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Suffredini E., and Caburlotto G. (2016) Vibrio: Types, Properties, and Determination. In: Caballero, B., Finglas,
P., and Toldr, F. (eds.) The Encyclopedia of Food and Health vol. 5, pp. 413-417. Oxford: Academic Press.
2016 Elsevier Ltd. All rights reserved.
microflora and, with the exception of V. cholerae, are not associated with fecal pollution or anthropic impact on seafood
production or harvesting areas, control measures can only
rely on adequate manipulation of the products (rapid refrigeration, uninterrupted cold chain, and proper cooking) and on
the monitoring of the environmental conditions that may
favor the increase of Vibrio levels.
Further to the three major human pathogens (V. cholerae,
V. parahaemolyticus, and V. vulnificus) and to the other species
(V. alginolyticus, V. fluvialis, V. furnissi, V. metschnikovii, etc.)
that are occasionally responsible for disease in susceptible
individuals, several Vibrio species (e.g., V. anguillarum,
V. harveyi, and V. salmonicida) express pathogenicity toward
marine animals (fish, shellfish, or mammals) with considerable economic loss in the aquaculture sector due to large
mortality episodes or to prolonged decrease of production.
Significantly, some Vibrio species display potential for both
severe diseases in humans and in aquatic animals, as is the
case of V. vulnificus, which causes mortality in eels and deadly
wound infections in aquaculture professionals (fishermen,
processors, etc.), or of V. parahaemolyticus, causative agent of
gastroenteritis in seafood consumers and responsible for the
early mortality syndrome in shrimps.
Vibrio cholerae
V. cholerae is the etiologic agent of cholera, an infectious disease responsible, in areas endemic for this microorganism, for
large epidemics and for a high number of deaths, especially in
the presence of inadequate hygiene standards and improper
treatment of wastewaters. The first description of a Vibrio-like
microorganism from patients affected by cholera dates back to
1854 with Filippo Pacini and first isolation of V. cholerae was
achieved in 1884 by Robert Koch. The modern history of
cholera is marked by seven pandemics, during which the disease spread at a global level with reiterated and geographically
shifting epidemic peaks.
V. cholerae includes more than 200 hundred serogroups
according to somatic antigen O, a thermostable polysaccharide
of the cellular wall. Serotypes O1 and O139 are associated with
cholera epidemics, whereas the other serotypes, generally
defined as V. cholerae non-O1/non-O139, are occasionally
responsible for sporadic cases of gastroenteritis, mild forms
of disease similar to cholera, or extraintestinal infections. Serogroup O1 can be further divided in two serotypes (Ogawa and
Inaba) and two distinct biotypes: the biotype Classic, deemed
responsible for the first six pandemics (18171923), and the
biotype El Tor, so called by the name of the quarantine center
where it was first isolated, responsible for the seventh ongoing
pandemic (1961). Biotype differentiation was based initially
on the characteristic hemolytic effect of El Tor variant on sheep
erythrocytes, but this feature became progressively less discriminating during the seventh pandemics as the nonhemolytic
http://dx.doi.org/10.1016/B978-0-12-384947-2.00724-8
413
Table 1
diseases
Species
Source
Disease
V. alginolyticus
Seafood,
seawater
Water or food
Water or food
Gastroenteritis, wound
infection, ear infection
Gastroenteritis
Gastroenteritis
Gastroenteritis, ear infection
Primary septicemia,
gastroenteritis
Gastroenteritis, ear infection,
wound infection
Gastroenteritis
Primary septicemia, wound
infection, bacteremia,
gastroenteritis
V. cholerae
O1-O139
V. cholerae non-O1/
non-O139
V. fluvialis
V. furnissii
V. hollisae
V. metschnikovii
V. mimicus
V. parahaemolyticus
V. vulnificus
Seafood
Seafood
Shellfish
Shellfish,
seawater
Shellfish,
seawater
Shellfish
Shellfish,
seawater
Vibrio parahaemolyticus
V. parahaemolyticus was first described as the etiologic agent of
the shirasu food poisoning in Japan. Late in 1950, a severe
foodborne outbreak (272 cases and 20 fatalities) was reported
in Osaka, with patients suffering from acute gastroenteritis,
severe abdominal pain, vomiting, and diarrhea. Epidemiological investigation led to identify the source of the outbreak to a
small, semidried fish (shirasu) and to the isolation by Dr.
Tsunesaburo Fujino of a new microorganism, Pasteurella parahaemolytica, later renamed Vibrio parahaemolyticus.
Since then, V. parahaemolyticus outbreaks have been repeatedly reported in the world, and at present, V. parahaemolyticus is
considered the first cause of the nonviral infections related to
shellfish consumption in the United States, Japan, and Southeast Asia. This microorganism, in fact, displays the features
common to Vibrio genus, as the natural occurrence in the
marine environment and the correlation with water temperature and salinity, and is therefore commonly present in seafood products, with densities usually lower than 103 cfu g 1,
although such levels may be exceeded in products harvested
from warmer seawaters. In addition, V. parahaemolyticus is also
characterized by a remarkable replication time (as short as
89 min in optimal conditions and in the range of
1218 min in seafood) that allows multiplication of the microorganism and rapid reaching of the estimated infectious dose
(105107 cfu for pathogenic strains) in raw, undercooked, or
cross contaminated seafood products that are incorrectly
stored.
As for V. cholerae, not all V. parahaemolyticus strains are
pathogenic. A strong correlation between human disease and
Vibrio vulnificus
V. vulnificus is the most clinically serious Vibrio infection in
developed countries, accounting in the United States for
almost the totality (95%) of the deaths associated with seafood
consumption. V. vulnificus is responsible, in fact, for severe
infections in susceptible individuals, with two distinct disease
syndromes, primary septicemia (with a case-fatality rate of
approximately 50%) and necrotizing wound infections.
Based upon the variation of their biochemical profiles,
three V. vulnificus biotypes can be distinguished. Strains
belonging to biotype 1 are most commonly associated with
human disease and can be isolated from seafood products
(finfish and bivalve mollusks), biotype 2 is usually considered
as an eel pathogen, while strains belonging to biotype 3, isolated in Israel in 1996, affect humans through wound infection
and bacteremia. V. vulnificus is a widespread inhabitant of
estuarine environments; its presence has been reported in seawater, shellfish, crabs, etc., and a strong correlation has been
detected between water temperature and its isolation.
Infections are usually sporadic, and in almost all of them, the
patient has an underlying chronic disease (e.g., cirrhosis or liveror blood-related disorders) leading to elevated serum iron levels;
other cases are reported in the presence of pathologies as chronic
renal or gastric diseases, immunosuppression, and diabetes.
Many studies have attempted discrimination of virulent and
avirulent strains by comparison of clinical and environmental
strains, but due to the high degree of genetic diversity, a definitive indicator has yet to be identified. V. vulnificus invasivity is
related to the high variety of virulence factors expressed by this
species: the polysaccharide capsule, which is produced by nearly
all strains and confers an opaque aspect to V. vulnificus colonies,
is essential for infection as it protects the microorganism from
phagocytosis and from the activity of the complement system;
the endotoxic lipopolysaccharides is responsible for the typical
415
Vibrio Detection
Cultural Methods
In routine analysis of food products, the detection of pathogenic vibrios mostly relies on conventional cultural methods,
followed by biochemical and, for V. cholerae, serological testing. Such approach, applied in internationally recognized
methods as ISO/TS 21872 and the FDA Bacteriological Analytical Manual, includes a preenrichment step in a nonselective
medium (usually alkaline peptone water with an appropriate
concentration of NaCl) and provides resuscitation of bacteria,
including cells in a VBNC state and cells stressed due to product refrigeration or freezing. Enrichment is then followed by
isolation of colonies on selective agar media, with thiosulfate
citrate bile salt agar (TCBS) being the most commonly used.
Random selection of colonies with characteristic features represents a critical point of the procedure, as species of interest in
food analysis (V. cholerae, V. parahaemolyticus, and V. vulnificus)
may be overgrown both during enrichment and on agar plates
by other vibrios as V. alginolyticus or by other genera, therefore
making isolation of relevant colonies less efficient. In this
context, the formulation of new selective and differential
media (e.g., chromogenic media) to use in association with
TCBS has progressively improved the chance to differentiate
the species targeted by the analysis. The isolation of colonies is
critical also under another prospective: as pathogenic strains of
Vibrio species often represent a small minority of the microbial
flora, the number and the proportion of characteristic colonies
selected for further testing may significantly affect the probability of detecting the pathogenic strains of a species (e.g.,
isolates belonging to the serogroups O1/O139 of V. cholerae
or tdh-positive/trh-positive V. parahaemolyticus strains). Further
to the isolation step, the total performance of the cultural
methods may be challenged also by the identification of the
isolates; the variability of the biochemical profiles of Vibrio
environmental strains, in fact, may negatively affect the accuracy of the biochemical identification by conventional tests,
dichotomous keys, or miniaturized tests, leading to inconclusive results or to misidentifications.
Conventional cultural methods for Vibrio analysis show,
overall, important characteristics that encourage their routine
adoption, including technical easiness, relatively low costs, and
integration with laboratorys workflows. On the other hand,
however, the cultural analytic process is time-consuming (45
days to obtain the identification of the microorganisms),
labor-intensive, and, lastly, unable to provide a characterization of the virulence factors associated with human illness. To
this aim, a progressive integration of the cultural and molecular analytic methods for Vibrio analysis has been pursued, as
testified by the introduction, also in the aforementioned
methods for official controls, of polymerase chain reaction
(PCR) protocols for sample screening, improvement of species
identification, and characterization of pathogenicity factors.
Molecular Methods
During the past years, nucleic acid detection and amplification
methods have significantly broadened the spectrum of diagnostic tools for the analysis of Vibrio species, and to date, a large
number of both conventional and real-time PCR protocols,
isothermal amplification (LAMP) procedures, and some hybridization methods have been published for the detection, identification, characterization, or enumeration of potentially
pathogenic species. One of the major advantages accorded by
nucleic acid-based techniques is related to the fact that, by
targeting directly genetic sequences responsible for or associated
with virulence of defined Vibrio species, they provide more
reliable information on the risk associated with the presence of
pathogenic vibrios in a product. A second advantage is given by
the substantial reduction of the time required for the analysis,
with detection of potentially pathogenic species completed, for
certain protocols, in 24 h or less. In this sense, the molecular
techniques represent the most suitable analytic approach for
industries and food producers to prevent release on the market
of products containing pathogenic Vibrio species, as well as for
control authorities to provide timely results, therefore ensuring
food safety and preventing outbreaks.
Molecular detection methods for Vibrio have been directed,
depending on the scope of the analysis, to either conserved
region of the genome (species detection or isolates
identification) or genes encoding or associated with virulence
(detection of potentially pathogenic strains). PCR targeting
total V. cholerae, for example, has largely relied on the detection
of conserved and widespread genes as toxR, which is involved
in the regulation cascade of virulence-associated genes; ompW
gene, which encodes an outer membrane protein and is conserved in both clinical and environmental isolates; and hlyA
gene, a nonclassical fragment of the hemolysin gene present in
O1, O139, and non-O1/non-O139 V. cholerae strains. Identification of pathogenic strains has, on the other hand, mainly
targeted genes associated with the CT production, including
ctxA and ctxB, or codifying for other virulence factors as zot, ace,
tcp, or st genes. PCR protocols, however, have also been successfully developed for rapid characterization of strains, for
example, with the identification of V. cholerae O1 epidemic
strains through ctxB allelic discrimination or by using rfb gene
for the molecular differentiation of O1 and O139 isolates.
Compared to V. cholerae, a multiplicity of sequences has
been proposed in the last years for the detection of
V. parahaemolyticus in food samples and seawater or for the
identification of isolates. The 16S ribosomal RNA gene, for
example, was repeatedly evaluated as a possible target for
molecular analysis, but high sequence homology with other
species (e.g., 99.7% with V. alginolyticus) and the presence
of intrastrain polymorphisms have hampered its use. GyrB
gene, encoding the B subunit of DNA gyrase, and fragments
of the hsp (heat shock protein) gene, being characterized
by a lower sequence homology with other Vibrio species,
were also suggested as possible targets for differentiation of
V. parahaemolyticus. Three other targets, however, have been
extensively used and compared, especially for the application
in food analysis: the tlh gene, codifying for a thermolabile
hemolysin (TLH) in V. parahaemolyticus and, at present, widely
used in the United States and Japan; the toxR gene, homologous to the regulator gene of V. cholerae and conserved with
low sequence identity in the whole Vibrionaceae family, whose
application is common in Southeast Asia and Europe; and the
pR72H sequence, a restriction fragment generated by HindIII
enzyme that includes a coding (phosphatidylserine synthase
gene) region and a noncoding region and that appears to be
conserved in V. parahaemolyticus strains. In contrast to methods
for V. parahaemolyticus identification, protocols aiming at the
detection of potentially enteropathogenic strains have
unequivocally targeted the tdh and trh genes, often in multiplex
format with each other or with one of the aforementioned
sequences for species identification.
For V. vulnificus, identification has been mainly based on the
cytotoxinhemolysin gene (vvh), thought to be common to all
biotypes, although potential problems of gene loss associated
with the use of this target have been reported. Alternative genomic loci, as the 16S rRNA gene, have been therefore investigated,
both for identification purposes and to achieve differentiation
between clinical and environmental strains; a 17-base sequence
variation in the 16S rRNA gene, in fact, was shown to have a
statistically significant correlation with clinical isolates. Other
genes and sequences, including the virulence-correlated gene
(vcg), a siderophore-encoding gene (viuB), and the capsular
polysaccharide operon, have demonstrated, however, comparable or higher discriminative power and have been widely
applied for V. vulnificus characterization.
In the analysis of foods in which Vibrio species may be
present, nucleic acid amplification-based methods usually provide results with high specificity and information relevant for
the characterization of pathogenicity potential of microorganisms. Nonetheless, a major drawback of these methods is that,
due to the low concentration of the targets and the high inhibitory effect of food matrices on PCR amplification, sensitivity
levels compatible with food analysis can be achieved only on
417
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Further Reading
Relevant Websites
Croci L and Suffredini E (2013) Real-time PCR detection of foodborne pathogenic
Vibrio. In: Rodrguez-Lazaro D (ed.) Real-time PCR in food science: current
technology and applications, pp. 135148. Norfolk: Caister Academic Press.
http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm2006949.htm
FDAs Bacteriological Analytical Manual (BAM).