Volume 10, lssue I 77

VIBRIO PARA HAEMOL YTZCUS AND RELATED HALOPHI LI C

VI BRI OS
Authors: Sam W. Joseph
Rita R. Colwell
Department of Microbiology
University of Maryland
College Park, Maryland
James B. Kaper
Center for Vaccine Development
University of Maryland Medical School
Baltimore, Maryland
Referee: A. von Graevenitz
Institute of Medical Microbiology
University of Zurich.
Zurich. Switzerland
I. I NTRODUCTI ON (HI STORY)
Thirty years have passed since the first isolation of Vibriopatahaemolyricuus. Volumes
of literature have been written about this organism and it has become recognized as a
major cause of food poisoning in areas of the world where seafood is a major staple of the
diet. The history of V. parahaemolyricus traces back to October 20and 21,1950, when an
outbreak of food poisoning occurred in the southern suburbs of Osaka, J apan. Of the 272
patients who suffered acute gastroenteritis, 20 died. The symptoms of the gastroenteritis
included acute abdominal pain, vomiting, and diarrhea, with watery and, in some cases,
bloody stools. The food suspected to have caused the food poisoning was a small, half-
dried sardine, Engradis japonica Houttuyn, called "shirasu" in J apanese. The sardine is
boiled in salt water and eaten when partially dried.'
Based on his expertise derived while assigned at Maymyo, Burma in 1944 as an army
surgeon sublieutenant, T. Fujino and his co-workers at the Research Institute for
Microbial Diseases (Osaka University) carried out the bacteriological investigation of
specimens from the intestinal tracts of the victims and the shirasu suspected to be the
source of the organism.*-' During his Burma tour of duty, he diagnosed two cases of
bubonic plague, by guinea pig inoculation, obtaining a pure culture of plague bacilli from
spleen and ascites. Thus, Fujino injected filtrates of the homogenized shirasu via the
intraperitoneal route into guinea pigs, the objective being to exclude chemical poisons
and pleuropneumonia-like, filterable bacteria as causative agents. Purulent peritonitis
was induced in a guinea pig. The saline homogenate of shirasu was centrifuged and the
supernatant was inoculated into various culture media and incubated at 37C under
aerobic and anaerobic conditions. Salmonella. Shigella. Clostridium, and Proteus were
not found. However, many colonies of what was thought to be hctobacillus and
Sruphylococcus were observed, along with many Gram-negative rods. The Gram-
negative rods formed whitish, opaque colonies and appeared microscopically to be
similar to Escherichia. In a smear of a single colony, Fujino observed a few Gram-
negative bacteria with swollen edges among the numerous Gram-negative rods. Attempts
to separate the two kinds of Gram-negative bacteria by transfer to fresh agar plates were
unsuccessful. Thus, hypothesizing that one of the two Gram-negative bacterial strains
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78 CRC Critical Reviews in Microbiology
was a pathogen and would inhibit growth of the other strain in vivo and grow more
rapidly, Fujino and his co-workers attempted separation of the two by animal passage. A
suspension of colonies containing the two kinds of Gram-negative bacteria was injected
intraperitoneally into mice. Several hours later, when symptoms of illness appeared in
the mice, samples of ascitic fluid were taken and inoculated into other mice. The animal
passage was repeated once again, and after 2% to 3 hr, symptoms appeared in the mice.
The ascitic fluid from the last set of mice to be passaged was inoculated onto blood agar
plates and the plates were incubated at 37C for 10 hr. Two types of colonies were
observed on the plates: a nonhemolytic, slender, Gram-negative rod, which on
subsequent testing in pure culture produced acid and gas in glucose and did not hydrolyze
gelatin, and a hemolytic, fat rod demonstrating bipolar staining. The latter isolated
produced acid - but not gas - from glucose, liquified gelatin, and proved pathogenic
for mice. The gas-producing strain was identified as Proreus morganii, but the
anaerogenic strain could not be identified at that time. The history of this series ofevents
in the isolation of V. parahaemolytica is nicely detailed by Fujino' and by Miwatani and
Takeda.6
The anaerogenic, unidentified bacterium of Fujino was tested further and found to be
actively motile by means of a single, polar flagellum, resembling Vibrio cholerae.
However, it did not react with V. cholerae antiserum and the long axis of the bacterium
was not curved. Based on these features and on its bipolar staining, the new species was
named Pasteurella parahuemolyticus [sic], n. sp.'
Fujino2 subsequently isolated the same bacterium from the intestinal contents of a
victim of food poisoning, and Fujino et al.' reported the discovery of the new pathogen at
the 25th Annual Meeting of the J apanese Association for Infectious Disease in 195 1. An
extensive, detailed description of Pasreurella parahaemolytica first appeared in
J apanese2 and later in English.'
In the fall of 1955 I. Takikawa,' from the National Yokohama Hospital, visited the
laboratory of Dr. Fujino. An outbreak offood poisoning had occurred at the Yokohama
Hospital, involving I20 patients, with no deaths. A Gram-negative rod with a single,
polar flagellum was isolated on 4% salt agar used to isolate Staphylococcus. The culture
appeared to be similar to Pasreurella parahaemolytica. but was halophilic, the salt-
requiring nature of P. parahaemolyrica not having been recognized previously. In the
hospital incident, the food implicated was brine cucumber (pickles) served to the patients
and it was speculated that the causative agent was a halophilic organism from mackerel
which had contaminated the cucumbers.7.*
The description of Pasreurella parahuemolyticus provided by Fujino et a].' was as
follows: "A rod-shaped organism. 1 to 3 p in length, with rounded ends, which is slightly
pleomorphic on blood agar. It shows a tendency toward bipolar staining, and when
stained with weak Giemsa solution for 15 min, gives a clear bipolar picture. Direct smears
from animal blood show that it has a thin capsule. It is monotrichal. A very few organisms
can be seen to move like Vibrio comma. When an 18-hr blood agar culture is examined
under dark field illumination one or two bacilli per field can be seen to move. Electron
microscopy shows that each bacillus has a single flagellum. Movement can also be seen in
direct preparations from mouse ascites. It liquifies gelatin, and shows a hemolytic ring in
blood agar. It grows aerobically on plain agar forming white, opaque colonies."
Takikawa7 reported that his isolate was closely related in its characteristics to the
organisms described by Fujino et al.,3 but he concluded that it should be considered a
member of the genus Pseudomonas." i.e., Pseudomonas enreritis. Thereafter, in J apan,
the organism was referred to as the so-called "pathogenic halophilic bacterium".
Miyamoto et aL9 stated that the organism should be placed in the genus Aeromonas
because of its fermentative utilization of glucose, and proposed a new genus,
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Volume 10, Issue I 79
Oceanomonas, for the halophilic, fermentative bacteria. Thus Miyamoto et aL9 proposed
that the clinical isolates of Fujino et aL3 and Takikawa" should be classified together
with their own strains isolated from feces and seawater in the new genus as 0.
parahaemolytica, 0. enteritidis. and 0. alginolytica.
Sakazaki and co-workers" first placed the strains into the genus Vibrio after
examination of 1072 such strains. Subsequently, K parahaemolyticus was divided into
two biotypes, which Zen-Yoji et a1.I' concluded should be separated into two species.
This was proposed by Sakazaki" in 1968, who designated biotype 2 as V. alginolyticus.
The main differences between the biotypes were acetoin production, sucrose
fermentation, growth in 10% NaCI, and swarming on agar plates containing 2 to 7%
NaCI. V. parahoemolyticus is negative for these characteristics and K alginolyticus is
positive.
The 8th edition of Bergey ' s Manual of Determinative Bacteriology" designated V.
alginolyticus as biotype 2 of V. parahoemolyticus. but the Subcommittee on the
Taxonomy of Vibrios of the International Committee on Systematic Bacteriology
recognized the separation of these two phena as distinct species." A third biotype was
discovered re~ently.'~''' Previously regarded as "lactose positive" vibrios, the nomen-
clature Vibrio vulnficus was proposed.18
Detailed reviews of the history of the taxonomy of K purahaemolyticus have been
provided by Colwell,' Cabassi and Mon,l9 Miwatani and Takeda,6 and Chatterjee.20 In
recent years, proposals have been made to reassign this species from the genus Vibrio to
the family Brucellaceae by Chatterjee" and to the genus Beneckea by Baumann et a1."
The definition of the genus Vi b r i ~' ' , ~~ includes straight rods and embraces V.
parahuemolyticus. since it produces a polar flagellum when grown in broth, even though
it may be peritrichously flagellated when grown on agar medium (vide infru). ,While V.
parahuemolyticus may require slightly higher concentrations of Na' than V. cholerae.
which requires only trace concentrations, V. cholerae demonstrates a salinity optimum of
approximately 1% NaCl as opposed to 2 to 3% NaCl for V. parahaemolyticus,"
differences not suitable for generic separation. In vitro DNA/ DNA hybridization data
also show that V. parahuemolyticus is more related genetically to K cholerae than to
other Beneckea species." Recent publications have demonstrated a concensus on
retaining this species in the genus Vi br i ~. ~'
11. TAXONOMY
A. Morphological, Cultural, and Biochemical Characteristics of Y. parahoemolyticus
I . Morphology
Viewed with the microscope, V. parahaemolyticus appears as a straight, sometimes
curved rod with rounded ends, pleomorphic, and usually occurring as single cells but
occasionally in chains. Undulating filaments and spheroplasts are often present." V.
parahuemolyticus is Gram-negative and occasionally a concentration of the Gram stain
appears at the polar extremities of young cells in the process of dividing.I9 The
flagellation of this organism has been extensively studied and most workers agree that V.
parahaemolyticus possesses a single, polar, sheathed flagellum when grown in liquid
medium and, in addition, unsheathed, peritrichous flagella when grown on solid
media.22.26-29 Ki mura et aL3' reported that formation of peritrichous, but not polar
flagella, was inhibited in media of pH 8.5 and higher.
2. Physiology and Resistance to Antibacterials
V. parahuemolyticus grows within a temperature range typical of mesophiles, with a
minimum growth temperature of 9 to IO'C, a maximum growth temperature of
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80 CRC Crirical Reviews in Microbiology
approximately 4 4 O C, and an optimum between 35 and 37' C."-3J Interacting effects of
pH, temperature, and salt concentration can result in slight alteration in the temperature
limit on growth. Be~chat'~ reported moderate growth occurred at 5C when the medium
was in the alkaline pH range. The pH range of growth of V. paruhaernolyricus is fairly
wide. Initial pH of media permitting growth ranges from pH 5 to 1 I, with an optimum
between pH 7.5 and 8.0. 1' *34J5 The ability of V. parahoemolyticus to grow at high pH has
been exploited in the development of selective isolation media (vide infru).
V. parahuemolyticus is a moderate halophile for which salt requirement and salt
tolerance have been extensively studied. Growth occurs at NaCl concentrations of 0. I M
to approximately 1.2 M, with an optimum salt concentration for growth of about 0.5 M
NaCI.1'*3'-39 Distilled water inactivates V. paruhaemolyricus, with a 90% reduction of
viable cells within 1 to 4 min." Other cations, e.g., Li' and K', can produce a sparing
effect on the specific requirements for sodium, but the minimal, essential requirement for
sodium is approximately 0.003 to 0.007 M Na', when the cells are grown in synthetic
media."*41 When nonmetabolizable LiCl, sucrose, and ethylene chloride are used to
replace sodium ion in a synthetic medium, each is capable of providing osmotic
regulation. In addition to this requirement, sodium ion appears to be necessary for
protein synthesis, which is inhibited in sodium ion-omitted medium containing osmotic
agents."*'"
In a study of a marine pseudomonad Drapeau and MacL ~od"~ found that washed
cells, when incubated with l'C-a-aminoisobutyric acid, were able to accumulate this
analogue inside the cell, without metabolizing it, and required the presence of Na' in the
medium. K', Rb', NH4', Li', and sucrose could not substitute for Na' in the transport
process .
In similar fashion Sakai and Sakai'"found that a group of marine bacteria, which they
term TH (terrestrial halophilic) type (and include V.pmahoemolyricus) is similar to their
M H (marine halophilic) type which is described as follows: 'M H-type bacteria seemingly
require Mg" for the oxidation of substrates, however, Na' only is able to play
the above physiological roles. Na'. in fact, prevents the lysis of cells and accelerates
cytochrome oxidase, the electron transport chain, and ATP formation of oxidative
phosphorylation. Na* also functions in the Na', K'dependent active transport of
nutrients into the cells. K'is accessonly needed only in the presence of Na'. Consequently
MH-type indispensably needs Na' as the sole cation of supporting growth. This type
belongs to psychrophilic bacteria because it lacks growth capacity at 37" C." The
exceptions which separate the TH type from the MH type are described as follows: "The
Na' requirement is less than that of MH-type for the prevention of lysis, the oxidation of
substrates, the electron transport system, the cytochrome oxidase, and growth. This type
is able to grow well at 37C and thereby belongs to mesophilic bacteria." The combined
effects of water activity, solute, and temperature on the growth of V. porohaemolyticus
have been studied with the limiting water activity for growth depending upon the solute
employed. "
An interesting physiological feature of V. parahuemolyricus is its extremely short
generation time. K at~h' ~ reported a generation time as short as 8 to 9 min, although 10 to
12 min is more commonly observed. UlitzurU studied 30 strains of V. parahoemolyticus
and observed two major groups, based on generation time. One group of strains
demonstrated a short generation time, ix., 12 to 14 min, and a second group, a longer
time of 20 to 25 min.
V. parahoemolyticus, like other vibrios, is a facultative anaerobe, possessing both
respiratory and fermentative metabolism. I t produces a catalase and cytochrorne
oxidase. It is sensitive to the vibriostatic agent O/ 129 (2, 4 diamino-6, 7-diisopropyl
pteridine) and is, in general, sensitive to chloramphenicol, gentamicin, kanamycin,
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Volume 10, Issue 1 81
nitrofurantoin, tetracyline, doxycyline, and streptomycin and is resistant to ampicillin,
carbenicillin, clindamycin, colistin, erythromycin, and penicillin.' ' J ~ ~ ~ ~ - ~ ~ V. para-
haemolyticus and V. alginolyticus from human and environmental sources, examined by
the minimal inhibitory concentration (MIC) method, are uniformly susceptible to
chloramphenicol and tetracycline within attainable serum levels. Most strains are
resistant to ampicillin and exhibit p-lactamase activity, which accounts for this
resistance. Occasional multiresistance is noted, but thus far plasmid-linked drug
resistance has not been shown. Susceptibility to gentamicin can be demonstrated with
agar diffusion, but examination for MI C in brain heart infusion broth, containing 2%
NaCl, yields inconclusive results because of diminished gentamicin activity.*8
3. Biochemical and Nutritional Characteristics
Several investigators have provided numerous biochemical and physiological
characteristics of V. parahaemolyticus. ' 1*r 9-5'
V. parahaemolyticus ferments glucose without the production of gas and does not
produce acetoin, i.e., it is Voges-Proskauer negative, ferments galactose, levulose,
maltose, mannitol, mannose, ribose, and trehalose, but does not ferment adonitol,
dulcitol, erythritol, inositol. lactose, melizitose, raffinose, rhamnose, salicin, sorbose,
sorbitol, sucrose, and xylose. Strain variation can be observed in the fermentation of
arabinose, cellobiose, and melibiose.
V. parahaemolyticus is positive for production of indole, and possesses lysine and
ornithine decarboxylase, reduces nitrates to nitrites, and degrades gelatin, chitin, starch,
casein, and lecithin. It is usually negative for arginine dihydrolase activity, hydrogen
sulfide production (vide infru), urease, phenylalanine deaminase, and luminescence. An
important, but variable, characteristic is hemolysis of blood, known as the Kanagawa
phenomenon (KP), measured by using a high salt-containing medium (vide infra).
I n general, strains of V. parahaemolyticus utilize the following compounds as sole
sources of carbon: D-glUCOnate, acetate, citrate, propionate, DL-malate, pyruvate,
fumarate, lactate, succinate, ketoglutarate, ethanol, propanol, L-serine, L-leucine, L-
glutamate, L-arginine, L-proline, L-tyrosine, L-alanine, L-arginine, and L-histidine. I t
cannot utilize phenol, catechol, malonate, oxalate, glutarate, tartrate, p-hydroxyben-
zoate, butanol, benzoate, palanine, L-ornithine, L-citrulline, or spermine."J 4J o
4. Deoxyribonucleic acid (DNA) Base Composition and Nucleic Acid Homology
The composition of chromosomal DNA of strains of V. parahaemolyticus is in the
range 44 to 46% guanine plus cytosine (G + C). DNA/DNA reassociation studies have
been performed by several investigators and intraspecific values are usually >90% (at 60
to 63C).24J z-54 Interspecific values between V. parahaemolyticus and V. cholerae range
from 16 to 56% (at 60C) with most values falling between 20 to 30%.52Js V. alginolyticus
shares 60 to 70% homology with K parahaemolyticus and strains of the "lactose positive"
vibrios share 40 to 50% homology with V. parahaemolyticus and V. al gi nol yt i cu~. ~~ V,
parahuemolyticus reveals only a low amount of homology with other marine vibrios,
specifically, V. anpillarum. Reassociation values between these two species are in the
range of 20 to 30% with the membrane filter whereas values of only 4 to 5%
homology were observed using the S 1 endonuclease assay, a more stringent method for
estimation of DNA homology.56
a PIasmids
Extrachromosomal elements of V. parahuemolyticus are evident but no specific
function has been assigned to them. Guerry and Colwel15' found that 40f 12strains from
human and environmental sources had multiple plasmid species of cryptic function.
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82 CRC Critical Reviews in Microbiology
Table 1
MINIMAL CHARACTERS FOR IDENTIFICATION OF
V. PARA H A EMOL YTICUS
Sign
Gram-negative. asporogenous rod
lndophenol oxidase
Glucose. acid under a seal of petroleum
Glucose. gas
D-rnannitol. acid
Sucrose. acid
Acctylmethylcarbinol
Hydrogen sulfide, black butt
L-lysinc dccarboxylax
L-arginine dihydrolase
L-omithine dccarboxylase
Growth in 1% tryptone broth
Growth in 1% tryptone broth with 8% NaCl
Growth in 1% tryptone broth with IO?6 NaCl
Growth at 42OC
From Hugh. R. and Sakazaki, R.. J. Con/: Publ. Healih Lab. Direct.. 30, 133, 1972.
With permission.
5. Minimal Characteristics for Identijication of V. parahaemolyticus and Biochemical
Variation
A list of minimum characteristics for identification of V. parahaemolyticus has been
reported by Hugh and Sakazaki and C0lwe11~~ (Table 1). A comparison of
characteristics for V. parahaemolyticus and related species and genera is provided in
Table 2. Individual characteristics should not be overemphasized for identification, since
strain variability is common. Conversely, even if a strain, particularly an environmental
isolate, fulfills all criteria, further biochemical characterization, even DNA/ DNA
hybridizations, sometime may be required for complete separation and identification. A
more complete characterization of V. parahaernolyticus, beyond a minimum key
character analysis, will occasionally distinguish an isolate from similar, but as yet
incompletely characterized, marine bacteria.
Exceptions to the list of minimal characteristics should be noted. For example, sucrose
fermentation is a primary differential characteristic and is the basic criterion employed in
most of the isolation methods recommended for V. parahaemolyticus (vide infra).
Nevertheless, many sucrose positive strains have been reported. ColwelldO found 6% of
the strains of V. parahaemolyticus examined to be sucrose positive. J oseph and
Gilmour6 also reported several such strains. Kampelmacher et a1.62 isolated sucrose
positive V. parahaemolyticus from mussels and Fujino et al. found 2% of their marine
isolates to be sucrose positive. Of 1484 strains of this species isolated from British coastal
waters, 6.9% was sucrose positive. Success in detecting- acid produced during
fermentation of sucrose by V. parahaemolyticus can vary, according to the medium and
concentration of sucrose used. Baross@ recommended the use of OF basal medium,6s
with 0.5% final concentration of added sucrose. Separation of V. parahaemolyticus and
V. alginolyticus must be based on several criteria, such as acetoin production, growth in
10% NaC1, swarming on agar plates, and methyl red reaction, in addition to sucrose
fermentation.
Lysine and ornithine decarboxylases are usually present in V. parahaemolyticus, but
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84 CRC Critical Reviews in Microbiology
many exceptions will be observed. Fujino et al. reported 4 to 5% of the strains tested to
be negative for ornithine and/ or lysine decarboxylase. The percentage of ornithine
positive strains isolated from samples collected in Tokyo has been reported to be as low
as 78%.
Production of hydrogen sulfide should be tested by reading the reaction occurring in
the butt of a tube of Kliglers Iron agar (KIA) or triple sugar iron agar (TSI). Sakazaki et
al. reported that none of 1702 strains examined were positive for H2S using TSI or SIM
media. ColwellW found nearly all of the strains of V. cholerae and V. parahaemolyricus
examined to be H2S, using more sensitive methods for detection of HIS production.
Twedt and co-~orkers ~ found nearly all of their cultures to be positive using SIM and
lead acetate agar (Difco). Indeed, hydrogen sulfide production on Russells Triple sugar
agar, but not on TSI, has been suggested as an aid in the identification of this species.67
Two other characteristics, although not included in the set of minimal characteristics
listed by Hugh and Sakazaki,* are noteworthy. Indole production is usually positive, but
indole negative strains have been implicated in several outbreaks of food poisoning in
Tokyo.66 V. parahaemolyticus is usually considered to be urease but
several investigators have reported other findings. Colwel16 found that 97% of V.
parahaemolyricus strains tested was urease +and Chitu et a1.,68 who examined 8 strains
of V. parahaemolyricus isolated from salted herring and roe, found 6 to be urease
positive. Sakazaki et al. reported 4% of the strains tested to be urease positive and
Kaper et al. (in preparation) reported 13 urease positive strains of 19 isolated from
shrimp. The isolation of urease positive strains from human infections is further
substantiated by the reports of Huq et al.69 and Lam and Y ~o. ~
B. Other Biochemical Studies
The cell envelope of V. parahaemolyticus has been characterized by Deneke and
Colwell and Tamura et al.,72*73 and has been shown to possess poly p-hydroxybutyric
acid and a highly branched a-Dglucan. Fatty acid composition was examined by
Rietschel et al., Oliver and C~l wel l ,~ and Beuchat and W~rthi ngton,~~ and the
predominant fatty acids were C12, C14, C16:l, C16, and C18:l. Using high pressure
liquid chromotography, Me11et al.,? also found C14:l, C13, CIS, C17, C18, C19, and C21.
Superoxide dismutase (SOD) and catalase levels in V. parahaemolyricus were examined
by Daily et al.,* who found only one detectable SOD enzyme in most of the strains
tested, as compared with the three SOD enzymes of E. coli. A c-type cytochrome, with
the capacity to bind carbon monoxide, was reported for the soluble fraction of cell-free
extracts of V. parahaemolyricus by Collins and Knowles (unpublished observations,
quoted by West et al.. The function of this cytochrome is unknown but evidence is
accumulating that such cytochromes are associated with organisms having complex,
branched respiratory Other enzymes of V. parahaemolyricus which have been
studied include phospholipase A, lysophospholipase and glycerophosphorylcholine
diesterase, lysophospholipase, lecithinase,82 aspartokinase, amylase, gel ati na~e,~
and acid and alkaline phosphatases.86
111. I SOLATI ON AND ENUMERATI ON OF V. PAWHAEMOLYTI CUS
V. parahaemolyticus has captured the interest of microbiologists in many fields,
especially medical, food, and environmental microbiologists. As a result, a variety of
methods have been developed specifically for the isolation, enrichment, and enumeration
of V. parahaem~ly~icus.~~*-~ Most of the methods involve direct plating of samples on
an agar medium, or initial enrichment in broth, followed by streaking and isolation on an
agar medium. Direct plating is sufficient, in most cases, for fecal specimens, but food
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Volume 10, Issue I 85
samples and samples collected from the environment frequently, if not always, require
enrichment. A few procedures require special incubation of media at elevated
temperatures (42 to 43C) or under anaerobic conditions to provide additional
selectivity.
Typical colonies of V. parahaemolyticus are picked and tested using a set of
biochemical, physiological, and serological tests. If immediate processing of a sample is
not possible, samples can be frozen, but the risk of stress and death of cells must be
recognized. Samples also can be transferred to a transport medium, such as Cary-Blair
medium" or that described by LeClair et al.9'
A. Enrichment Broth
Initial contributions to methodology for isolation and enumeration of V. parahaemo-
lyricus were made by J apanese investigators who developed selective media employing
agents or conditions such as Teepol (a neutral detergent), bile salts, dyes, high salt
concentrations, and alkaline pH. Glucose-salt-Teepol broth (GSTB), devised for
isolation of V. parahaemolyticus, contains methyl violet and Teepol and is adjusted to
pH 9.4.93 GSTB has been modified subsequently by substitution of lauryl sulfate for
Teep01.~~ Arabinose-ethyl violet broth" contains ethyl violet and is adjusted to pH 8.6. A
collaborative study reported by Petersenm3 described Horie broth as yielding MPN
values ten times greater than GSTB. This medium was modified by Kaper et by
substitution of galactose for arabinose. Other enrichment broths proposed for V.
parahuemolyticus include the SWYE medium of Kaneko and Colwe1I9' and salt-colistin
broth (SCB) of Sakazaki." A salt-polymyxin B broth (SPB) was proposed by
Kampelmacher et al.," and a more recent version of SPB, used by Sakazaki (personal
communication), contains polymyxin B (500 pg/m!l), salt (2% NaCl), and nutrient broth. A
survey of several broths proposed for V. parahaemolyticus by Nakanishi and MuraseIoo
showed that SPB provided the best recovery when raw fish was examined for the
organism.
A simple medium, containing Teepol and 3% NaCl in a phosphate buffer, was reported
by Chun et a1.'" and Teepol has also been employed in a water blue-alizarin yellow
br~th. ~' Kri sten~en'~~*'~' utilized a meat broth amended with 7% NaCl and 0.3% alkyl
benzene sulphonate, to which starch and chitin had also been added. Trypticase Soy
broth (TSB), amended with 7%NaCI, has been used by Vanderzant and Nickelsonmas an
enrichment, with alkaline-saline peptone wateriM as a second enrichment after
incubation for 8 to 12 hr. Sakazaki" has used the tellurite-bile salt broth of MonsurIo5 for
secondary enrichment. Bismuth sulphite phenol red (BSPR) broth, containing sucrose,
NaCI, mannitol, and bismuth sulphite, was employed by Thompson and Trenh~l m"~ for
isolation of V. parahuemolyticus. A recent study of V. parahaemolyticus in Dutch
mussels'07 included a comparison of five enrichment broths. The conclusion was that
enrichment in the 5% NaCl meat broth of Kampelmacher et incubated at 37C
provided the best recovery of V. parahaemolyticus.
B. Plating Media
A wide variety of plating media, many of which were originally developed for isolation
of V. cholerae, has been employed for recovery of V. parahuemolyticus. Undoubtedly,
the most commonly employed agar medium is the thiosulfate citrate bile salts sucrose
(TCBS) agar of Kobayashi et al.''' This medium inhibits most of the other bacterial
species comprising the fecal flora primarily because of the presence of bile salts, sodium
citrate, and the highly alkaline pH of 8.6. Good differentiation of Vibrio species is
provided by the sucrose fermentation reaction. V. cholerae and V. alginolyticus ferment
sucrose, producing yellow colonies, indicated by color change in the brom thymol blue
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86 CRC Critical Reviews in Microbiology
and thymol blue included in the medium. V. parahaemolyticus, generally, is not a
sucrose-fermenting organism on TCBS agar, and therefore produces a bluish or blue-
green colony. Although TCBS is a medium useful for isolation of V. cholerae and V.
parahaemolyticus, the selectivity of the medium is such that it may not always suppress
growth of other organisms, such as Proteus spp., Aeromonas spp., or Staphylococcus
spp. Furthermore, differentiation among species of pathogenic and other, as yet poorly
characterized, species of Vibrio found in the aquatic environment is not always achieved
and additional tests, comprising a follow-up screening, are required. Modifications of
TCBS have been proposed, such as elevation of the NaCl content,Iw addition of bile salts
derivatives,"' or alkyl benzene sulphonate. lo* Significant variation in selectivity can be
observed when the several brands of TCBS agar are compared.
Other media have also been devised which purport to be selective. Several plating
media utilize sucrose fermentation as a differential characteristic. For example brom
thymol blue (BTB) Teepol agar of Akiyama et al.93 includes Teepol for selection and
sucrose fermentation (via the brom thymol blue-thymol blue indicator) for differentia-
tion. Sakazaki" modified BTB Teepol by substituting sodium heptadecyl sulphate
(Tergitol 7) for Teepol and found it to bemore selective. Teepol can also be replaced by
lauryl sulphate.99 Polymyxin-tylosin sucrose salt (PTSS) agar can provide differentiation
of Vibrio spp. on the basis of sucrose fermentation, utilizing antibiotics, rather than
detergent, for selectivity.62
Water blue, alizarin yellow agar (WA)"' incorporates water blue and alizarin yellow in a
medium suitable for distinguishing between V. parahuemolyticus and V. alginolyricus. In
addition to dyes, WA contains beef extract, peptone, sodium chloride, Teepol, and
sucrose. Fermentation of sucrose by V. alginolyticus, when it is grown on WA, results in a
reduction in the pH of the medium, thereby causing the medium to assume a blue hue,
caused by a change in the dye, water blue, induced by the altered pH. Alkalinization of
the medium by sucrose negative strains of V. parahaemolyricus results in an orange-
yellow color, caused by the pH effect on the alizarin yellow dye.
A fermentable carbohydrate other than sucrose, such as arabinose, was employed by
Horie et al.,"' who devised an arabinose ammonium sulfate cholate (AAC) medium
which contains sodium cholate at a pH of 8.6. Unfortunately, there is a tendency for the
medium to provide an underestimate of the true incidence of V. parahaemolyticus
because arabinose fermentation is a variable characteristic of the species (see Table 2).
Sodium cholate was used by Watkins et al."' to inhibit the growth of Gram-positive
organisms on the primary isolation medium. Copper sulfate has been used to inhibit V.
alginolyticus, a species closely related to V. parahuemolyticus, with galactose included as
a fermentable carbohydrate."'
A nonselective medium was devised by Baross and Li ~ton"~*"' employing starch
hydrolysis as a differential Characteristic. The Baross and Liston medium contains no
selective agents and the pH of the medium is adjusted to 7.5. Inoculated plates are
incubated in an anaerobic jar for 36 to 48 hr at 37C. For samples containing large
numbers of Bacillus spp., penicillin (20 U/mP) is added to achieve selection of Vibrio
SPP-
Twedt and Novelli'I8 carried out a systematic study of media and media constituents
proposed for isolation of V. parahaemolyricus. Penicillin inco6orated into a medium of
'alkaline pH proved to be more useful than a variety of selective agents that had been
suggested by investigators for V. parahaemolyticus. These included potassium tellurite,
sodium deoxycholate, Teepol, and 6% NaCl, all of which were considered to be useful in
the isolation of V. parahaemolyticus. With starch hydrolysis as the differentiating
characteristic, the final formulation included peptone (2'39, yeast extract (0.2%), corn
starch (0.5%), NaCl (3.0%),-@nicillin (2 U/mP), and agar (1.5%), pH 8.0. Subsequently,
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Volume 10, Issue I a7
Vanderzant and Nickelsonw modified the medium of Twedt and Novelli slightly, using
7% NaCl and 1% corn starch, finding results superior to those from other media
recommended for recovery of V. parahuemolyticus.
Because of the high cost and great difficulty in obtaining TCBS agar in India,
investigators there have devised a less expensive and simpler plating media. De et
described VP agar, similar to TCBS and containing sodium taurocholate and sodium
lauryl sulphate. Sucrose Teepol tellurite (SIT) agar, described by Chatterjee et a1.,*
contains peptone, beef extract, Teepol, potassium tellurite, sucrose, bromthymol blue,
and agar. In the U.S. TCBS is not difficult to obtain but not all laboratories, especially
clinical laboratories, routinely stock this medium. Some workers have reported
satisfactory results using more common media, such as mannitol salt agar and xylose
lysine deoxycholate (XLD) agar amended with salt and starch, for isolation of V.
parahaemolyticus. In addition, well-trained and observant technical staff have noted
aberrant biochemical and morphological characteristics of cultures grown on conven-
tional bacteriological media and, in their follow-up, detected the presence of V.
parahuemolyticus.
C. Recovery of Stressed Cells
Problems of heat and cold stress on V.parahaemolyticus have been examined in detail
by food micr~biologists.~*-~ This aspect of the biology of V. parahaemolyticus is
interesting since seafood is preserved by chilling or freezing and most seafood is cooked
well, or at least heated, before consumption. V. parahaemolyticur is widely recognized as
being sensitive to cold when it is present in food and water.27- In addition, effects of
heat and cold stress of V. parahaernolyticus are altered significantly by the medium and
composition of the diluent to which they are exposed during and after tress.^'**^^'-^^^
Vanderzant et al. evaluated several procedures for isolation of V. parahaemolyticus
which were applied to recovery of stressed cells. They concluded that TSB containing 7%
NaCI, when used with modified agar of Twedt and N~vel l i ,~ was more effective, but less
selective, than GSTB and TCBS agar. Beuchat reported that TSB to which 7% NaCl
had been added and GSTB were inferior to arabinose ethyl violet broth9 and to water
blue-alizarin yellow broth for enrichment and efficiency of recovery of cold- and heat-
stressed V. parahaemolyticus.
D. Characterization of Isolates
Pure cultures obtained using any of the above methods can be characterized and
identified using a scheme similar to those mentioned above. Biochemical properties of V.
parahaemolyticus can be determined using conventional media supplemented with NaCl
at approximately 1 to 3%final concentration. While V. parahaemolyricus grows well on
ordinary blood agars and on Mueller-Hinton agar, as well as most media containing
NaCI, there are particular media, e.g., MR-VP and decarboxylase media, to which NaCl
must be added in approximately 2% concentration for significant growth to occur.135 A
specific medium, Wagatsuma medium, is employed to test for hemolysis of blood. The
reaction observed on Wagatsuma medium is termed the Kanagawa phenomenon
(KP).6736 This medium contains: yeast extract, 5 g/P; peptone, 10 g/P; mannitol, 5 g/P;
K2HP0,, 0.5 gin; NaCI, 70 g/ Q; agar, 15 g/P; and crystal violet, 1 mPof a O.l%solution.
Freshly drawn and washed human blood cells are added to the cooled, prepared medium.
Results of the hemolysis test are not valid if the medium is held longer than 24 hr after
i noc~l ati on. ~*~~ A new method for testing the Kanagawa phenomenon, using a liquid
medium, was reported by Ohashi et a]. Chun et al.8 reported that variable KP
reactions will occur on Wagatsuma medium, depending upon the presence or absence of
fermentable carbohydrates in the medium.
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E. Enumeration of V. pmoliaemolyticus
The two most widely used methods for enumeration of V. parahaemolyticus in food or
water samples are the most probable number (MPN) and the membrane filtration (MF)
methods. Direct plating, rather than MF, can be used if the number of cells in thesample
are high enough. MPN methods utilize enrichment broths, such as those discussed (vide
supra). Replications of three or five tubes can be used, with inoculation of at least three
different sample volumes. An example of the MPN would include a series of lo-, 1 and
0.1-mP volumes of sample inoculated into three tubes of broth for each volume or
dilution employed. Sample volumes larger than 10 mP can be concentrated using a
filtering system fitted with a 0.45-pm membrane filter. The filter with trapped cells,
can be placed into enrichment broth tubes, and the procedure carried out in replicate.
lsolation media, which are often replicate plates containing any of several acceptable
media, are inoculated from tubes showing growth. These are incubated as for the
nonquantitative isolation. Nearly all enrichment broths and plating media described for
V. parahaemolyticus have been used in the MPN procedure by investigators whose work
has been cited herein. At the present time, no universally accepted, standard method
exists for the enumeration of V. parahaemolyticus, i.e., there is none which is the
equivalent of the test for acid and g as formation in lactose broth and presumptive,
confirmed, and completed tests employed for enumeration of coliforms. Thus, the
inoculated plates are incubated and colonies must be picked for purification and
characterization. The extent of the testing procedure employed in the characterization of
strains, as well as the "presumptive" or "confirmed" appellations remain subjective. These
are determined by the governmental agency, individual laboratory, or the investigator
conducting the study. However, Hugh and Sakazak?' have published a list of minimal
characters suitable for the identification of V. parahaemolyticus and this has proved
effective in many instances (Table 2).
A multitest, presumptive identification medium specific for V. parahaemolyticus has
been developed and it offers a relatively quick and inexpensive aid in biochemical
characterization and screening of large numbers of isolates suspected to be V.
parah~ernolyticus.~~ This medium, prepared in tubes, gives a characteristic overall
reaction for V. parahaemolyticus, derived from arginine dihydrolase (-), mannitol
fermentation (+), sucrose and lactose fermentation (-), HI S production (-), gas
production (-), and indole production (+).
The membrane filtration technique, which has been successfully employed to
enumerate other taxonomic groups of bacteria, was first devised for vibrios by Horie et
al. "' Enumeration of V. parahaemolyticus can be effected with an arabinose, ammonium
sulfate, sodium cholate (AAC) medium at pH 8.6. After filtration of the sample, the
membrane is placed on the AAC medium and incubated at 42OC. Growth at 42C has
proven to be an important differential characteristic of V. parahaemolyticus. Yellow
colonies appearing on the filter, incubated on the surface of the AAC medium. are
arabinose fermenters, and therefore are presumed to be K parahaemolyticus.
Unfortunately, as noted above, there is a tendency for underestimation of the true
incidence of V. parahuemolyticus, when this method and medium are used, because
arabinose fermentation is a variable characteristic of the species.
Many of the agar plating media discussed herein have been adapted for use with
membrane filters, and TCBS agar is one that is most commonly employed. Watkins et
al."' reported a method involving membrane filtration which was specifically designed
for enumeration of V. parahuemolyticus. The primary isolation medium for the method
was based on the ability of V. parahaemolyticus to grow in the presence of 3% NaCI, at
high pH, i.e., 8.6, and at 41OC. Sodium cholate in the medium served to inhibit growth of
Gram-positive organisms. Galactose provided the source of carbohydrate for V.
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Volume 10, Issue I 89
parahoemolyticus. V. alginolyricus, a closely related species, was inhibited by copper
sulfate. Attempts were made to achieve rapid identification of V. parahoemolyticus,
without transfer of each individual colony to other test media. For example, the
membrane filters, on which the colonies were growing, were transferred successively to
galactose and sucrose fermentation media, with the oxidase test performed as the final
step. The procedure as described by the authors can be done within 30 hr, yielding 95%
accuracy of identification.
IV. ECOLOGY OF V. PARAHAEMOLYTICUS
A. Geographical Distribution
V. parahuemolyticus is, indeed, widespread in occurrence. First recognized in
japan,' 1.95.139 it has also been isolated from samples collected in Korea,Ia Thailand.Io4
Indonesia,47 Vietnam,14' China,'42 India,'43-146 Iran,147 Rus~i a,'~' and A ~stral i a. ' ~~* ' ~
The occurrence of V. parahaemolyricus in Europe has been reviewed by Leistner and
Hechelmann,'" and countries of isolation include the nether land^,^^.^^"" Great
Britain,63"s2 Denmark, 102,153 Germany,'5'''l'4 Italy,'9*'5s Scotland,6' Spain,'" and the
Black, Baltic, North, and Mediterranean Seas."' Reports from Africa include isolation
of V. parahoemolyticus in Togo'56 and Madaga~car."~ In the Western Hemisphere, V.
parahuemolyticus has been isolated in
In the U.S. V. parahuemolyticus was reported by Ward'60 on the basis of serological
relatedness of V. parahaemolyricus-like organisms in sediment samples collected from
the Southeastern coast. Since then a report of the occurrence of this species from nearly
every coastal state has been made, including New Hampshire,94 Massachusetts,I6' Rhode
Island,I6' Maryland,97.'63-'64 Virginia,'63 North Car~l i na,'~' South Carolina,'66 Flor-
ida,'67 the Gulf Coa~t, ' ~' - ' ~~ Oregon,I7' Wa~hi ngton,"~"~ Hawaii,m and A1a~ka.I ~~
V. parahuemolyticus can be isolated throughout the estuarine environment. Water,
sediment, suspended particulates, plankton, fish, and shellfish samples have been shown
to harbor the organism. The principal features which influence the ecology of V.
parahuemolyticus are salinity, seasonality, and association with higher organisms. V.
parahuemolyticus is most commonly an inhabitant of estuaries and is infrequently found
in freshwater or full-strength seawater. The seasonal cycle is temperature dependent,
with higher numbers evident during warmer summer months. V. parahaemolyricus is
associated with a number of higher organisms, notably plankton and shellfish.
Panama,'59 and the U.S.
B. Seasonal Variation
In most of the geographical areas where V. parahaemolyricus is known to occur, the
incidence of the organisms follows a distinct seasonal cycle, with highest counts recorded
in the summer and fall and lowest counts in the winter. This phenomenon was first noted
in J apan by Miyamoto et al.'39 and has been confirmed by a number of other J apanese
investigators including Nishio et a1.173v174 and Shin et al.175 In other countries, the
seasonality of the organisms subsequently was recorded, including
Australia,'50 and the U.S.%97,116,117,1W176 Interestingly, Thompson and Vander~ant'~~
reported that a seasonal cycle for V. parahuemolyticus could not bt detected in the Gulf
of Mexico, but noted that temperatures were higher year round than in other
environments studied, with the lowest temperature only 11.6OC. In the Chesapeake Bay
and other areas, the organism is absent from the water column during the winter months,
but can be isolated from sediment throughout the inter.^"'^In other environments it
remains in the water column, but at greatly reduced numbers.63
I n tropical countries, the seasonal cycle of V.parahaemolyricus is correlated with rainy
and dry seasons. In Vietnam, highest numbers are found in rainy months (March and
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90 CRC Critical Reviews in Microbiology
April) and lowest numbers are found in the dry season (December to February).'"
Curiously, the opposite was found in Togo (West Africa) and in Indonesia where highest
counts were found at the end of the dry season (April) and the lowest in the rainy season
( J ~ne) . ~~. " ~ Salinity measurements, not recorded during the Vietnam or Indonesian
study, are available for Togo, where salinity was found to be highest during the dry
season, i.e., greater than 12% per thousand, when counts of V. parahaemolyticus also
were highest. During the rainy season, a salinity of 1.6 to 4.2% per thousand was
recorded, which is less than optimum for V. parahaemolyricus. At that time, few isolates
were recovered.'56
Besides temperature and salinity, seasonal variation of V. parahaemolyticus can also
be influenced by interactions with plankton and higher organisms. Kaneko and
~~l ~~1197. 164. 178 studied the seasonal cycle of V. parahaemolyticus and zooplankton in
Chesapeake Bay and reported that from late spring to early summer, vibrios over-
wintering in sediment enter the water column by attachment to zooplankton. Interaction
between sediment, water, and zooplankton was found to be essential. As temperatures
increased and vibrios proliferated, V. parahuemolyticus was readily isolated from the
water column. Miyamoto and K ~r oda' ~~ suggest that a Bdellovibrio lethal for V.
parahaemolyticus may also play a role in the seasonal cycle of the host. These
investigators found that Bdellovibrio can lyse V. parahaemolyticus at temperatures as
low as 5OC, but not at higher temperatures, i.e., 35OC. Thus, at lower temperatures,
during fall and winter months when Bdellovibrio can actively lyse the host, V.
parahaemolyticus does not readily proliferate. Obviously, the seasonal cycle of V.
parahaemolyticus may be influenced by a variety of factors and a complex interaction of
these, especially temperature, salinity, adsorption, attachment, plankton, parasites, etc.
C. Correlation with Environmental Parameters
The correlation of V. parahaemolyticus and its Occurrence in the environment with
indices of pollution is not at all clear. Several investigator^""'^^'^^reported greater
concentrations of V. parahaemolyricus in polluted waters vs. nonpolluted waters. On the
other hand, Thompson and Vander~ant,'~~ Sutton,15' Kaneko and C0lwe11,~' and J onas
et a1.'65 report no significant correlation with counts of V. parahuemolyti& and pollu-
tion indices, such as total counts of coliforms, fecal coliforms, or Escherichia coli. These
variances can be resolved if other factors associated with pollution are considered, such
as the concentration of nutrients and suspended particulates, rather than coliform
counts. Coliform indices usually correlate well only with occurrence of allochthonus
bacterial pathogens, such as Salmonella spp. and not with the autochthonous,
potentially pathogenic bacteria, such as V. parahaemolyticus.'".'8'
Watkins and Cabelli'62 reported that adsorption of V. parahaemolyticus to
particulates is greater in water of lower salinity and that the numbers of V.
parahaemolyticus may be indirectly related to pollution because of concurrent input of
particulates. Otherwise there is no direct correlation with pollution. In studies carried out
in Chesapeake Bay, the occurrence of V. parahaemolyticus did show a positive
correlation with coliforms in the area of the Chesapeake Bay surrounding Baltimore
Harbor, but was not as highly correlated as was Salmonelluspp.~o the coliform count.'"
In a subsequent survey covering the entire Chesapeake Bay, multivariate regression
analysis of the data revealed that salinity and dissolved oxygen concentration were most
closely correlated with the incidence of V. parahuemolyticus. The frequency of
occurrence, i.e., total number of V. parahaemolyticus, increased with increasing salinity
and decreasing dissolved oxygen (DO) concentration, the latter most likely reflecting
increased nutrient concentration in eutrophic areas of the Bay.'83
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Volume 10, lssue 1 91
V. parahuemolyticus has been isolated from freshwater in several areas. Well water
from Indonesia was found positive in several instances where samples from J ava were
e~ami ned.~' In Calcutta, India, V. parahaemolyticus has been isolated from water sam-
ples and fish taken from essentially fresh water in the River Hooghly, approximately
50 miles upriver from the Bay of Bengal.145 V. parahuemolyticus is widely distributed in
this area, being found in 40% of pond water samples "having practically no sa1inity"and
fed chiefly by rain water.lE4 In the Chesapeake Bay, V. parahuemolyticus has been
isolated from the Upper Bay, as well as the uppermost reaches of the J ames River and the
Potomac River."' Sayler et al."' examined the Upper Chesapeake Bay and recovered
several Isolates from samples in low salinity areas. An isolate was obtained from
suspended sediment where the water temperature was 4.3OC and the salinity was below
the detectible limits of the salinometer. Tidal transport of V. parahaemolyticus, no
doubt, plays an important role in the occurrence of the organism up-river of estuaries.
Ayres and reported higher concentrations of V. parahaemolyticus in muddy
sediment than in sand or gravel sediment, indicating the influence of organic matter on
Occurrence and survival of this species. The transfer of vibrios from sediment to the water
column and from the water column to marine animals was examined for "V. parahae-
molyticus biotype 2". i.e., V. alginolyticus. by Gauthier and Clement.'86 These
investigators reported that transfer of vibrios via sediment was very important in
persistence in the water column and marine animals and that colonization of water from
sediments was not observed at temperatures less than 16OC. As noted above, adsorption
of V. parahaemolyticus to particulates is greater in lower salinity waters,16' a finding
consistent with observations made in Chesapeake Bay'" and in Calcutta. Thus, the
Occurrence of V. parahaemolyricus in brackish or freshwater environments can be
concluded to be significantly affected by the occurrence of particulate matter, nutrient
concentration, and salinity.
D. Occurrence in the Open Sea
V. parahaemolyricus has been isolated rarely from pelagic regions of the world oceans.
It appears to be limited to inshore coastal and estuarine areas. Aoki et al.142 reported the
isolation of V. parahaemolyricus from the open sea of J apan, but neither Horie et al.9s9i14
nor Miyamoto et al.I3' found V. parahaemolyticus in water samples collected from
pelagic areas around J apan. The inability to isolate V. parahuemolyficus from pelagic
areas was also reported by Varga and Hirtle"* in Canada and Bockemuhl and Triemerls6
off the coast of Africa. Baross and Liston"' noted that the incidence of V.
parahaemolyticus in seawater decreased with depth off the Washington coast and very
low numbers were found in deeper sediments. Kaneko and Colwe11''' collected samples
along four transects on the continental shelf off the southeastern U.S. and did not isolate
classical V. parahaemolyricus from any of the water, sediment, or plankton samples.
However, a number of vibrios very similar to V. parahaemolyticus was recovered. The
absence of V. parahaemolyticus from the open ocean is most likely a result of low water
temperature, high salinity, and low nutrient concentration, since V. parahaemolyricus
unlike other marine bacteria, is cold sensitive and does not survive well in low nutrient
waters. Another major factor to be considered when examining th? incidence of V.
parahaemolyticus in the open ocean is hydrostatic pressure, the effect of which was
studied by Schwartz and Colwell,"' who reported that V. parahaemolyticus was unable
to grow at any hydrostatic pressure simulating the deep ocean environment, i.e., from 200
to 1000 atm of pressure. Thus, the inability of V. parahaemolyticus to tolerate elevated
hydrostatic pressure supports the conclusion that neritic or estuarine waters are habitats
of V. parahaemolyticus.
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E. Association with Higher Organisms
V. pnrahoemolyricus is now known to be associated with a variety of higher organisms
living in &he marine and estuarine environment. including plankton, fish, and shellfish.
Shellfish. in particular, are important because of the possibility of human disease arising
from their ingestion. Fishbein et reported the isolation of V.parahaemolyticusfrom
30 different marine species, including clams, oysters, lobsters, scallops, sardines, shrimp,
squid, crab, eel, and various species of fish. From 1969 to 1972, 546 out of 635 (86%)
seafood samples examined by the Food and Drug Administration were found to be
positive for V. parahaem~l yt i cus, ' ~~ perhaps a biased result because many of the samples
were collected during outbreaks of enteric disease. Nevertheless, other equally extensive
studies have been reported which document the presence of V. parahuemolyticus in
Counts of V. parahaemolyticus clams, mussels, and oysters.
in oysters can be as high as 1300 per gram of tissue.'70 More typically, the numbers are 10
per gram. Isolation of V. parahuemolyticus from crabs has been reported by Fishbein et
al,,la9 Krantz et al.,lw Barrow and Miller,'s2 Colwell et aI.,I9' and De et al.,14' and from
shrimp by Vanderzant et a1.,1689192 Fishbein et a].,*' De et al.,145 Felsenfeld and Cabirac,I7'
and J oseph et Concentrations of V. parahaernolyricus in crabs can be as high as 10'
per gram of meat.
V. parahuemolyticus has not been isolated from fish as frequently or as readily as from
filter feeding invertebrate^."^^"^Nevertheless, a wide variety of marine and freshwater
fish has been shown to harbor the org~nism.6*'99''4~"46~170~193-'9~
In addition to the commensal, or symbiotic, association of V. parahuemolyticus with
higher organisms, a pathogenic relationship is possible also. Krantr et isolated V.
parahaernolyiicus from lethargic and moribund crabs, and Brinkley et al.I9' and Tubiash
et al.IP6 reported an association of V. parahoemolyticus with disease in lobsters and
bivalve mollusks, respectively. Vanderzant et a1.16a,192 and Vanderzant and Ni ckel ~on'~~
reported the death of shrimp in mariculture caused by infection with V. parahaemolyri-
cus. An outbreak of disease in a Mexican shrimp hatchery has also been shown to be
associated with V. parahaemolyricus (D. Danald, personal communication). Pathogenic
properties of V. parahuemolyticus may be as important in the mariculture of shrimp or
other invertebrates, as V. anguillarum has proved to be in fish hatcheries.
62.94.99.106,107,117.149,l50.l56,l~7,l77
F. Kanngawa Phenomenon Positive Y. pwuhernolyticus in the Environment
An important, as yet, unanswered question concerning V. parahuemolyticur is the
exact relationship between the Kanagawa phenomenon (KP), pathogenicity of the
organism, and the environmental reservoir of the organism. There is a significant
correlation between the KP and pathogenicity of V. parahuemolyticus, but the reason is
not yet known. Sakazaki et al.197 reported that 96.5% (2655 out of 2720) of strains
isolated from human patients was KP+ while only 1% (7 out of 650) of isolates from the
environment was KP+. Other investigators have reported similar findings. Thompson
and Vanderzant"' found only 4 KP+ strains out of 2218 total isolated from water and
sediment in Galveston Bay. Spite et a1.'99found one isolate in an oystcr from U.S. waters.
Three strains out of 251 water, fish, and shellfish strains examined in were KP+.
Ayres and Barrow63 found no KP+strains out of 1484 isolates obtained from British
coastal waters. Leistner and H~chel man'~' found only 1 KP? strain out of 1708 from
European waters. Sutton'so concluded that 2%(22 out of 986) of the strains isolated from
oysters growing in Australia was KP+. An ecological study of KP+ strains found in
J apan was reported by Wagatsuma.2w who recovered KP+ strains from samples of mud,
seawater, and oysters. However, isolation of KP+strains was rare in the absence offood
poisoning in a nearby community, whereas KP- strains were always isolated, regardless
of documentation of food poisoning cases in the community.
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Volume 10. Issue I 93
These findings are best explained perhaps by a process of natural selection of KP+ strains
in the intestine and better survival of KP- strains in the environment. Sakazaki and co-
workers demonstrated that KP+vibrios do, in fact, multiply more rapidly than KP-
strains in ligated rabbit loops and postulated, therefore, that selective multiplication of
KP+organisms occurred in the intestine, even if KP- strains were predominant in the
ingested food sample. Conversely, KP- strains survived longer in seawater than KP+
strains and grew better at 25OC than the KP+strains. Interestingly, KP+strains grow
better at 37C and under acid conditions.
Thus, the phenomenon of KP+ V.parahaemolyricus isolation almost exclusively from
human patients and KP- strains from seawater and seafood may be only a result of
selection and environmental pressure, with selection occurring in the intestine for KP+
strains and in the marine environment for KP- strains, a provocative phenomenon yet to
be elucidated.
V. RELATIONSHIP OF V. PARAHAEMOL YTICUS WITH
BACTERIOPHAGE AND BDELLOVIBRZO
Bacteriophages specific for V. parahaemolyticus were isolated from fecal and seawater
samples in J apan by Nakanishi et a~~ Sklarow et examined Atlantic coastal
sediment and recovered a bacteriophage specific for V. parahaemolyticus which was
morphologically similar to phage isolated from Washington coastal waters.M Baross, et
al.17~206307 recovered bacteriophages active against V. parahaemolyricus from 177 to 643
samples of marine molluscan shellfish, crustaceans, seawater, and sediment collected
from Washington and Oregon waters. Titers of V. parahaemolyticus bacteriophage were
found to increase with increasing water temperature during the warm months of t.he year.
The titer of bacteriophage was discovered to be proportional to the increase in the
numbers of mesophilic vibrios, but not with the incidence of V. parahaemolyticus.
Lysogenic bacteriophage could be induced from an agardigesting vibrio and it was
speculated that bacteriophage may, in fact, be important in explaining the variability of
marine vibrios, with respect to phenotypic characteristics. and possibly even in animal
and human path~genicity.~ Interestingly, no bacteriophage active against V.
alginolyticus has been reported, even though V. alginolyticus occurs in larger numbers in
the marine environment than does V. parahaemofyri~us.~
Marine Bdellovibrio capable of lysing V. parahaemolyticus have been isolated from
Chesapeake Bay and Osaka Bay.79.20*-210 Isolates of marine Bdellovibrio from both U.S.
and J apanese waters are reported to have a broad host range, and at least in Chesapeake
Bay, demonstrate a seasonal cycle similar to that of the host.2
VI. PATHOGENICITY
A. General
Diarrheal disease caused by K parahaemolyricus is a food-borne infection related
chiefly to the ingestion of seafoods. The organism is autochthonous in waters of low
salinity, e.g., estuaries, and appears to have an essential role in theecology of coastal
marine environment^.^^"" Its involvement as a pathogen for man, therefore, is usually
inadvertent through contact with contaminated and improperly handled seafoods. V.
parahaemolyricus is not regarded as an acutely infectious organism, although there is
little doubt that it is capable of causing serious illness. Results of experiments in which
human volunteer subjects were fed broth cultures of clinical i ~ol ates~ ~ ~ and an accident
in which a laboratory worker ingested approximately lo5 viable cells97 make it quite
clear that V. parahaemolyticus, occurring in sufficiently large numbers, can cause acute
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gastroenteritis in man. Sanyal and Senzi3 confirmed the enteropathogenic properties of
KP+ strains in human volunteer experiments. Significantly, symptoms of gastroenteritis
appeared rapidly in individuals who had ingested at least 2 X 10' to 3 X lo7 CFU of KP+
V. parahaernolyticus. Volunteers, receiving KP- V. parahaemolyricus in concentrations
ranging from 4 X lo9 to 1.6 X 10" CFU, did not have diarrheal symptoms. Earlier studies
had shown also that ingestion of lo9 cells of KP- strains by human volunteers had
seemingly no ability to induce diarrheal Thus far, there is no indication,
from either epidemic or sporadic cases, that enteropathogenicity is associated with
particular serotypes.*" A11 serotypes have been isolated from humans, seawater, and
seafishes. Longitudinal studies in the same localities reveal that particular serotypes do
not predominate in human illness and marine environments from 1 year to the next.
Serotypes frequently isolated from seawater and seafish during shorter periods tend to
predominate in cultures from patients.'"
The nature of the illness caused by V. parahaernolyricus leads one to conclude that an
exotoxin, i.e., enterotoxin, should be the responsible. identifiable substance. Interest-
ingly, feeding experiments showed that only administration of live cells produced
intestinal tract-related symptomatology, belying the possibility of preformed exotoxin in-
gestion with incriminated foods. Further evidence for infection lies in the fact that lo* to
10I2 organisms are required to establish disease, and in the acute stages, lo6 to 10' organisms
per milliliter of feces can be It is not yet known whether the pathological
effects of the organism are due to toxin production, direct damage to the intestinal tract
from microbial invasion, both, or neither. The ability to multiply in the intestinal tract is
generally associated with the KP reactivity of the vibrio, which commonly is considered
to be the essential index of capacity for enteropathogenicity in humans. This hemolytic
characteristic, described in greater detail below, is found in approximately 95% of clinical
isolates and is rarely associated with environmental isolates. Human volunteer and
animal experiments generally tend to substantiate this view, although the significance of
the few cases caused by KP- strains remains partially unexplained. The suspicion is that
these strains produce hemolysin but in very small quantities. This has been borne out, in
fact, by using a serological method of detection, which reveals that, indeed, hemolysin is
produced by some presumed KP- strains.13' The suggestion has been made that V.
parahuemolyricus produces an cnterotoxin.'I6 The possibility that enterotoxin is
produced and contributes to the disease process to some extent, regardless of KP
reaction, has yet to be clearly establi~hed.''~
B. Toxin Assessment
1. Mouse Inocularion
Studies using inoculation in adult and suckling mice generally confirm that large
numbers of vibrios are required to cause lethality. Fujino* observed the lethal toxicity of
V. parahaemolyricus to mice and guinea pigs. Zen-Yoji et al."' used mouse foot pad
measurements to show that KP+ and KP- strains were uniformly toxic and caused
highly edematous reactions. In contrast, V. alginolyticus strains were relatively
benign, producing significantly less swelling. Concentrated cell-free filtrates introduced
via foot inoculation showed lethal toxicity, whereas all lysates provoked only slightly
edematous responses. Attempts to detect an E. coli-like heat-stable toxin (ST) in suckling
mice were inconclusive. Recently, J ohnson and Calia,'" using concentrated filtrates,
demonstrated weak ST-like responses in the same system.
Whole cells of V. parahaernolyricus administered per os, SC, or I P resulted in mouse
lethality caused by septicemia.' Sakazaki et al." found that 0.5 mP of subgroups I and 2,
now V. parahaemolyricus and .V. alginolyricus, injected IP into mice resulted i n lethality
within 24 to 48 hr. Lethality is dose dependent. Two different KP+ clinical strains
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Volume 10, Issue I
inoculated into NMRI mice yielded LDso values at concentrations 2 10' but were
nonlethal at lesser concentrations.'" Inoculations of cell-free filtrates of over 200
separate clinical isolates, even at IOX concentration, did not cause death of NMRI
mice,221 further emphasizing the apparent requirement for large numbers of vibrios to
cause significant disease.
2. Rabbit Inoculation
The rabbit ligated ileal loop (RIL) model has been used by several groups of
investigators to describe the enteropathogenic character of V. parahaemolyticus.201*
Whole cultures of V. parahaemolyticus and V. alginolyticus introduced into RIL can
elicit "enteritis" reactions"." Both KP- and KP+ strains of V. parahaemolyticus may
cause dilatation in RIL. Generally, KP+ strains are positive more frequently in the RIL
system, than are KP- strains.2'4225~z2* Cell filtrates are not capable of eliciting a fluid
accumulation in RIL, although based on early studies, Sakazaki et al.'lS suggested that
an enterotoxic substance was produced by V. parahaemolyricus and played a role in
inducing gastroenteritis. This appeared to be substantiated when heatingat 100C for 30
min eliminated activity of tenfold concentrated cell-free filtrates. Recently, J ohnson and
Calia233 have shown that concentration of filtrates produces solutions containing >20%
NaCI and reported that media containing NaCl2495 induced positive responses in RIL.
They concluded that cell-free filtrates of V. parahaemolyticus, even if concentrated
tenfold, after dialysis, were not capable of causing dilatation i n RIL, when NaCl
concentrations were <4%. Twedt and Brown2" investigated the enteropathogenicity of
KP+and KP- whole cultures, cell lysates, and cell-free filtrates, concluding that whole
culture and cell lysates of KP+ strains cause fluid accumulation in RIL.
Calia and J ohnson234 further related enteropathogenicity to KP by oral administration
of broth cultures to suckling rabbits, which showed bacteremia within a few hours after
disruption and penetration of the epithelium. KP- strains apparently were unable to
stimulate such activity. On the other hand, J oseph et al.'35 found that the whole culture of
a KP- human isolate injected directly into the externalized ileum of anesthetized
suckling rabbits resulted in diarrhea and bacteremia within 5 hr of inoculation. Scanning
and transmission electron microscopy revealed focal necrosis and attachment of vibrios.
Attachment was not observed in apparently unaffected areas leading to the hypothesis
that tissue necrosis and bacterial adherence were closely associated.
Histologic studies of RI L using fluorescent antibody suggested invasion into the
underlying epithelium of the intestine of rabbit^.'^' In some instances, V. parahaemoly-
ficus strains do not elicit fluid accumulation in RIL regardless of KP reaction, which
suggests that substances or mechanisms other than hemolysins are involved in causing
gastrointestinal disease.
216,222-233
.
C. Tissue Culture Responses
Adherence of bacterial cells to mammalian and other cell lines can correlate with the
ability to cause infections involving the epithelial surfaces of the intestinal, respiratory,
urinary, and genital tracts. '36237 Although investigations of this nature with V.
parahaemolyricus are limited, there are some reports which provide further characteri-
zation of the activity of their whole cells and cell-free filtrates in tissue culture systems.
Cellular destruction of HeLa cell monolayers occurs after 3 hr challenge with live KP+
vibrios and 2 hr after challenge with culture filtrates regardless of KP reaction of the
strain. Similar exposure of L cells results in loss of response of the nuclei to Giemsa stain,
but without cellular destruction."' CarruthersZJ 8also observed cytotoxicity to HeLa cells
in vitro by V. parahaemolyricus. Although cytotoxicity occurred with both KP+ and
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96 CRC Critical Reviews in Microbiology
KP- strains, the effect was more rapid and complete with the KP- strains. It was
subsequently noted that V. parahaemolyticus adhered rapidly to suspension-grown
HeLa cells and to human fetal intestinal cells (HFI). KP-strains did not adhere to HeLa
cells and adhered to HFI cells at a much slower rate than did KP+ strains. Adherence
appeared to depend upon interaction between the cell surface of the bacteria and the
epithelial cell, a carbohydrate on the outer membrane of the bacterial cell wall apparently
necessary for the Using clinical strains of KP+ and KP- V.
parahaemolyticus, Sochard and J oseph were unable to differentiate between either
group similarly exposed to HeLa cells.2a Gingras and Howardz4 also were unable to
detect significant differences in vitro, by radioassay. in the adherence of KP+and KP-
strains of V. parahoemolyticus to human intestinal cells.
Culture filtrates of V. porahaemolyricus isolates from human gastrointestinal
infections, when exposed to Y-1 adrenal cells, do not cause morphologic change^.^^^-^@ In
contrast, Honda et al.27 have isolated a heat-labile factor from the culture filtrates of
KP+ strains which causes morphological changes of Chinese hamster ovary (CHO) cells,
the premise being that this factor is an enterotoxicsubstance separable from the cardio-
toxic hemolysin.
Oishi et al.245 have recently described the presence of exohemagglutinins (HA) from
marine vibrio strains, which had taxonomic properties similar to V. parahaemolyticus.
HA showed remarkably high hemagglutinating titers for a wide spectrum of
erythrocytes. The more widely reactive HA were not easily inhibited, whereas the HA in
the group with narrower spectra, were specifically and completely inhibited by L-fucose
and D-arabinose. The physiologic significance of HA is not clear, but they may eventually
emerge as an important feature of adherence, an essential step in pathogenicity.
Clinical examination of a few patients in India, Bangladesh,246 and the U.S.,247 who
were suffering from V. parahaemolyricus dysentery-like diarrhea, suggested that
invasion of the bowel may occur, i.e., blood and mucus in the stool, polymorphonuclear
leukocytes seen by microscopy, and superficial ulceration of colonic mucosa seen by
sigmoidoscopy. These observations are significant when compared with similar,
experimental findings in rabbit^.^^^'^^' Boutin et a1.,232 usinga direct fluorescent antibody
method, studied invasiveness as a step in pathogenesis during host organ-bacterial
interaction. All strains tested, KP+ and KP-, penetrated into the lamina propria of the
ileum and were eventually isolated from the spleen. However, not all were able to cause
fluid dilatation of RI L. KP- strains caused only occasional positive responses.
Interestingly. V. parahaemolyricus does not exhibit a positive Sereny test, i.e.,
penetration of the corneal epithelium of guinea Attempts to demonstrate
invasiveness by V. parahaemolyricus and other closely related vibrios with the HeLa cell
techniques described by Mehlman et reinforced the impression that, by this
experimental method, as well, V. parahaemolyticus penetration or invasion of tissue is
still a questionable phenomenon.240
J oseph et aL2 found that a KP- strain injected into the ileum of suckling rabbits
caused focal necrosis and appeared to adhere in damaged areas, but not on apparently un-
effected tissue, leading to speculatioh that tissue damage, adherence, and invasion were
interrelated, possibly with toxicity. Further examination showed that adherence was
possibly mediated by lateral appendages (flagella), and played i n important role in the
stimulation of fluid accumulation in RI L and lethality to mice by the whole, intact
Similar investigations reported simultaneously by Shinoda et al.2s
substantiated these results.
D. Permeability Factor
The permeability factor (PF) or skin toxin, found to be capable of producing increased
vascular permeability in the skin of experimental animals, has been described for V.
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Attempts to show a similar response to V. parahaemolyticus have given cholerae.
variable results. Unconcentrated filtrates tested by Bhattacharya et al.254 were
nonreactive, but after 1OX concentration, three of four strains had positive PF reactions.
Unlike cholera toxin, they failed to produce enterotoxicity in RIL. KP- strains can
exhibit a heat-labile, trypsin-sensitive PF as well. However, Ghosh et a1.,226 using
similar methods, were unable to discern PF in six clinical strains. Presumed
enteropathogenic toxin, separated from culture filtrate and examined for PF, was
positive for three hemolytic strains of V. parahaemolyricus, but negative for three
nonhemolytic strains.224 Further characterization showed dissimilarity of activity in
comparison with choleragen.
252-254
E. Hemolysin
1. Background
Fujino assigned the species designation to V. parahaemolyticus because the organism
resembled Pasteurella hemol yt i ~a. ~ That he so named the organism was fortuitous. since
beta hemolysis by the organism under particular conditions has now assumed significance
as a corollary of enteropathogenicity. Beta hemolytic activity on blood agar by V.
parahaemolyticus was noticed by Fujino during his early characterization of the
reported a close correlation between
pathogenicity for humans and subsequent isolation of KP+ strains of V. parahaemo-
lyticus. Wagats~ma~ subsequently modified the medium used by Kato et al.256 to
provide improved hemolysis. The medium has since been termed Wagatsuma agar
(WA) and the hemolytic reaction is called the Kanagawa Phenomemon (KP), which is
characterized by the appearance on WA of a clear halo of hemolysis, with distinct
outlines under or around the periphery of the colonies after 18 to 24 hr incubation at
37oc.
Kato et a1.256 and Miyamoto et
2. Detect ion
evaluated Wagatsuma agar (WA) in a study of 3370 vibrio cultures
from patients and from sea sources. On WA 96.5% of the cultures from humans gave a
KP+reaction, whereas only I % of the cultures from marine sources was positive. The
results appeared to confirm the impression that KPS reactions were linked closely to
pathogenicity for humans. Interestingly there was no correlation between serotypes and
Kanagawa test reactions. A comparative study by Twedt et a1. essentially substantiated
this observation except that strains from nonhuman sources were curiously KP+at a
higher frequency than that found by Sakazaki for seafood strains.I9
Chun et a1.138*259*260 have surmised that the composition of WA, especially the NaCl and
carbohydrate concentrations, may tend to potentiate hemolysis quantitatively. Sak-
azaki2 showed that adding 0.2% glucose to blood agar after it was sterilized resulted in
inhibition of hemolytic activity by most vibrios originating from sea fish. Similarly, KP
reactions observed in a liquid medium, suggested to be superior to WA, can be modified
by changing the surface volume of the broth.I9
Methods now available for serologic detection of hemolysin show greater sensitivity in
detecting KP than does WA.37*261 These methods may contributt to better quantifica-
tion, thus clarifying occasional reports of outbreaks or sporadic cases caused essentially
by KP- strain^.'^'-^^^In a retrospective analysis of presumed KP- strains from human
origin and seafoods, Ohashi et al.,137 using the reversed passive hemagglutination (RPHA)
test, found that hemolysin could bedetected at 2 ng/mQquantities of hemolysin, whereas
detection by WA required 1 pg/mP. Their survey showed that questionable KP+ strains
could be easily detected, and that occasionally some strains presumed to be negative by
WA are in fact weakly KP+. Honda et found that a modified Elek test and an
Sakazaki et
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98 CRC Critical Reviews in Microbiology
immunohalo test correlated well with KP responses on WA. These findings agreed well
with those of Ohta,26' who used Oudin's immunodiffusion test and observed that
amounts as small as I pg/ mQ of toxin could be detected, as compared with 3 pg/ mQ by
the WA plate. Maximum toxin yield was obtained when the organism was cultured in 2%
peptone water with 5% NaCl at 35C for 18 hr with reciprocal shaking at approximately
I20 strokes per minute. Considering the difficulties in the preparation of WA and the
requirement to use it in the fresh state, the serological methods offer an appealing
alternative.
3. Separarion, Purificarion, and Characterization
At least four hemolytic constituents have been described for V.parahaemolyticus, i.e.,
a heat-stable direct he~nol y si n, ~~~~~~ a heat-labile direct hemol y ~i n, ~~~- ~~~ phospholipase
A, and lysophospholipase.80." I n addition, V. parahaemolyticus produces lecithinasen2
and glycerophosphorylcholine diesterase." The relationship of phospholipases and
lysophospholipases has not been studied extensively; thus, their roles in enteropatho-
genicity, if any, have not been clarified. Phospholipase is an indirect hemolysin since
addition of lecithin to blood agar results in larger, better-defined zones of hemolysis.
Conversely, direct hemolysins lyse erythrocytes and do not require added substituents
such as phospholipid^.^^^
There were several reports from 1964 to 1969 which resulted from investigations on the
hemolytic activity of V. parahaemolyticur.254257~2'1-2'' The correlation of KP with
pathogenicity for humans was derived from some of those efforts.2569257v27' The
significance of hemolysin was further delineated by the demonstration of antihemolysin
titers in patients affected with gastroenteritis caused by V. p a r a h a e mo l y t i c u ~ . ~ ~ ~ ~ ~ ~
a. Separation and Purification
There is diversity among procedures used for the isolation and purification of
thermolabile direct hemolysin, but descriptions of the ultimate product indicate that each
is reliable for the A simplified method for the purification has been
proposed. 28'
b. Characterization
K a~amura* '~ first studied the nature of the hemolysin produced by V. parahaemoly-
ticus and reported that it was not a protein and was heat stable. A subsequent report
described hemolysin as a protein, which was free of polysaccharide and lipid.272 The heat-
labile and heat-stable hemolysins were briefly described by Fujino et following a
study of V. parahaemolyticus WP-I. Heat inactivation at either 55 or 60C for 10 min
inactivated the direct hemolysin significantly, with no direct hemolysis observed in titers
> 1:8. The results indicated that both toxins were present. In one of the few attempts to
characterize the thermolabile direct hemolysin, Miwatani et isolated and partially
purified a hemolysin which was destroyed by heating at 70 or 100C for 10 min. They
noted that crude hemolysin was subject to the Arrhenius effect.269 The heat-labile
substance is usually found in KP- strains, but not consistently in filtrates of KP+ strains,
which characteristically possess thermostable hemolysin.'"
Further attempts at purification using more efficient methbds revealed that the
substance responsible for the Kanagawa phenomenon (KP) was the thermostable
hemo~ysin.266~78J82-Z~5 Obara266J 84 suggested that the hemolysin might be responsible for
human pathogenicity. The final product, after partial purification, had a 2686-fold
increase in specific activity, was unable to hemolyze erythrocytes of several animal
species, and had a minimum lethal dose of 0.62 pg nitrogen per mouse. The toxic and
hemolytic properties were thermostable after treatment at 100C for 10 Zen-Yoji
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Volume 10. Issue I
et al. proposed the name enteropathogenic toxin for this substance, presumably
because of its lethality for mice.278 Sakurai et al.267 showed that additives such as lecithin
had no effect on their purified toxin and termed it thermostable direct hemolysin.
Honda et al.2B0 identified the thermostable direct toxin with the lethal toxin for mice
described by Ueyama et a1.86
Several biological properties have been attributed to thermostable direct hemolysin
(TDH) after either partial or complete p ~ r i f i ~ a t i o n , ~ ~ ~ ~ ~ ~ ~ - ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ including entero-
toxi ~i ty,~ ~* ~~ ~ytotoxi ci ty, ~~~~~ and cardi ot~xi ci ty. ~~-~~ TDH has been the most
extensively investigated of the hemolytic substances produced by V. parahaemolyticus.
The molecular weight of TDH has been reported variably as 45,000,277J 79*288 66,000,277
I 18,000,267 30,000,270 and 42,000 daltons.280 A recent study suggests that TDH has a
molecular weight of 42,000 daltons and is composed of two subunit molecules of
approximately 21,000 daltons each.287 I t was postulated that the previously proposed
molecular weight of 118,OOO was inaccurate because of aggregation of TDH when
heating to 55C.267J 87
When purified, TDH is characterized as a protein free of carbohydrate, lipids, and
It can be inactivated by trypsin or pepsin,279 is organic phosphorus.
resistant to heating at l00OC for 30 min at pH 6.0,2797283*2*8J95 and is activated by Ca .
Although TDH is inactivated by heatingat 60C for 5 min, reheatingat 100C for 30 min
reactivates it to some extent.288J 9 Conversely, toxic activities of 100C heated toxin are
markedly diminished by heating at 60 C.29 Ohta showed by polyacrylamide gel (PAG)
disc electrophoresis that heating at 60C reduced aggregation of the toxin to an
insoluble, nontoxic form, and reheating at 100C resulted in partial reversal with the
release of soluble active toxin, concluding that the paradoxical behavior of the toxin was
due to conformational changes of the molecular composition of the toxin.295.
Amino acid analysis reveals that 43% of the total amino acids is acidicamino acids, and
I 1 % is The toxin has only one N-terminal phenyl-alanine,279 which
probably applies to each of the presently identified subunits.287 TDH has a sedimentation
coefficient of 5.35279 or 6.95, and an isoelectric point of 4.9326277 or 5.02.288
267,278-28OJ83JS7
++283
4. Effect in Animals
The amount of TDH required for a positive reaction in RIL is much higher than the
effective dose of choleragen, reported to be 2.0 pg, causing skepticism regarding the role
of TDH in enteropathogenicity of V. parahaemolyricus.280 Skin responses are dissimilar
with those caused by choleragen in that diffusive dye permeation is absent.
Histopathologically, there is edema, erythema, and induration peaking at about 8 hr
after inoculation.288 The minimal cutaneous response dose in the guinea pig skin test is
about 2.5 pg. Miyamoto et and Ohashi and Shimada296 found that capillary
permeability activity measured by size of the bluingzone reached maximum reaction in 3
hr with 5 pg of injection. In comparison choleragen elicited continuing enlargement until
and sometimes after 24 hr exposure.
Lethality for mice has been established,278J 83 the minimum lethal dose being0.62 pg of
nitrogen per mouse. The LDSO for mice is approximately 1.5 pg to 12.8 pg. Heating of the
purified toxin at 60 C results in complete inactivation of hemolytic activity, mouse
lethality, and guinea pig skin rea~tivity.~ Mice injected I P with 60C toxin at
concentrations 50 X greater than LDm can survive, whereas mice injected with IOOC
treated toxin die within 15 min.279 Peroral challenge of suckling mice with live organisms
causes neither death nor diarrhea. To the contrary, hemolysin in small doses does cause
diarrhea in suckling mice, but is not Autopsy of diarrheal cases revealed
edematous changes in the lamina propria and looseness of interdigitations of the
intercellular junctions suggesting fluid accumulation. The intestine was characterized by
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100 CRC Critical Reviews in Microbiology
destructive changes in the mucosa showing structural damage of endoplasmic
reticulum, swelling of the mitochondria, loss of distinct cristae, and vacuolization in the
cytoplasm as well as disarrangement of microvilli. Destructive changes were not evenly
distributed, and cells showing destructive changes were often attended with almost intact
neighboring cells.277J 96 In surviving animals, marked inflammatory response and round
cell infiltration were the major findings. Oral challenge with rather large doses of TDH
(approximately 50 pg) causes mice to develop diarrhea and Histologic
examinations revealed findings very similar to those described above.
Honda et a1.2*0 found that I V injection of 5 pg of TDH killed mice very rapidly,
whereas death was not engendered by 0.5 pg within 3 days after similar injection.
Injection by the IP route was not as efficient in killing mice. When injected with TDH, the
animals became motionless and sometimes showed signs of cramping.
A positive response, accompanied by neutrophil infiltration and bloody exudate, is
elicited in RI L by injecting 200 pg of TDH.2797288 The intestinal tissue is characterized by
conspicuous erosive lesions and desquamation of necrotic mu~o s a. ~~~
RI L inoculated with 100 pg shows similar, but less intensive, histological changes in
spite of failure to excite dilitation. In contrast, choleragen and clostridium exotoxin only
require 0.2 and 30 p g , respectively, to cause di l i tati ~n.~'~ Based on determinations of
effective ileal loop dose of K P + K parahemolyticus tested in the presence and absence of
competitive V. alginolyticus in RIL, Twedt et al. suggested that pathogenicity of KP+
strains may involve the participation of some virulence mechanisms in addition to the
Kanagawa hemol y~i n. ~~~
Monkeys injected in the duodenum with 5 and 10 mg of toxin exhibited watery
diarrhea and catarrhal lesion, accompanied by a mucoid exudate accumulation in the
jejunum. With 25 mg of toxin the monkeys died between 5 and 10 hr afterinjection. By
comparison major pathologic findings in the human were conspicuous thinning and
markedly diminished tonus of the small intestinal wall and a great abundance of bloody
mucoid exudate.
5. Effect of Exposure 10 Hear
The toxic and hemolytic properties are thermostable at 100C for I 0 min,269 a
phenomenon reported for staphylococcal a-toxin and now described as the Arrhenius
effect.
reported on the peculiar heat stability of TDH. It was inactivated by
heating at 60C for 5 min but not significantly inactivated by heating at 100C for 30
Reheating the 60C heated toxin at 100C for 5 to 30 min restored about
30% of its initial activity. On the other hand 100'C-treated toxin (30 min), which held
about 50% of the initial activity, was almost entirely inactivated upon subsequent heating
at 60"c for 5 min.279
Changes in immunological behavior and biophysical properties of toxin after heating
were noted. In double immunodiffusion tests, 60C inactivated toxin did not give a
visible precipitin line against corresponding antiserum, whereas exposure to 100" C for 30
min gave a clearly sharp line. After reheating at 100" C for 30 min, the 60" C preheated
toxin gave a faint line of pre~i pi tati on.~~~* ~~' Conversely, 100C for 30 min exposed
hemolysin, when reheated at 60C for 5 min, and gave a very faint line which differed
sharply from the previous reaction.279
Takeda et al. found that a temperaturedependent inactivating factor of the TDH was
associated with the hemolysin, but could be separated from it by DEAE-cellulose column
chromatography. The factor was nondialyzable and inactive without heat treatment. The
factor was thermolabile and lost activity on heating at or above 60C.299 Destruction of
hemolysin by inactivating factor suggests proteolytic activity. The inactivating factor is
stimulated by NaCl or MgC12 and has maximum activity at approximately pH 8.0.300
Zen-Yoji et
min.177.279.298
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Volume 10, Issue 1 101
6. Interaction with Erythrocytes
TDH lyses erthrocytes of rats, dogs, humans, monkeys, guinea pigs, chickens, rabbits,
mice, and sheep, but not those of h o r ~ e ~ . ~ ~ ~ ~ ~ ~ ~ * ~ ~ ~ Apparently equine erythrocytes do not
possess the binding sites required by TDH for adsorption.m1 Lysis of equine erythrocytes
and destruction after exposure to 6OoC for 10 min are characteristic for V. cholerae
hemolysin and separate it from TDH. The amount of hemolysin required to form a
clear, hemolytic zone of 5 mm in diameter on Wagatsuma agar is 0.1 pg per
Sakurai et al.)O1 studied the interaction between TDH and human erythrocytes. Lysis
was dependent upon temperature. Although there was no detectable hemolysis at 0 to
4 O C, adsorption was discovered at low temperatures. Therefore, hemolysis is probably a
two-step procedure, with adsorption cccumng initially followed by hemolysis which is
enhanced by the presence of 25 mM divalent cations at a reaction pH of 6.0 to 8.0.
7. inhibition of Gangliosides
The hemolytic activity of TDH is inhibited by a mixture of gangliosides, but not by
G M1.280*302 Neuraminidase-sensitive gangliosides, especially G T ~ ganglioside, do inhibit
TDH biological activity. This further explains why equine erythrocytes seemingly are
uneffected by TDH, since their membranes apparently do not contain either of the
neuraminidase gangliosides GTI or D D ~ .
When injected intravenously into rats, TDH causes rapid death. Electrocardiograms
(ECG) used to monitor heart function detected irregularities after IV injection of the
toxin. Subsequent exposure of cultured mouse. heart cells to 0.1 mg or more of TDH
caused cessation of beating rhythm leading to the conclusion that TDH has cardiotoxic
activity, probably the reason for death of animals. This effect could be blocked by first
exposing TDH to a mixture of gangliosides.
As a result of their findings, Honda et al. proposed to chifnge the name of thermostable
direct hemolysin (TDH) to cardiotoxin*. They were able to show changes in ECG of
humans suffering from V. parahaemolyticus food infections and also reported the
presence of antihemolysin thenm4
3 0 3
8. Effect on Tissue Cultures
Morphological damage and cessation of cardiac contractions occurs in cultured fetal
mouse and fetal rat myocardial cells after exposure to TDH, but not in cultured
embryonic chick ventricular cells.294
Cytotoxic effects of TDH have been observed with HeLa and L cells.216 When FLcells
were treated with TDH, observations with scanning electron microscopy showed the
occurrence of gradual morphological changes in the microvilli of the cell after 5 and 10
min exposure, followed by complete absence of the microvilli after 30 min. There was
degradation of the cytoplasm ofthe cells and complete disintegration of the nuclei at 60
min.Os
9. Hemolysin Production through Generic Interconversion
The peculiar discrimination between KP+and KP- strains of Y. poruhaemolyticus
especially according to source, human and environmental, respectively, has prompted
introspection into the possibility of genetic interconversion. Burstyn et al.m6 have
isolated auxotrophic and KP- mutants. Cys- and Arg- mutants of KP+ strains were
found to be KP-. Revision to prototrophy was not accompanied by a concomitant
return to the KP+phenotype. The suggestion was made that environmental stimuli
might influence insertion/ excision events of an IS-like element governing the expression
of the hemolysin gene, which might explain the organisms ability to respond t o changes
in the environment.
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TABLE 3
VlBRIO PARAHAEMOLYTICUS
ANTIGENIC SCHEMA
0 group antigens
I
2
3
4
5
6
7
8
9
10
I I
I2
K type antigens
I ,25,26,32.38,4 1,5638
3.28
4.5.6.7.29.30.3 I ,33,37.43.45.48.54,57,59
4.8.9.10, I I , I2,13.34.42,49,53.55
15,17.30.47
18.46
19
20.2 1.22.39
23.44
24
36,40,50,5 I
52
VII. SEROTYPE CLASSIFICATION
Serological typing of V. parahaemolyticus is based on two major antigenic structures:
the somatic 0 antigen and the capsular polysaccharide K antigen. The H or flagellar
antigens are common to all strain^,^' and thus far have not been used for serotyping,
which depends upon the detection of specific 0 and K antigens among the 12 recognized
0 and 59 K antigenic component^^^'^'^^^(Table 3). The K antigens are thermolabile and
are susceptible to heating at 100C for 1 to 2 hr, while the 0 antigens are thermostable.
This antigenic schema was suggested by Sakazaki et al. after a study of 2720 strains
isolated from patients with gastroenteritis. Their analysis showed that V. parahaemoly-
ticus was serologically distinct from other vi bri ~s."~
This specificity of antigens was the subject of inquiry which led to the finding that V.
parahaemolyticus, V. alginolyticus, and V. anguillarum have closely 'identical H
antigen^,'^'"'^and V. parahaemolyticus and V. alginolyticus possess, in common, some
0 and K antigen^.*^*"^Twedt et al. have shown that some strains of V.parahaernolyticus
produce heterologous reactions due, in part, to the presence of other K antigens or cross-
reaction with other 0 antisera.'"
There do not appear to be significant correlations between V. parahaemolyticus and
other vibrios and in those cases where heterologous K antigens are apparent, definitive
typing can be accomplished with the 0 antigen.'" Although numerous environmental
and some clinical isolates are untypable by the K antigen, the majority of the clinical
strains usually can be categorized according to the 0 type.'12
As noted by Fishbein and Wentz, serological typing alone is not sufficiently specific to
be diagnostic. Also, the Sakazaki antigenic schema was based on strains isolated from
human infections and does not necessarily embrace those from the environment which
are frequently ~ntypabl e.'~ They point out that the Committee on Serotyping of V.
parahaemolyticus has adopted the position to enlarge the schema only by including
additional human ser~types.'~'
There has been interest in analyzing the chemical structure of the major antigens of V.
parahaemolylicus. Preparation of the K antigen has been described by Terada3I3 and
Zen-Yoji et al.,309 and purification achieved by Omoriet al.'" using V. parahaemolyticus
A55 (05:K 15). The substance was characteristically acidic, containing considerable
amounts of hexosamine and acetyl groups but no hexoses or pento~es.~" Kudoh
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Volume 10, Issue I 103
discovered the presence of numerous sugar components with varying percentages of each
in several purified K
Immunochemical studies performed on phenol-water extracted material of boiled cells
by Toni et al. indicated that the 0 antigen is a lipopolysaccharide containing glucose,
galactose, glucosamine, heptose, phosphorous, fatty acid ester, and nitrogen com-
pounds. Galactosamine was found in five of ten samples. When tested by precipitation,
the specificity of this material coincided with that of the O-cell agglutination.
Depending upon growth conditions, V. parahuemolyticus exhibits both polar
monotrichous and lateral flagella, a characteristic shared with V. alginolyticrcs. Shinoda
et al. demonstrated the antigenic difference between the two structures. Antisera against
purified flagella preparations of each did not cross-react when tested with opposing
antigens. The polar monotrichous flagellin exhibits homogeneity among numerous
marine vibrios, whereas the lateral flagellin cross-reacts only with antisera against V.
algino~yticus.~~
There are 59 K antigens and 12 0 antigens recognized and undoubtedly others
eventually will be i n~l uded. ~~- ~~ The predominance of serotype varies within particular
geographical locations from year to year and between diverse areas as well. There seems
to be no significance attached to any particular serotype and correlation with virulence is
lacking.
VIII. CLINICAL DESCRIPTION
Diarrhea is the chief illness caused by V. parahaemolyricus in humans. There are
reported instances of extraintestinal illnesses including wounds, ear infections, and
septi ~emi a. ~~~~~~ ~~ The mechanism by which V. parahaemolyticus is able to cause
gastroenteritis is still not well understood. The presence of leukocytes and superficial
ulceration of the colonic mucosa in the stool implies an invasive mechanism which has
not been substantiated in the laboratory. I t is clear that when present in ingested food,
this organism is capable of multiplying rapidly and causing debilitating diarrheal disease.
The symptomatology and salient features of this disease are exemplified by an.outbreak
aboard a U.S. Naval vessel where 87 of 276 persons were affected by contaminated food.324
The time of onset ranged from 4 to 30 hr with 10 cases occurring after 30 hr. The majority
were between 12 and 24 hr, and the mean time was 23.6 hr. The main clinical
manifestations were nausea and vomiting (26%), early fever (23%), cramping (86%), and
diarrhea (100%). The disease subsided in 3 to 5 days in 50% of the individuals, 5 to 7 days
in 30%, and persisted in 10 to 20% for more than 7 days with symptomatic treatment.
The majority of the cases were moderately affected and were able to continue their
duties to some degree. Diarrheal fluid was characteristically liquid, brown, with negative
white blood cell smears and negative stool guaiac in moderate cases. In markedly severe
cases, diarrhea was subsequently watery with mucus, blood, and tenesmus. Although
these were findings in an outbreak which occurred in a primarily young adult, male
population, they appear to minor the observations made during several outbreaks in the
US. and other countri e~. ~~
While most clinical cases are diagnosed in coastal regions, there tire instances when V.
parahuemolyticus-associated infections are seen in areas remote from ~eashores.~
Although V. paruhaemolyticus-associated diarrhea is generally self limiting, there are
cases of severe disease where antibiotic treatment is indicated. In such instances, isolation
and identification of this organism is required because unlike many other agents of
diarrheal disease, it is resistant to penicillin and its congeners, such as ampicillin. The
antibiotic generally recommended in such cases is tetracycline, although the ultimate
benefit of antibiotic therapy in this disease has not been thoroughly evaluated.
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IX. EPIDEMIOLOGY
V. parahaemolyticus food-borne outbreaks were originally described in J apan where
an estimated 70% of the attacks of food poisoning in the summer months is attributed to
the presence of this organism - hence the term summer diarrhea. The first
documented outbreak reported outside of J apan occurred in the state of Maryland in the
U.S. in 1971 among groups of picnic participants who had ingested contaminated
~rabs. ~ * ~~ Twelve other outbreaks occurred in the U.S. from 1969 to 1972, between J une
and October, in the Atlantic, Pacific, and Gulf Coast States and Hawaii.326 Five were
reported from the state of where the vehicle was crab and one each from
Massach~setts,~~~ Louisiana,ZA329 New J ersey, Washington,327 Texas,327 Ha~ai i , ~
and Florida.I2 Representative strains from several of these outbreaks and elsewhere in
the U.S., with very few exceptions, showed the same characteristics as those isolated in
J apan.334 In seven of the outbreaks, V. parahaemolyricus was isolated either from the
stools of infected patients or from suspected food, or in some cases, from both sources.
Etiology in the remaining five was based on the circumstances and environment
surrounding the ~utbreak.~ One isolated case following ingestion of conch meat in
California was reported in 1973. Although fish is usually the primary vehicle of
infection in J apan, the outbreaks in the US. involved shellfish, i.e., crab, shrimp, lobster,
and oysters.
Barker et al.J 26 proposed three pathways by which seafood could become
contaminated with sufficient numbers of V. parahuemolyticus to cause illness in humans.
Each pathway is based on the condition that a minimal number of organisms is present
initially and, therefore: (1) if food is allowed to remain unrefrigerated for a sufficient period
of time before ingestion without cooking, or (2) is insufficiently cooked, or (3) is
recontaminated after cooking, then illness can occur. These three mechanisms serve to
show that the majority of outbreaks and sporadic cases of gastroenteritis can be
prevented by attention to corrective measures.
There is usually some association between contact with seafood or seawater and the
presence of V. parahaemolyticus in human illness, although on occasion, patients deny
having had contact with either source for extended periods of time prior to the onset of
symptomatology. Other than anecdotal information, there seems to be no indication of
person to person spread through family contacts of patients or with food handlers, in
whom approximately 2.5% was found in summer months in J apan to harbor small
numbers of organisms insufficient to cause enteritis. Carriage could not be detected in the
winter months.12
The distribution of V. parahuemolyticus-caused gastroenteritis is widespread, having
been reported elsewhere on the North American Continent from Canada;33336 Europe
from the United K i ngd~m, ~ ~- ~ ~ Denmark, and the Soviet Uni ~n; ~ ~ Panama in
Central
and other Asian countries including Taiwan, Thailand,I4 India,4J o-52 Bangla-
d e ~h , ~~~ K~rea, ~-~ Viet Nam,14 Ok i na~a, ~~~* ~ ~ Mal ay~i a,~ and I ndone~i a. ~- ~~~~ ~ ~~
Additionally, there have been three shipboard o~tbr eaks. ~~* ~~ ~~~*
The annual incidence of gastroenteritis in these distributions varies in range from 2.646
to 33%.12 One study in Calcutta revealed that 33% of the patients examined and from
whom V. parahaemolyticus was isolated had no history of having eaten seafood within 7
days of the onset of symptoms. Approximately 15% of the healthy contacts of infected
patients was detected to be carriers of pathogenic V. parahaemolyticus. Another study in
showed that cases occurred throughout the year with peak incidences during
the premonsoon summer months. The rationale given was that V. parahuemolyticus
gastroenteritis may be virtually endemic in Calcutta. Similarly, this disease was noted
New Zealand;* Africa;44 Territory of
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Volume 10, Issue 1 105
throughout the year in Indonesia,46 but in all likelihood, persisted because of the year-
round tropical climate, which provided optimal conditions for proliferation of the
organism. This is in contrast to the marked seasonality of the disease in the U. S. 326 and
J apan.I2
A longitudinal study by R~mans ~ of human gastroenteritis caused by V.
parahaemolyticus in Indonesia provided informative data on this disease as it occurs in
the tropical environment. There was a 2:l ratio of infected males to females, which was
similar to the earlier finding of J oseph et al? The majority of cases was in the 20- to 49-
year age group. Symptomatology included diarrhea (98%), vomiting (73%), abdominal
pain (61%), nausea (45%), and fever>38C (29%). Records were not maintained for the
presence of headaches and chills. The primary symptomatic difference noted between
these patients and those suffering from cholera was the higher incidence of vomiting and
shock in cholera patients (90 and 50%, respectively, as compared with 73 and 24%).
Conversely, the V. parahaemolyticus-infected groups of patients had higher rates of
abdominal pain, nausea, and fever (61, 45, and 29%, respectively, vs. 21, 6, and 6%).
X. V. ALGZNOLYTZCUS
V. alginolyticus, originally. classified as biotype 2 of V. parahaemolyticus, was
reclassified as a separate species because of several different characteristic^'^(see Table
2). The ecological niche occupied by V. alginolyticus is similar to that of V.
parahaemolyticus, but very little specific ecological work has, as yet, been performed. V.
alginolyticus has rarely been implicated in intestinal disease and its pathogenicity
previously had been q~esti 0ned.I ~~ Within recent years, however, a number of cases have
been reported of superficial V. alginolyticus infections of the ear, eye, hand; leg, lung,
blood, and burns which, in most instances, had been exposed to sea~ater . ~~ - ~~ ~ At least
100 cases of disease associated with this organism have been in the
u .s. ,365,366268,369371-373 England,374 A~stral i a,~ ~~ Romania,376 and Bel gi ~m.~ In a
study carried out in Western Australia, V. algino/yticus was isolated from 20 of 36
samples (56%) taken from infected superficial wounds contaminated with seawater.370
In one of the few ecological studies reported, V. alginolyticus was isolated in the
Netherlands from 6% of water samples, 4% of mussels, and 7% of oyster samples during
J anuary to March.6 In the warmer months of August and September, Golten and
Scheffers found V. alginolyticus to comprise 85% of 50-mP samples collected from
Dutch coastal waters. This species has also been reported to occur in Danish, J apanese,
Alaskan, and Indonesian
Tubiash et al.Ig6 reported V. alginolyticus associated with bacillary necrosis of larval
and juvenile bivalve mollusks. Other investigators have isolated V. alginolyticus from a
variety of fish, shrimp, crabs, oysters, and lam^.^^^^^^-^^^In general, V. alginolyticus
appears to be present in larger numbers in seawater than V. parahaemolyticus021o and,
in fact, a proportional relationship between V. parahaemolyticus and V. alginolyticus
has been s~ggested.~ J oseph et found that V. parahaemolyticus showed
proportionally higher numbers than V. alginolyticus during the rainy season (November
to May) in the tropical waters and seafoods of J ava Bay, while V. alginolyticus was more
predominant in the dry season (J une to October).
Using the agar diffusion method, Larsen and Farid386 studied V. alginolyticus from
environmental sources of different geographical areas and some human pathogenic
strains for their sensitivity to 29 different antimicrobial agents. All strains were sensitive
to gentamicin, neomycin, sulfaisodimidin, sulfamethoxazole-trimethoprim, rifamycin,
nalidixan, and linco-spectin and all except one to trimethoprim, tetracycline,
chloramphenicol, nitrofurantoin, and tobramycin. Between 80 and 94% of tested strains
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was sensitive to clindamycin, cephalotin, kanamycin, tylosin, and spiramycin, while
ampicillin, vancomycin, and novobiocin were effective to approximately 50%. A high
percentage of resistance was found to methicillin, carbenicillin, and lincomycin. All
strains were resistant to penicillin and fucidin. No variation in susceptibility pattern
between environmental strains isolated from different areas was detected. Good
correlation was found among environmental and human pathogenic strains.
XI. V. VULNIFICUS (LACTOSE-POSITIVE VIBRIOS)
Another species of Vibrio recognized as a pathogen and receiving attention recently is
the lactose-positive(L+) vibrio, V. vulnijicus. Strains of this group closely resemble V.
parahaemolyticus and many were originally and understandably identified as V.
parahaemolyticus. Blake et al.7*2 studied the clinical characteristics and epidemiology
of disease associated with V. vulnijicus and reported two distinct clinical presentations.
In the first instance, illness began with septicemia, often within 24 hr after raw oysters had
been eaten, wherein the organisms had passed through the intestinal mucosa and into the
portal system. Eleven of 24 such patients, most of whom had preexisting hepatic disease,
died due to intractable shock secondary to Gram-negative sepsis. A separate case report
by Kelly and McCormick described a patient with acute bacterial myositis caused by V.
vulnificus, in whom there was no obvious portal of entry through the skin. In the
second group, characteristically, a wound became infected after exposure to seawater or
an injury caused by the handling of crabs. There was one death in the group of 15 patients
examined. A notable feature of both clinical presentations was the lack of vomiting or
diarrhea, unlike the symptoms associated with V. cholerae- or V. parahaemolyticus-
related infections.
Histologic features of bullous skin lesions and rapid onset of refractory shock, with
complete blockage of the heart in a patient with fulminant V. vulnificus sepsis, suggested
that potent bacterial toxins are involved in the pathogenesis of disease caused by this
organism.82
Most cases of V. vulnificus infection occurred during the warm months of the year in
coastal states. In addition to earlier cases occurring in or near U.S. coastal waters, where
the etiologic agent was originally reported as V. parahaemolyti~us,~~~.~~ recent
reports have also documented cases in BelgiumJ 8 and J apan.86 Raw seafood serves as
a major dietary staple in J apan and undoubtedly will prove to be the source of additional
cases as clinicians in that country become aware of this organism.
Few studies have been done on these strains other than those described in the clinical
reports cited above. Poole and Oliver87 studied V. vufnflcus in animal models and
reported severe local infections with gross tissue necrosis and rapid bacteremia. However,
rabbit ileal loop studies showed no fluid accumulation to suggest production of an entero-
toxin. Likewise, Bowdre et al? showed that neither filter sterilized supernatants nor killed,
whole cells were able to cause local edema or lethality in mice, whereas 10 viable cells
injected intraperitoneally or subcutaneously (SC) into mice resulted in hemoconcen-
tration and edema at the site of SC inoculation. Carruthers and Kabatfound that 1 1 V.
vulnijicus strains were less susceptible to the bactericidal activity of normal human serum
or serum treated with magnesium-ethyleneglycol-bis (B-aminoethyl ether)-N,N-tetraace-
tic acid than were 6 Vibrioparahaemolyticus strains. This suggests that V. vulnificus has
more potential for systemic invasion than does V. parahaemolyticus.
Ecological studies of these strains have not yet been reported; thus, seasonality,
environmental niche, and relationships to V. parahaemolyticus, V. alginolyticus, and
other species are still not known. Following isolation of V. vulnijicus from sputum and
blood of a resuscitated, drowning victim, attempts were made to determine the
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Volume 10, Issue 1 107
distribution of this organism in the waters around Galveston Island, where the accident
occurred. Seawater, sampled from over a period of 4 weeks, yielded positive cultures in
36% of the examinations made from 21 sites.390
Studies conducted on the survival of V. VulnijinrS in oyster homogenates held at 4 C
indicated a rapid and dramatic decrease in viability not attributable to either cold shock
or the oyster homogenate alone but to a combination of the a finding not observed
with V. parahuemolyticus.
Hollis et a1.I6 studied the biochemical characteristics of 38 isolates and were able to
differentiate them from V. parahaemolyticus on the basis of salt tolerance and lactose
fermentation (Table 2). Several strains were analyzed by DNA/ DNA hybridization by
Clark and Stcigerwalt, who concluded that this group constituted a species distinct
from V. parahaemolyticus and V. alginolyticus. Representative strains exhibited
hybridization values of 36,45,15, and 32% with V. alginolyticus, V.parahaemolyticus, V.
cholerae biotype El Tor and V. cholerae non 0-1, respectively, when examined at 60 C.
These values are comparable to those obtained by Reichelt et al.* for strains of L+
vibnos. Reichelt et al.2 have already suggested a nomenclature for this species, viz.,
Beneckea vulnijku, thereby removing them from the genus Vibrio. Clark and
Steigerwalt5 placed them in the genus Vibrio and considered them to be a Vibrio species.
suggested revival of the formal taxon, Vibrio vulnifcus.
XII. V. FLUVZALIS (GROUP F; EF6 VIBRIOS)
V. fluvialis (group F vibrios),@ yet another species of Vibrio implicated in
gastrointestinal di~ease,~~-~ was also called EF6.396 This organism was responsible
for an epidemic involving more than 500 patients in Bangladesh396 and has also caused
diarrheal disease in Bahrain, Bangladesh, and I nd~nesi a. ~~*~~ Several strains from
Indonesia and Bangladesh were found to be resistant to several antibiotics, including
chlorarnphenic~l.*~~* Like V. purahaemolyticus, these strains are widely distributed in
the marine and estuarine en~i ronment.~~ ~~
J ensen et a1.400 proposed a set of readily determinable phenotypic properties for the
differentiation of strains of group F from other related species. They concluded that there
were two biotypes based on formation of gas during D-glucose fermentation and the
utilization of glutarate as the sole source of carbon and energy. In another taxonomic
study, Lee et al.O showed that group F strains all fell in one closely knit cluster distinct
from all the species of Vibrio, Aeromonas, Plesiomonas, and Photobacterium studied.
Group F strains could be divided into two biovars, I and 11, both of which are present in
aquatic, particularly estuarine, environments throughout the world. Biovar I strains have
also been isolated from humans with diarrhea. They concluded that group F is a synonym
of group EF6 and that the strains within these groups should be classified in a new species
named V. fluvialis.
It is emphasized that this organism can be misidentified as Aeromonas because of
similar biochemical reactions in identification schema and with V. alginolyticus,
especially because of its tolerance to 8 to 10% NaCl concentrations.**93*399401
Differentiation of V.fluvialis and V. alginolyticus can be accomplished according to the
criteria in Table 2. Aeromonas can be differentially separated from V. fluvialis on the
basis of indole production, ability to utilize propionate and ketoglutarate growth in
the presence of 6% NaCl, minimal inhibitory concentrations of vibriostatic agent O/ 129,
and at 5C as shown in Table 4.
The mechanisms(s) of pathogenicity of V.fluvinlis is yet to be understood clearly. Thus
far, studies performed with whole cultures399 and with concentrated filtratesa2 suggest
the presence of an enterotoxin-like substance. Kreger and L ~ckwood~ found that V.
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I08 CRC Critical Reviews in Microbiology
TABLE 4
USEFUL FEATURES FOR DIFFERENTIATION OF
AEROMONAS HYDROPHILA AND
VIBRIO FLU VIA LIS
Chsnctcnrtic A. hydrophila V. fluvialis
-
lndolc , +
Propionare utilization -
a Ketoglularate utilization -
+
+
Growth
+
-
6% NaCl
5 O c +
-
01 129 MIC 2 320 pg/mP <320 pg/ rnP
% G + C 57 - 63 50
fluvialis possessed at least four toxic activities including cytolytic activity against
mammalian erythrocytes, cytotoxic activity for Chinese hamster cells, lethal activity for
mice, and a permeability factor (Po activity in rabbit skin. A nonhemolytic cytotoxic
factor was also observed. This organism has been shown to cause cytotoxic, and in some
instances, cytotonic-like reactions of mouse Y-1 adrenal cells.399 Sanyal et isolated
a group of organisms tentatively designated as group F vibrios from human diarrheal
cases and from marine and estuarine environments. Live cells of 23 strains isolated from
different sources were tested for their enterotoxicity in the adult rabbit ileal loop model
and all but two caused fluid accumulation comparable to that obtained with V. cholerae
569B. An inoculum of lo' viable bacteria gave rise to gut loop reaction. Culture filtrates
of the 19 strains tested, including one giving negative reaction with live cells, also caused
accumulation of fluid in loops.
The sugar composition of the 0-antigenic lipopolysaccharides isolated from group F
vibrios has b'een described. 2-Keto-3deoxy-octonate was totally absent from the
lipopolysaccharides. As common component sugars, glucose, galactose, L-glycero-D-
mannoheptose, and glucosamine were present. The group F vibrios examined were
found to be divided into two groups, designated tentatively as groups I and 11, on the
basis of the pattern of the sugar composition of their lipopolysaccharides. Additional
sugar components, mannosamine. quinovosamine and two unidentified amino sugars,
FI and F2, were present in group I. Rhamnose, galactosarnine, an unidentified amino
sugar, F3, and a relatively high content of D-glycero-D-mannoheptose were found in
group 11.'~'
ACKNOWLEDGMENTS
The studies performed by one of us (SWJ) were supported by Naval Medical Research
and Development Command work units nos. MF51.524.000.0040B and ZF5 1.
524.009.0057. The authors are sincerely grateful for the assistance provided by Agnes
Ginn and Carol Adkins in the preparation of this manuscript.
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Volume 10, Issue I 111
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56. Schiewe, M. H., Crosa, J. H.. and Ordal, E. J., Deoxyribonucleic acid relationships among marine
vibrios pathogenic to fish. Con. 1. Microbiol.. 23, 954, 19.77.
57. Cuerry, P. and Colwell, R. R., Isolation of cryptic plasmid deoxyribonuclcic acid from Kanagawa
posiiive strains of Vibrio porohoemolyricus, Infecr. Immun.. 16. 328. 1976.
58. Hugh, R. and Sakaukl. R., Minimal number of characters for the identification of Vibrio species.
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69. Huq, M. I., Huber, E., and Kibryia, C., Isolation of urease producing Vibrioporohoemol~:ricus strains
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72. Tamurr, T., Fujino. T., Kondo, M., and Kotani, S., Occurrence of poly-beta-hydroxybutyric acid
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87. Sakauki, R., Control of contamination with Vibrio puruhuemolyricus in seafoods and isolation and
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Volume 10, Issue I 113
109. Morris, C. K., DeWitt, W. E., Gangarm, E. J., and McCormack. W. M., Enhancement by sodium
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290. Oban, Y., Yanui, S.. Niikawa. T., Miyamoto, Y., Ohashi, M., and Shinoda, T., Histopathological
changes in the small intestine of suckling mice challenged orally with purified hemolysin from Vibrio
parahaemolyricus. i n Inr. Symp. Vibrioparahaemolyricus. Fujin0.T.. Sakaguchi. G. . Sakazaki. R.. and
Takeda, Y.. Eds., Saikon Pub]. Co., Tokyo, 1974. 253.
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direct hemolysin produced by Vibrio parahoemolyficus on FI cells. Infect. Immun.. 13. 876. 1976.
292. HondaJ ., Goshi m, K., Takeda, Y.,Sugino. Y..and Mi ratmi , T., Demonstration of the cardiotoxicity
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294. Gosbima, K., Hondn, T., Hirata, M., Kikuchi, K., fakeda, Y., and Miwrtani, T., Stopping of the
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298. Miwahni, T., raked., Y., Sakuni. 1.. Y oshhra, A., and Tag& S., Effect of heat (Arrhenius effect) on
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299. Takeda, Y., Hod. Y., !hkUrd, J., and Miwahni. T., Effect of heat on direct hemolysin of Yibrio
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Jap. 1. Med. Sri. Bi ol .. 28. 90. 1975.
300. Takcda. Y., Hori, Y., Toga, S., Sakurai, J., and Mi wat~i , T., Characterization of the temperature-
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301. S8kur.i. J., B.h.nar, M. A.. J inguji, Y.,rnd Miwatani, T., lnteraction of thermostable direct hemolysin
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302. Takeda. Y., Takeda, T., Honda, T., Sakuni , J., Ohtomo. N.,and Miwatani,T., Inhibition of hemolytic
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303. TI kedl. Y.. Taked8, T., Homda. T., and Miwatani, T., Inactivation of the biological activities of the
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304. Honda, T., faked.. Y., and Miwat8ni.T.. Role of the cardiotoxin produced by Vibrioparahuemol~lticus
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305. Sakurai, J., Honda, T., J inguji, Y., Arita. M.. and Miwatani, T., Morphological changes of FL cells
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3 10. Sakauki , R., Iwanami, S., and Tamura, K., Studies on the enteropathogenic, facultatively halophilic
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312. Miwrtmi, T.. Shinoda, S., Tamura. T., Nishimunc, H., Tomaru, A., Yoshihnrn, A., and Fujino, T.,
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314. Omori, C., Iwao. M., lida. S.. and Kuroda. K.. Studies on K antigen of Vibrio puruhuemolyricus. 1.
Isolation and purification of K antigen from Vibrio puruhuemo/vricus A55 and some of its biological
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antigens and their sugar constituents, Jup. J. Bucreriol., 24,331, 1969.
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77. 1969.
318. Shinoda, S., Honda, T., Tikeda, Y., and Miwatani, T., Antigenic difference between polar
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332 Center for Disease Control, Gastroenteritis - Florida, Morbid. Mor r ul ., 21.6. 1972.
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