted to new hosts through brackish water (4), where they survive as freeliving microorganisms for at least a month (9). The two biotypes also share many genotypic and phenotypic traits, including cytotoxin-hemolysin genes, immunologically related outer membrane proteins, soluble intracellular proteins, etc.…”

Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar

“…Our investigations have revealed that biotypes 1 and 2 of V. vulnificus exhibit a high level of phenotypic homology, sharing many biochemical traits, immunologically related outer membrane proteins (OMPs), the expression of several virulence factors, and virulence for mice (2,5,9,(13)(14)(15)(16). Further, we have demonstrated the survival of the eel pathogen in seawater as a free-living microorganism, as well as its water-borne transmissibility (3,11). However, the two biotypes still can be differentiated based on biochemical and serological properties, as well as by eel pathogenicity (16).…”