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VIBR IO P A R A HAEMOL YTZCUS A N D R E L A T E D H A L O P H IL I 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 N T R O D U C T I O N ( H I S T O R Y )

Th irty years have passed sinc e the first isolation of Vibriopatahaemolyricuus.Volumes

of literature have been w ritten a bo ut this organism a nd it has become recognized as a

ma jor cau se of food p oisoning in area s of the world where seafood is a majo r staple of the

diet. Th e history of V parahaemolyricus traces back to O ctobe r 20a nd 21,1950, when an

out bre ak of food poisoning occurred in the southern su bur bs of Osaka, Jap an . Of the

272

patien ts who suffered acute gastroenteritis,

20

died. The sym ptom s of the gastroenteritis

included acute ab dom inal pain, vomiting, and diarrhea, with watery and , in som e cases,

bloody stools. Th e food suspected to ha ve caused the foo d poisoning was a small, half-

dried sardine, Engradis aponica Ho uttu yn, called "shirasu" in Japanese. Th e sardine is

boiled in salt water and eaten when partially dried.'

Based on his expertise derived while assigned at Maym yo, B urma in 1944 as an arm y

surgeon sublieutenant,

T.

Fujino and his co-workers at the Research Insti tute for

Microbial Diseases (Osaka University) carried

o u t

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, ob taining a pure c ultu re 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 pleuro pneum onia-like, filterable bacteria as causative agents. Pu ru len t peritonitis

was induced in a guinea pig. Th e saline hom ogenate of shirasu w as 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

hctobac i l lus

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 w ith swollen edges am ong the num erous G ram-negative rods. Attempts

to separate the two kinds of Gram-n egative bacteria by transfer to fresh agar plates were

unsuccessful. Thus, hypothesizing that one of the two G ram-neg ative bacterial strains

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was a pathogen and would inhibit growth of the other strain in vivo and grow more

rapidly, F ujino an d his co-workers attempted sep ara tion of the two by anim al passage. A

suspension of colonies co ntai nin g the tw o kinds of Gram -negative ba cteria was injected

intraperitoneally into mice. Several h ou rs later, when sym ptom s of illness appeared in

the mice, samples of ascitic fluid were tak en an d inoculated into oth er mice. The anim al

passage was repeated once again, and after 2% o

3

hr, sym ptom s appeared in the mice.

The asc itic fluid f ro m the last set of mice to be passaged was inoculated onto blood aga r

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

subsequen t testing in pur e culture produced acid an d gas in glucose an d did not hydrolyze

gelatin, and a hemolytic, fat rod demonstrating bipolar staining. The latter isolated

produced acid - ut not gas rom glucose, liquified gelatin, an d proved pathogenic

for mice. The gas-producing strain was identified as

Proreus morganii, but the

anaerogen ic strain could not

be

identified a t tha t time. T he history of this series ofe ven ts

in the isolation of V .parahaemolytica is nicely detailed by F ujino' an d by Miw atani and

Takeda.6

The anaerogenic, unidentified bacterium of Fu jino was tested fu rther and found to be

actively motile by means of a single, polar flagellum, resembling

Vibrio cholerae.

However, it did no t react with

V. holerae

ant iserum and the long axis

of

the bacterium

was not curved. B ased o n these features an d o n its bipola r staining, the new species was

named Pasteurella parahuem olyticus [sic], n. sp.'

Fu jino 2 subsequently isolated the sam e bacterium fro m the intestinal contents of a

victim of foo d poisoning, an d Fujino et al.' rep orte d the discovery of the new pathogen a t

the 25th Annual Me eting of the Japan ese Association fo r Infectious Disease in

195

1.

An

extensive, detailed description of Pasreurella parahaemolytica first appeared in

Jap an ese 2 and later in English.'

In

the fall of

1955 I.

Takikawa,' from the Nation al Yokoh am a Hospital, visited the

laboratory of Dr. Fuj ino. An outbreak o ffoo d poisoning had occurred at the Yokoham a

Hospital, involving

I20

patients, with no deaths. A Gram-negative rod with a single,

polar flagellum was isolated on 4% salt ag ar used to isolate Staphylococcus. The culture

appeared to be similar to Pasreurella parah aem olytica. but was halophilic, the salt-

requiring na ture of

P. parahaemolyrica

not having been recognized previously. In the

hospital inciden t, the foo d implicated was brine cuc um ber (pickles) served to the patients

and it was speculated tha t the causative agent was a halophilic orga nism 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, w ith rou nd ed 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 anim al blood show th at it has a thin capsule. It

is

monotrichal.

A

very few organism s

can be seen to mo ve like

Vibrio comm a.

When an 18-hr blood agar culture is examined

und er d ark field i l lumination o ne o r two bacill i per field c an be seen to move. Electron

microscopy shows th at each bacillus has a single flagellum. Mo veme nt c an also be seen in

direct pr epa ratio ns from mouse ascites. It liquifies gelatin, an d show s a hemolytic ring in

blood agar. It grows aerobically on plain ag ar f orm ing white, op aqu e colonies."

Ta kik aw a7 rep orte d tha t his isolate was closely related in its characteristics to the

organisms described by F ujin o et al.,3 but he concluded tha t it sho uld be considered a

member of the genus Pseudomonas." i.e., Pseudom onas enreritis. Thereafter , in Ja pan ,

the organism was referred to

as

the so-called "pathogenic halophilic bacterium".

M iyam oto et aL9 stated tha t the organism shou ld be placed in the genus Aeromonas

because of its fer me ntat ive utilization of glucose, and proposed a new genus,


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Oceanomonas, for the halophilic, fermen tative bacteria. Th us M iyamoto et aL9proposed

that the clinical isolates of Fujino et aL 3 and Takikaw a" 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.

Sa ka zak i and co-workers" first placed the strains into the genus Vibrio after

examination of

1072

such strains. Sub sequently,

K

parahaemolyticus was divided into

two biotypes, which Zen-Yoji et a1.I' concluded should

be

separated into two species.

This was propose d by Sakazaki" in 1968, wh o designated biotype 2 as

V.

alginolyticus.

The main differences between the biotypes were acetoin production, sucrose

fermentation, growth in 10% NaCI, and swarm ing 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 sep aration of these two phen a a s distinct species." A third biotype was

discovered re~ently.'~' ' ' reviously regarded as "lactose positive" vibrios, the nomen-

clature Vibrio vuln ficus was proposed.18

Detailed reviews of the history of the taxonomy of

K

purahaemolyticus have been

provided by Colwell, ' Cabassi and M on, l9 Miwatani and Take da,6and C hatterjee.20 n

recent y ears, pro posa ls have been made t o reassign this species from the genus Vibrio to

the family Brucellaceae by Chatterjee" an d to the genus Beneckea by Baumann et a1."

The definition of the genus

V i b r i ~ ' ' , ~ ~ncludes straight rods and embraces

V.

parahuemolyticus.

since it produces a p ola r flagellum when grown in broth, even tho ugh

it

m ay be peritrichously flagellated when gro wn on a ga r medium (vide infru). ,While V.

parahuemolyticus may require slightly higher concentrations of Na' than V. cholerae.

which re quires only trace concen trations,

V .

cholerae demonstrates a salinity op timum of

approximately 1 NaCl as opposed to 2 to 3% NaCl for V. parahaemolyticus,"

differences not suitable for generic separation. In vitro D N A / DN A h ybridization d ata

also show that

V.

parahuemolyticus

is

more related genetically to

K

cholerae than to

other Beneckea species." Recent pub lications have demo nstrate d a concensu s on

retaining this species in the genus V i b r i ~ . ~ '

11.

TAXONOMY

A. Morphological, Cultural, and Biochemical Characteristics of Y.parahoemolyticus

I . Morphology

Viewed with the microscope, V. parahaemolyticus appe ars 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 G ra m stain

ap pe ar s at the p olar extremities of y ou ng cells in the process of dividing.I9 Th e

flagellation of this organism has been extensively studied and mo st 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

K i

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 m aximum growth tempera ture of



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approximately 4 4 O

C,

an d a n opti m um between 35 and 37' C."-3J In teracting effects of

pH , temperature, an d salt concen tration can esult in slight alteration in the temp eratu re

limit on g rowth . B e ~ c h a t ' ~eported moderate grow th occurred

at

5C when the medium

was in the alkaline pH range. T he pH r ange

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

T he ability of

V. arahoemolyticus

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. Gro wth occurs at NaC l conce ntra tion s

of

0. I M

to approxim ately 1.2 M i th an opt imum sal t concentrat ion for growth of about 0 .5 M

NaCI.1'*3'-39Distilled water inactivates

V. aruhaemolyricus,

with a

90%

reduction

of

viable cells within 1 t o 4 min." Ot he r cations, e.g., Li' an d K', can produce a sparing

effect on th e specific requirements fo r sodium, but th e minimal, essential requirement

for

sodium is appro xim ately 0.003

to

0.007

M

Na', wh en the cells ar e grow n in synth etic

media."*41 When nonm etabolizable LiCl, sucrose, an d ethylene chloride ar e used to

replace sodium ion in a synthetic medium, each is capable

of

providing osmotic

regulation. In ad dition t o this requirement, sod ium ion ap pea rs to be necessary for

protein synthesis, which is inhibited in sodium ion-omitted medium containing osmotic

agents."*'"

In a s tudy of a ma r ine pseudomonad D rapeau and M a c L ~ o d " ~ound that washed

cells, when incubated w ith l'C-a-aminoisobutyric acid, were ab le t o accum ulate this

analogue inside the cell, with ou t metabolizing

it,

an d required the presence of Na' in the

medium.

K',

Rb', NH4', Li', an d sucro se could not su bs titu te fo r Na' in the tra ns po rt

process

.

In similar fashion Sa ka i and Sakai'"found tha t a gr ou p of marin e bacteria, which they

term T H (terrestrial halophilic) type (and include V.pmahoemolyricus) is similar to their

MH mar ine halop hilic) type w hich is described a s follows:

M

H-type ba cteria seemingly

require

Mg

for the oxid ation of substrates, however, Na' only is able to play

the ab ov e physiological roles. Na'. in fact, prevents th e lysis of cells an d accelerates

cytochrome oxidase, the electron tran spo rt chain, and A T P form ation of oxidative

phosphory lation. Na* also functions in the Na', K'dependent active tran spo rt of

nutrien ts in to the cells. K'is accesso nly needed only in the presence of Na'. Con sequ ently

MH -type indispensably needs Na' as the sole cation of su pp or tin g growth. This type

belongs t o psychrophilic bacteria because it lacks grow th capacity a t 37" C." Th e

exceptions which sep arate the T H type from th e M H type are described as follows: "The

Na' requirement is less than that of MH -type fo r the prevention of lysis, the oxid ation of

substrates, the electron tran spo rt system, the cytoch rom e oxida se, an d growth. T his type

is able to grow well a t 37C an d thereby belongs to mesophilic bacteria." Th e combined

effects of water activity, solute, and temp erature o n th e grow th of V .porohaemolyticus

have been studied with t he limiting water activity for g rowth d epend ing up on th e solute

employed.

An interesting physiological feature of V.

parahuemolyricus

is its extremely short

g en eratio n tim e. K a t ~ h ' ~eported a generation time

as

shor t as 8 to 9 min, althoug h 10 to

12 min is mo re com mo nly observed. UlitzurU studied 30 strains of

V.

parahoemolyticus

and observed two major groups, based on generation time. One group

of

strains

dem onstrated a short generation time, ix . , 12 to 14 min, and a second grou p, a longer

time of

20 to

25 min.

V. parahoemolyticus,

like other vibrios, is a facultative anaerobe, possessing both

respiratory an d fe rm enta tive 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 ch loram phen icol, gentamicin, kan amy cin,



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nitrofurantoin, tetracyline, doxycyline, and streptomycin an d is resistant to ampicillin,

carbenicillin, clindamycin, colistin, erythromycin, and penicillin.'

J ~ ~ ~ ~ - ~ ~

para-

haemolyticus

and

V.

alginolyticus

from hum an and environmental sources, examined by

the minimal inhibitory concentration (MIC) method, are uniformly susceptible to

chloramphenicol an d tetracycline within attai nab le serum levels. Most strains are

resistant to ampicillin and exhibit p-lactam ase activity, which accou nts f o r this

resistance. Occ asional multiresistance is noted, bu t thus fa r plasmid-linked d ru g

resistance has not been shown. Susceptibility to gentamicin can be demonstrated with

agar diffusion, but examination for MIC 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, an d de grades gelatin, chitin, starch,

casein, and lecithin. It is usually negative for arginine dihydrolase activity, hydrogen

sulfide production (vide infru), urease, phenylalanine deam inase, and luminescence. An

im po rta nt , but variable, characteristic is hemolysis of blood, kno wn as the K anagawa

phenomenon (KP), measured by using a high salt-containing medium (vide infra).

In

general, strains

of V .

parahaemolyticus utilize th e following co mp ou nds 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, p al an in e, L-ornithine, L-citrulline, or spermine."J4Jo

4.

Deoxyribonucleic acid

(DNA)

Base Composition and Nucleic Acid Hom ology

The composition of chromosomal DNA

of

strains

of V.

parahaemolyticus is in the

range 44 o 46% guan ine plus cytosine (G

+C).

D N A /D N A reassociation studies have

been performed by several investigators an d intraspecific values ar e usually >90% (at 60

to 63C ).24Jz-54nterspecific values between

V .

parahaemolyticus and V cholerae range

from 16 to 56% (at 60 C) w ith mo st values falling between 20 to

30 .52Js

alginolyticus

shares

60

o 70% homology with K parahaemolyticus and strains

of

the "lactose positive"

vibrios share 40 to 50 homology with V parahaemolyticus and V. a l g i n o l y t i c u ~ . ~ ~,

parahuemolyticus reveals only a low amount of homology with other marine vibrios,

specifically,

V.

anpi l larum. Reassociation values between these tw o species ar e in the

range of 20 to 30% with the mem brane filter whereas values of only 4 to

5

homology were observed using the S endonuclease assay, a m ore 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' fo un d that 40 f 12s train sfrom

human and e nvironme ntal sources had multiple plasmid species of cryptic function.



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82


Table 1

MINIMAL CHARACTERS

FOR

DENTIFICATION OF

V .

PARA

H A

MOL 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 d ccarbo xylax

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.

Minima l Characteristics f o r Identijication

of

V. parahaem olyticus and Biochemical

Variation

A

list of m inimum characteristics for identification of V. parahaemolyticus has been

repor ted by Hugh and Sakazaki and C0lwe11~~Table 1). A comparison of

characteristics fo r

V.

parahaemolyticus and related species and genera is provided in

Table 2. Individual characteristics sho uld not be overem phasized f or identification, since

stra in variability is co mm on. Conversely, even if a strain , particularly a n environmen tal

isolate, fulfil ls all cri teria, fu rther biochemical cha racteriz ation, even D N A / DN A

hybridizations, sometime m ay be required for c om plete separation an d identification.

A

more complete characterization of V. parahaernolyticus, beyond a minimum key

chara cter analysis,

will

occasionally distinguish an isolate from similar, but as yet

incompletely characterized, marine bacteria.

Exceptions

to

the list of minimal characterist ics shou ld be noted. F or example, sucrose

fermentation is a primary differential characteristic an d

is

th e basic criterion employed in

most of the isolation methods recommended for

V .

parahaemolyticus (vide infra).

Nevertheless, man y sucrose positive strain s have been r ep orte d. ColwelldO oun d 6% of

the strains of V. parahaemolyticus examined to be sucrose posit ive. Joseph and

G ilm our 6 also re ported several such s train s. K am pel ma che r et a1.62 isolated sucrose

positive

V. parahaemolyticus

from mussels and F ujin o et al. fou nd

2% of

their marine

isolates to be sucrose positive. Of

1484

strai ns of this species isolated fro m British coastal

waters,

6.9%

was

sucrose positive. Success in dete ctin g- acid produced du rin g

fer me ntat ion of sucrose by

V.

parahaemolyticus can vary , accord ing to the medium and

concentrat ion of sucrose used. B aross@ recom mend ed the use of

OF

basal medium ,6s

with 0.5% final conc entration of added sucrose. Se pa ratio n of V. parahaemo lyticus and

V . alginolyticus

must be based on several criteria, such

as

acetoin prod uction, grow th 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. parahaem olyticus, but



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C R C

Critical Reviews in Microbiology

ma ny exceptions will be observed. Fujin o et al. repo rted

4

to

5

of the s train s tested to

be negative for ornithine a n d /

or

lysine decarboxylase. The percentage of ornithine

positive strains isolated fro m sam ples collected in Tok yo has been rep orted to be as low

as 78%.

Production of hyd rogen sulfide should be tested by reading the reaction occurring in

the butt of a tube of Kliglers Iron agar (KIA) or triple sugar iron aga r

(TSI).

Sakazak i et

al. reported that non e of 1702 strains examined were positive for H2Susing TSI or S I M

media. ColwellW fou nd nearly all of the stra ins of

V. cholerae

and V .

parahaemolyricus

examined to be

H2S,

using more sensitive methods for detection of

HIS

production.

T wedt and c o - ~ o r k e r s ~ou nd nearly all of their cultures to be positive using SIM and

lead acetate ag ar (Difco). Indeed, hydroge n sulfide pro duc tion o n Russells Triple sugar

agar, but not on

TSI, h a s

been suggested as a n aid in the identification of this species.67

Two o ther cha racteristics, althou gh no t included in the set of minimal characteristics

listed by H ugh an d Sakazaki,* are noteworthy. Ind ole pro duc tion is usually positive, bu t

indole negative strain s have been implicated in several outbr eak s of foo d 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 stra ins tested was urease + and Ch itu et a1.,68who examined 8 strains

of V. parahaemolyricus isolated from salted herring and roe, found 6 to be urease

posit ive. Sakazaki et al . reported 4% of the stra ins tested to be urease positive and

Kaper et al. (in prep ara tion ) reported 13 urease positive strain s of 19 isolated from

shrimp. The isolation of urease positive strains from human infections is further

substant iated 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 De neke and

Colwell and T am ur a et al . ,72*73nd has been shown to possess poly p-hydroxybutyric

acid and a highly branched a- D glu ca n. Fa tty acid com position was examined by

Rietschel et al . , Ol iver and C~lwel l ,~n d B euchat an d W ~ r t h i n g t o n , ~ ~nd the

predominant fatty acids were C12, C14, C16:l , C16, and C18:l . Using high pressure

liquid chr om otogr aph y, Me11 et al.,? also fo und C 14 :l, C13, CI S, C 17, C18, C19, and C 21.

Sup erox ide dismutase

(SOD)

nd catalase levels in

V. parahaemolyricus

were examined

by Daily et al.,* who found only one detectable SOD nzyme in most of the strains

tested, as compared with the three

SOD

enzymes of

E.

coli.

A c-type c ytoch rom e, with

the capacity to bind carbo n mo nox ide, was repo rted for the soluble fraction of cell-free

extracts of

V.

parahaemolyricus by Collins and Knowles (unpublished observations,

quo ted by West et al.. Th e function of this cyto ch rom e is un kn ow n but evidence is

accumulating that such cytochromes are associated with organisms having complex,

branched respiratory Oth er enzymes of

V. parahaemolyricus

which hav e been

studied include phospholipase

A ,

lysophospholipase and

glycerophosphorylcholine

diesterase, lysophospholipase, lecithinase,82aspar tok inase , amylase , ge la t ina~e,~

and acid and alkaline phosphatases.86

111. I S O LA T I O N A N D E N U M E R A T I O N OF V. P A W H A E M O L Y T I C U S

V . parahaemolyticus has captured the interest of microbiologists in many fields,

especially medical, fo od , and env iron m enta l microbiologists. As a result, a variety of

me thod s have been developed specifically

for

the isolat ion, en richment , and enum erat ion

of

V.parahaem~ly~icus.~~*-~

ost of the me tho ds involve direct plating of samples on

an aga r medium, o r initial enrichment in broth, followed by s t reaking and isolat ion on a n

agar medium. Direct plating is sufficient, in most cases, for fecal specimens, but food



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samples and samples collected from th e environment frequ ently, if not always, require

enrichment.

A

few procedures require special incubation of media at elevated

temperatures

(42

to 43C)

o r

under anaerobic conditions to provide additional

selectivity.

Typical colonies of V.

parahaemolyticus

are picked and tested using a set

of

biochem ical, physiological, an d sero logical tests. If imme diate 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 tran spo rt medium , such as Cary-Blair

medium"

or

that described by LeClair et al.9'

A. Enrichment Broth

Initial contributions to methodology f or isolation and en ume ration of V.parahaemo-

lyricus

were m ade by Japa ne se 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 GS TB has been m odified subsequen tly by substitution of lauryl sulfate f o r

T e e p 0 1 . ~ ~rabinose-ethyl violet broth" con tains ethyl violet an d is adjusted to pH

8.6.

A

collaborative study reported by Petersenm 3 described Horie bro th

as

yielding MPN

values ten times greater tha n GSTB . This medium was modified by Kape r et by

substitution of galactose for arabinose. Other enrichment broths proposed for V.

parahuemolyticus

include the SWYE medium of K ane ko a nd Colwe1I9' an d salt-colistin

broth (SCB) of Sakazaki." A salt-polymyxin B broth (SPB) was proposed by

Ka mp elma che r et al.," an d a mo re recent version of

SPB,

sed by Sakazaki (personal

comm unication), contains polymyxin B (500 pg/m l), salt

(2%

NaCl), and nu trient broth.

A

survey of several broths proposed for V.parahaemolyticus by Naka nishi an d 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 pho sph ate buffer, was reported

by Ch un et a1.'" an d Teep ol has also been employed in a wa ter blue-alizarin yellow

b r ~ t h . ~ ' r i s t e n ~ e n ' ~ ~ * ' ~ 'tilized a meat broth amended with 7% NaCl and

0.3%

alkyl

benzene sulphonate, to which starch and chitin had also been added. Trypticase Soy

bro th (TSB), amen ded with 7%NaCI, has been used by Vand erzant and N ickelsonmas an

enrichment, with alkaline-saline peptone wateriM

as

a second enrichment after

incubation for 8 to 12 hr. Sakazak i" has used the tellurite-bile salt br oth of Mon surIo5 or

secondary enrichment. Bismuth sulphite phenol red (B SP R) bro th, containing sucrose,

NaCI, manni tol, and bismuth sulphite, was employed by Thompson and T r e n h ~ l m " ~or

isolation of V. parahuemolyticus.

A

recent study of

V.

parahaemolyticus in Dutch

mussels'07 included a comp arison of five enrichment broths. Th e conclusion was tha t

enrichment in the 5% NaCl meat broth

of

Kam pelmach er et incubated at 37C

provided the best recovery of

V

parahaemolyticus.

B. Plating Media

A wide variety of plating media, m any of which were originally developed fo r isolation

of

V. cholerae,

has been employed fo r recovery of V.

parahuemolyticus.

Undoubtedly,

the m ost comm only employed a gar m edium is the thiosulfate citrate bile salts sucrose

(TCB S) aga r of Kobaya shi et al.''' This medium inh ibits mo st of the othe r bacterial

species comp rising 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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and thymol blue included in the medium. V. parahaemolyticus, generally, is not a

sucrose-fermenting organism on TC BS agar, a nd the refore prod uces a bluish or blue-

green colony. Although TCBS

is

a medium useful for isolation of

V.

cholerae

and

V

parahaemolyticus, the selectivity of the m edium is suc h tha t it may not always suppress

growth of other organisms, such as Proteus spp., Aeromonas spp.,

or

Staphylococcus

spp. Furth erm ore, differentiation am ong species of pa thoge nic and other, as yet poorly

characterized, species of

Vibrio

foun d in the aq uat ic environ me nt is not always achieved

and additional tests, comprising a follow-up screening, are required. Modifications of

TC BS have been proposed, such as elevation of the NaCl conten t, Iwadd ition of bile salts

derivatives,"' o r alkyl benzene sulpho nate. lo Significant variation in selectivity can be

observed when the several brand s of TCB S aga r are com pared.

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 ncludes Teepol for selection and

sucrose ferm entation (via the b rom thymol blue-thymol blue indicator) for differentia-

t ion. Sakazaki" modified BTB Teepol by sub sti tuting sod ium heptadecyl sulpha te

(Tergitol

7)

for Teepol and found i t to be m or e selective. Tee pol ca n also be replaced by

lauryl sulphate.99 Polymyxin-tylosin sucrose salt (P T S S) a ga r can provide differentiation

of Vibrio spp. on the basis of sucrose ferm entation , util izing antibiotics, rather tha n

detergent, fo r selectivity.62

W ater blue, alizarin yellow ag ar (WA)"' incor pora tes water blue and alizarin yellow in a

medium suitable fo r 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 gro w n on WA , results in a

reduction in the pH of the medium, thereby cau sing the me dium t o assume a blue hue,

caused by a chan ge 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 c arboh ydra te other than sucrose, such a s arabinose, was employed by

Horie et al.," ' who devised an arabinose am m on ium sulfate cho late (AAC) medium

which contains sodium c holate at a pH of 8.6. Un fortuna tely, there is a tendency for the

medium to provide an underestimate of the true incidence of

V.

parahaemolyticus

because arabin ose ferm entation is a variable chara cterist ic of the species (see Table 2).

So diu m cholate was used by Watkins et al."' to inh ibit the grow th of Gram-posit ive

organisms on the prim ary isolation medium. Co pp er su lfate has been used to inhibit

V

alginolyticus, a species closely related t o V. parahuemolyticus, with galactose included as

a ferm entab le carbohydrate." '

A nonselective medium was devised by B aross an d L i~ to n "~ * " ' mploying starch

hydrolysis as a differential Characteristic. The Baross and Liston medium contains no

selective agents and the pH of the medium

is

adjus ted to

7.5.

Inoculated plates are

incubated in an anaerobic jar for 36 to 48 hr a t 37 C.

For

samples conta ining large

numb ers 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 a nd media consti tuents

proposed for isolation of V. parahaemolyricus. Penicil lin in co 6o ra ted into a medium

of

'alkaline pH proved to be more useful than

a

variety of selective agents that had been

suggested by investigators fo r V. parahaemolyticus. Th ese included potassium tellurite,

sod ium deoxycholate, Teepol, and 6% NaC l, all of which were considered t o

be

useful in

the isolation of

V. parahaemolyticus.

With starch hydrolysis as the differentiating

characteristic, the final formulation included pep tone

(2'39,

yeast extract

(0.2%),

co rn

starch (0.5%), Na Cl (3.0% ),-@nicillin (2 U/mP ) , and ag ar (1.5%), pH 8.0. Subsequently,



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Vanderzant and Nickelsonw modified the medium of Twedt a nd Novelli lightly, 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 TC BS a nd containing sod ium taurocholate and sodium

lauryl sulphate. Sucrose Teepol tellurite (SIT) agar, described by Chatterjee et a1.,*

contains peptone, beef extract, Teepol, potassium tellurite, sucrose, bromthym ol blue,

and agar. In the U.S. CB S is not difficult to obtain but n ot all laboratories, especially

clinical laboratories, routinely stock this medium. So me workers have reported

satisfactory results using more com mon media, such

as

mannitol salt agar and xylose

lysine deoxych olate (XLD ) agar amended with salt an d 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

Prob lem s of heat and cold stress on V.parahaemolyticushav e been examined in detail

by food micr~biologis ts .~*-~his aspect of the biology of V parahaemolyticus is

interes ting since seafood is preserved by chilling

or

freezing and m ost seafood is cooked

well, or a t least heated, before consump tion. V.parahaemolyticur iswidely recognized a s

being sensitive to cold when it is present in fo od an d water.27 - In add ition, effects of

heat a nd cold stress of

V.

arahaernolyticus

are altered significantly by the m edium and

composition of the diluent to which they are exposed during and after tress.^'**^^'-^^^

Va nde rzan t et al. evaluated several proc edur es fo r isolation of V.parahaemolyticus

which were applied t o recovery of stressed cells. They co ncluded that TS B containing 7%

NaCI, when used with modified agar of Twedt and N ~ v e l l i , ~as more effective, but less

selective, tha n G STB and TC BS agar. Beuchat reported t ha t TSB to which

7

NaCl

had been add ed a nd GST B were inferior to arabinose ethyl violet broth9 an d to w ater

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 m entioned ab ove. B iochemical prop erties of V.

parahaemolyticus can be determined using conventional media supplemented with N aCl

at approximately

1

to 3% final concentration. While V arahaemolyricus grows well on

ordinary blood agars and on Mueller-Hinton aga r, as well as most media containing

NaCI, there are particular media, e.g., M R -V P an d decarbox ylase media, to which NaCl

must be added in approximately 2%

concentration fo r significant growth to occur.135

A

specific medium, Wagatsuma medium, is employed

to

test fo r hemolysis of blood. The

reaction observed on Wagatsuma medium is termed the Kanagawa phenomenon

(KP).6736his medium contains: yeast extra ct, 5 g/P; peptone, 10g/P; mannitol, 5 g / P ;

K2HP0 , ,

0.5

gin ; NaCI, 70 g /Q ; agar,

15

g/ P; an d c rystal violet,

1

m Po f a O.l%solution.

Freshly dra wn and washed hum an blood cells are add ed to the cooled, prepared medium.

Results of the hemolysis test are not valid if the medium is held longer than

24

hr af ter

i n o c ~ l a t i o n . ~ * ~ ~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 W agatsuma m edium, depend ing u pon th e presence or absence of

fermentable carbohydrates in the medium.



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E. Enumeration of V.pmoliaemolyticus

The two most widely used me thods for enum eration of

V.

arahaemolyticus in food o r

water samples are the most probable number (M PN ) and the mem brane f il tration ( M F)

methods. Direct plating, rath er than M F, can be used if the num ber of cells in thesam ple

are high enough. M PN me thods uti lize enrichment broths, such a s those discussed vide

supra). Replications of three or five tubes can be used, with inoc ulation of a t least three

different sample volumes. A n exam ple of the M P N 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. S am ple volumes larger than 10 m P can be concentrated using a

filtering system fitted with a 0.45-pm membrane filter. The filter with trapped cells,

can be placed into enrich me nt broth tubes, and the proc edur e carried out in replicate.

lsolation media, which a re often replicate plates conta ining any of several acceptable

media, are inoculated from tubes showing growth. These are incubated

as

for the

nonquan titative isolation. Nearly all enrichme nt bro ths and plating media described fo r

V. parahaemolyticus

have been used in th e M P N proc edure 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 a s formation in lactose broth and presumptive,

confirmed, a nd completed tests employed f or enum eration of coliforms. Thus, the

inoculated plates are incubated and colonies must be picked for purification and

characterization. T he exten t

of

the testing proc edur e employed in the characterization of

strains, as well a s the "presumptive"

or

"confirmed" appellations remain subjective. These

are determined by the govern me ntal agency, individual labo rato ry,

or

the investigator

conduc ting the study. H owever, H ugh 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, presump tive identification med ium specific fo r

V. parahaemolyticus

has

been developed and it offers a relatively quick and inexpensive aid in biochemical

characterization an d screening of large nu mb ers of isolates suspected to be

V .

parah~ernolyticus.~~his med ium, prepa red in tubes, gives a cha rac teris tic overall

reaction for

V. parahaemolyticus,

derived from arginine dihydrolase (-), manni tol

fermentation

(+),

sucrose and lactose fermentation (-), HIS product ion

(-),

gas

production

(-),

and indole product ion (+).

The membrane filtration technique, which has been successfully employed to

enumerate oth er tax ono mi c groups of bacteria, was first devised for vibrios by Horie et

al.

'

numeration of

V. arahaemolyticus

can be effected w ith a n arabinose, amm onium

sulfate, sodium cholate (AAC) medium at pH 8.6. After fi l tration of the sample, the

membrane is placed on the A AC medium an d incubated a t 42OC. Gr ow th at 42C has

proven to be an important differential characterist ic

of

V. parahaemolyticus. Yellow

colonies appe aring on the fi lter, incubated on the surface of the AA C medium. a re

arabin ose fermenters, an d therefore ar e presumed to be

K

parahaemolyticus.

Unfortunately, a s noted abov e, there is a tendency for und erestim ation of the true

incidence of V. parahuemolyticus, when this method and medium are used, because

arabin ose ferme ntation is a variable characteristic of th e species.

Many of the agar plating media discussed herein have been adapted for use with

membrane f i lters, and TC BS ag ar is one that is most com monly employed. W atkins et

al."' reported

a

me thod involving m em brane fi l tration w hich was specifically designed

for enumeration of V. parahuemolyticus. The primary isolation medium fo r the method

was based on the ab ility

of V .parahaemolyticus

to grow in the presence of

3%

NaCI, a t

high p H , i.e., 8.6, and a t 41OC. S od ium cho late in the medium served to inh ibit grow th of

Gram-posit ive organisms. G alactose provided the source of carb ohy dra te

for

V.



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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 a nd sucrose fermentation media, with the oxidase test performed as the final

step. Th e procedure a s described by the auth ors can be done within

30

hr, yielding 95%

accuracy of identification.

IV.ECOLOGY OF V . P A R A H A E M O L Y T I C U S

A. Geographical Distribution

V

parahuemolyticus is, indeed, widespread in occurrence. First recognized in

japan,

.95.139

it has also been isolated from samples collected

in

Korea,Ia Thailand.Io4

Indonesia,47 Vietnam,14' China,'42 India,'43-146ran,147R u s ~ i a , ' ~ 'nd A ~ s t r a l i a . ' ~ ~ * ' ~

The occurrence of

V.

parahaemolyricus in Europe has been reviewed by Leistner and

Hechelmann,'" and cou ntries of isolation include the

nether land^,^^.^^""

Great

Britain,63"s2 Denm ark,

102,153

Germany,'5'' 'l '4 Italy,'9*'5sScotland,6' Spain,'" and the

Black, Baltic, No rth, and M editerra nea n Seas."' Re ports fro m Africa include isolation

of V . parahoemolyticus in Togo'56 n d M a d a g a ~ c a r . " ~n the Western Hemisphere,

V.

parahuemolyticus has been isolated in

In the U.S. V. parahuemolyticus was reported by W ard'60 o n the basis of serological

relatedness of V. arahaemolyricus-like organisms in sediment samples collected from

the Southeastern coast. S ince then a r epo rt of the occurrence of this species from nearly

every coastal state has been m ade, including New Ham pshire,94Massachusetts,I6' Rhode

Island,I6' Maryland,97.'63-'64Virginia, '63 North C a r~ l i n a , ' ~ ' outh Carolina, '66 Flor-

ida,'67 th e Gulf C o a ~ t , ' ~ ' - ' ~ ~regon,I7' W a ~ h i n g t o n , " ~ " ~aw aii,m a nd A 1 a ~ k a . I ~ ~

V. parahuemolyticus can be isolated throughout the estuarine environment. Water,

sedimen t, suspende d particula tes, plank ton, 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 com monly an inh abit ant of estuaries and is infrequently fou nd

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, '59and the U.S.

B. Seasonal Variation

In most of the geographical areas where V. parahaemolyricus

s

known to occur, the

incidence of the organism s follows a distinct seasona l cycle, with highest counts recorded

in the summ er and fall and lowest coun ts in the winter. This phen om enon w as first noted

in Jap an by M iyamoto et al. '39 and has been confirmed by a number of other Japanese

investigators including Nishio et a1.173v174nd Sh in et al.175 n other countries, the

seasonality of the organisms subsequently was recorded, including

Australia,'50 and the

U.S. 97,116,117,1W176

In te re stin gly , Th o m ps on a nd V a n d e r ~ a n t ' ~ ~

reported that a seasonal cycle for

V.

parahuemolyticus

could no t b t 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 fro m the water column during the winter mon ths,

but can be isolated from sediment throu gh out the inter.^"'^ In other environm ents it

remains

in

the water column, but at greatly reduced numbers.63

In tropical countries, the s easo nal cycle of V.parahaemolyricus s correlated with rainy

and dry seasons. In Vietnam, highest numbers a re found in rainy months (M arc h and



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April) and lowest numbers are found

in

the dry season (December to February). '"

Curiously, the opposite was found in To go (West A frica) and in Indonesia where highest

cou nts were found a t the end of the dry season (April) and the lowest in the rainy season

( J ~ n e ) . ~ ~ . " ~alinity 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. D urin g the rainy season, a salinity of 1.6 t o 4.2% per thousand was

recorded, which is less tha n o ptim um fo r V. arahaemolyricus. At th at time, few isolates

were recovered.'56

Besides tem per atu re and salinity, seasonal va riatio n of V. parahaemolyticus can also

be influenced by interac tions with plank ton a nd higher organisms. K anek o and

~ ~ l ~ ~ 1 1 9 7 . 1 6 4 . 1 7 8tudied the season al 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 atta chm ent to zoop lankto n. Interaction

between sediment, water, an d zoo plank ton was found to be essential . As temperatures

increased and vibrios proliferated, V. parahuemolyticus was readily isolated from the

water co lum n. M iyam oto and K ~ r o d a ' ~ ~uggest 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 a t 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 va riety

of

factors a nd a com plex interaction of

these, especially tem peratu re, salinity, ad sorption , a ttach me nt, plank ton, parasites, etc.

C. Correlation

with

Environmental Parameters

Th e 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 wa ters vs. nonp ollu ted waters. On the

o th er ha nd , T h om p so n a nd V a n d e r ~ a n t , ' ~ ~utton,15' Ka neko a nd C 0lwe11,~' nd Jon as

et a1.'65 re po rt no significant correla tion with cou nts of V. arahuemolyti& an d pollu-

tion indices, such as total cou nts of coliforms, fecal coliforms,

or

Escherichia coli. These

variances can be resolved if other factors associated with pollution ar e 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 wi th the autochthonous,

potentially pathogenic bacteria, such as V parahaemolyticus.'".'8'

Watkins a nd Cabelli'62 reported tha t ads orp tion 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 co ncu rren t inp ut of

particulates. Otherw ise there is no direct corr elat ion with pollution. In studies carried ou t

in Chesapeake Bay, the occurrence of

V.

parahaemolyticus did show a positive

correlation with coliforms in the area of the Chesapeake Bay surrounding Baltimore

Ha rbor, but was not a s highly correlated

as

was Sa l m one l l u sp p .~ohe coliform count.'"

In a subsequent survey covering the entire Chesapeake Bay, multivariate regression

analysis

of

the da ta revealed that salinity an d dissolved o xyge n conc entration were most

closely correlated with the incidence of

V. parahuemolyticus.

The frequency of

occurrence, i.e., tota l nu mb er of

V.

parahaemolyticus, increased with increasing salinity

and decreasing dissolved oxygen (DO) concentration, the latter most likely reflecting

increased n utrient concen tration in eutrop hic area s of the Bay.'83



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V. parahuemolyticus has been isolated from freshwater in several areas. Well water

from Indonesia was found positive in several instances where samples from Ja va w ere

e ~ a m i n e d . ~ 'n Calcutta, India, V.

parahaemolyticus

has been isolated from w ater 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 ar ea , being found in

40

of pond wa ter sample s "having p ractically no sa1inity"and

fed chiefly by rain water.lE4 In the Ch esap eak e B ay, V. parahuemolyticus has been

isolated fr om the U pper Bay, as well as the uppermost reaches of the James River and the

Po tom ac River."' Sayler et al."' examined the Uppe r Chesa peake Bay an d 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

do ub t, plays an imp orta nt role in the occurrence of the organism up-river of estuaries.

Ayres an d reported higher concentrations of V. parahaemolyticus in muddy

sediment than

in

sand or gravel sediment, indicating the influence of organic matter on

Occurrence an d survival of this species. The transfer of vibrios fro m 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 G auth ier and Clement.'86 These

investigators reported that transfer

of

vibrios via sediment was very important in

persistence in the water column and marine anim als and th at colonization of water from

sediments was not observed a t temperatures less tha n 16OC. As noted ab ov e, ads orp tion

of V. parahaemolyticus to particulates is greater in lower salinity waters,16' a finding

consistent with observations made in Ch esap eake Bay'" and in Ca lcutta.

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

conc entration , and salinity.

D. Occurrence

in

the

Open

Sea

V.parahaemolyricus

has been isolated rarely from pelagic regions of the world ocea ns.

I t appears to be limited to inshore coastal an d estuarine areas. Aoki et al.142eported the

isolation of V. parahaemolyricus from the op en sea of Japa n, but neither Horie et al .9s9i1 4

nor Miyamoto et al.I3' found

V. parahaemolyticus

in water samples collected from

pelagic areas around Japan. The inability to isolate

V parahuemolyficus

from pelagic

areas was also reported by V arga an d H irtle"* in Ca na da an d Bockemuhl and Triemerls6

off the co as t of Africa. Baross and Liston"' noted th at the incidence of V .

parahaemolyticus in seawater decreased with dep th off the W ashington co ast and very

low num bers w ere foun d in deeper sedime nts. K anek o an d Colwe11''' collected samples

along fo ur transects on the con tinental shelf off the southeastern U.S. and did not isolate

classical

V.

parahaemolyricus from any of the water, sediment,

or

plankton samples.

However, a num ber of vibrios very similar to V. parahaemolyticus was recovered. Th e

absence of

V. parahaemolyticus

from the open oce an is most likely a result of low water

temperature, high salinity, an d low nutrient c onc ent ratio n, 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 Schw artz and Colwell,"' who reported th at

V parahaemolyticus

was unable

to

grow at a ny hydrostatic pressure simulating the de ep ocean env ironment, i.e., from 200

to

1000

atm of pressure. Thus, he inability of V. parahaemolyticus to tolera te elevated

hydrostatic pressure supp orts the conclusion tha t neritic o r estuarine waters are habitats

of V. parahaemolyticus.



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92


E. Association with Higher Organisms

V .pnrahoemolyricus is now known t o be associated with a variety of higher organisms

living in &hemarine an d estu arin e environm ent. including plan kto n, fish, an d shellfish.

Shellfish. in particular, a re im por tan t because of the possibility of hum an disease arising

from their ingestion. Fishbein et repo rted the isolation of V.parahaemolyt icusfrom

30 different marine species, including clams, oysters, lobsters, scallops, sardines, shr im p,

squid, crab, eel, and various species of fish. From 1969 to 1972, 546 ou t of 635 (86%)

seafood samples examined by the Food and Drug Administration were found to be

positive fo r V. p a r a h a e m ~ l y t i c u s , ' ~ ~erh aps a biased result because man y of the samples

were collected d urin g outbre ak s of enteric disease. N evertheless, oth er equally extensive

studies have been reported which document the presence of V. parahuemolyticus in

Coun ts o f

V.

parahaemolyticus

lams, mussels, and oysters.

in oysters c a n be as high a s 1300per g ram of tissue.'70 Mo re typically, the n um ber s ar e

10

per gram. Isolation of

V.

parahuemolyticus from c rabs has been rep orted by Fishbein et

al ,,la9Krantz et al.,lw Barrow an d Miller,'s2 Colwell et aI.,I9' and D e et al.,14' an d from

shrimp by Vand erzant e t a1.,1689192ishbe in e t a].,*' De et al.,145 elsenfeld a nd Cabirac,I7'

and Joseph et Co ncen trations of V. parahaernolyricus in crab s can be as high as

10'

per gram of meat.

V.

parahuemolyticus

has not been isolated from fish as frequently or a s readily as from

filter feeding

invertebrate^. ^^ ^

Nevertheless, a wide variety of marine and freshwater

fish has been shown to harbor the

o r g ~ n i s m . 6 * ' 9 9 ' ' 4 ~ 4 6 ~ 1 7 0 ~ 1 9 3 - ' 9 ~

In addition to the com mensal, or symbiotic, association of

V

parahuemolyticus with

higher organisms, a pathogenic relationship is possible also . K ra nt r et isolated

V.

parahaernolyiicus

from lethargic and mo ribund 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,192n d V anderzant a nd N i c k e l ~ o n ' ~ ~

reported the death of shrim p in ma ricu lture caused by infection with V. parahaemolyri-

cus. An outbre ak of disease in a Mexican shrim p hatchery has also been shown to be

associated with

V .

parahaemolyricus

(D.

Danald, personal comm unicat ion). Pathogenic

properties of V parahuemolyticus may be a s important in the maricul ture of shr imp

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 Phenomeno n 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 enviro nm ental reservoir of the organism. The re

is

a significant

correlation between the K P and pathogenicity of

V

parahuemolyticus, bu t the reason is

not yet known. S akaz aki et al .19 7 eported that 96.5% (2655 out of 2720) of strains

isolated from human patients was K P + while only

1% (7

ou t of 650) of isolates fro m the

environment was KP+. Oth er investigators have reported similar findings. Thom pson

and Vanderzant"' foun d only

4

KP+ st rains out of 2218 total isolated fro m water and

sediment

in

Galveston Bay. Sp ite et a1.'99foundone isolate in an oy stcr from U.S. waters.

Three strains o ut

of 251

water, fish, and shellfish stra ins exam ined in were KP+.

Ayres and Barrow63found no

KP+

strains ou t of

1484

isolates obtain ed from Brit ish

coas ta l waters . Lei s tner and H~chelman '~ 'ound only

1

KP? strain ou t of 1708 f rom

European waters. Su tton'so concluded that

2%(22

ou t of

986) of

the strains isolated fr om

oysters growing in Australia was

KP+.

An ecological study of K P+ strains fou nd in

Ja pa n was reported by Wagatsuma.2w who recovered KP + strains from sam ples of mu d,

seawater, and oysters. H owever, isolation of K P+ strains was rare in the absence offo od

poisoning

in

a nearby co mm unit y, whereas K P- strains were always isolated, regardless

of docu me ntation of food poisoning cases in the com mu nity.



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93

These findings are best explained perha ps by a process of natura l selection of

K P +

strains

in the intestine and better survival of KP- strains in the environment. S akazaki a nd co-

workers dem onstrated tha t K P+ vibrios do, in fact, multiply more rapidly tha n

KP-

strains in ligated rabbit loops and postulated, therefore, that selective multiplication of

KP+ organisms occurred in the intestine, even

i f

KP- strains were pred om inant in the

ingested food sample. Conversely, KP- strains survived longer in seaw ater tha n 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

hum an patients and KP- strains from seaw ater and seafood may be only a result of

selection and environmental pressure, with selection occurring in the intestine for

K P+

strains and in the m arine environm ent for

KP-

strains, a provocative ph enom enon yet to

be elucidated.

V.

RELATIONSHIP O F

V.

P A R A H A E M O L

YTICUS

WITH

B A C TE R IO P H A G E A N D BDELLOVIBRZO

Bacterioph ages specific fo r

V.parahaemolyticus

were isolated from fecal and seaw ater

samples in Japan by Nakanish i et a ~ ~ klarow et exam ined Atlantic coastal

sediment and recovered a bacteriophage specific for

V. parahaemolyticus

which was

morphologically sim ilar to phage isolated fro m W ashington c oastal waters.M Baross, et

al.17~206307ecovered bacteriophages active against V.parahaemolyricus from 177 to 643

samples of marine molluscan shellfish, crustaceans, seawater, and sediment collected

from W ashington and Oregon waters. Titers of

V.

parahaemolyticus

bacteriophage were

fou nd to increase with increasing water temp erature du ring the warm months of t.he year.

The titer of b acteriophage was discovered to be pr opo rtion al to the increase in the

numbers of mesophilic vibrios, but not with the incidence of V. parahaemolyticus.

Lysogenic bacteriophage could be induced from a n agar dig es tin g vibrio and it was

speculated tha t bacteriophage may, in fact, be impo rtan t in explaining the variability of

marine vibrios, with respect to phenotypic characteristics. and possibly even in animal

and human pa th~ ge nic i ty . ~ nterestingly, no bacteriophage act ive against V.

alginolyticus has been reported, even though V.alginolyticus occurs in larger num bers in

the marine environment than does

V. p a r a h a e m o f y r i ~ u s . ~

Marine

Bdellovibrio

capable of lysing

V.

parahaemolyticus

have been isolated from

Chesap eake Bay and Osaka Bay.79.20*-210solates of marine

Bdellovibrio

from both

U.S.

and J apa nes e waters are reported to have a broad host range, an d a t least in Chesa peake

Bay, demonstrate a seasonal cycle similar to th at 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 ap pea rs to have a n essential role in the ec olo gy of coastal

marine

environment^.^^

Its involvement as a pathogen f or m an, there fore, is usually

inadvertent through contact with contaminated and improperly handled seafoods. V.

parahaemolyricus is not regarded a s an acutely infectious organism, althoug h there is

little doubt that it is capable of causing serious illness. Results of experiments in which

human volunteer subjects were fed broth cul tures of clinical i ~ o l a t e s ~ ~ ~nd a n accident

in which a labora tory worker ingested approx imate ly

lo5

viable cells97make i t quite

clear that V .parahaemolyticus, occurring in sufficiently large num bers, can cause ac ute



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94 C R C Crirical Reviews in M icrobiology

gastroenteritis in man. Sanyal and Se nz i3 onfi rmed the enteropathogenic properties of

KP + strains in hum an volunteer experiments. S ignificantly, sym ptom s of gastroenteritis

appeared rapidly in individuals who had ingested at least

2

X

10' to

3

X

lo7

CFU

of KP+

V.

arahaernolyticus. Volunteers, receiving KP-

V.

parahaemolyricus in concentrations

ranging from

4

X lo9 o 1.6X 10 C F U , did not have diarrh eal symptoms. Earlier studies

had shown also tha t inge stion of lo 9 cells of

KP-

strains by human volunteers had

seemingly no ability to induce dia rrh eal Th us far , there is no indication,

from either epidemic

or

sporadic cases, that e nte rop ath oge nici ty is associated with

particular serotypes.*" A11 serotypes have been isolated from humans, seawater, and

seafishes. Lo ngitudinal studies in the same localities reveal th at p artic ula r serotypes d o

not predom inate in hu ma n il lness and marine enviro nm ents fro m

1

year to the next.

Serotypes frequently isolated from seawater and seafish during shorter periods tend to

predominate in cultures from patients.'"

Th e nature of the illness caused by

V. arahaernolyricus

leads one to conclude that a n

exoto xin, i .e., entero toxin, should

be

the responsible. identifiable substance. Interest-

ingly, feeding experiments showed that only administration of live cells produced

intestinal tract-related sym ptom atolog y, belying the possibility of preform ed exo toxin in-

gestion with incriminated foods. F ur the r evidence fo r infection lies in the fact tha t l o* o

10I2organisms are required to establish disease, an d in the acute stages,

lo6

o 10' organisms

per milliliter of feces can be

I t

is not yet known whether the pathological

effects of the organism ar e due to toxin production, direct dam ag e to the intestinal tract

fro m microbial invasion, both, or neither. The ability to multiply in the intestinal tract is

generally associated with the K P reactivity of the vibrio, which co m mo nly is considered

to

be

the essential index

of

capacity for en teropa thoge nicity in hum ans. This hemolytic

characteristic, described in gre ate r detail below, is fou nd in appr oxi ma tely 95% of clinica l

isolates and is rarely associated with en viro nm ent al isolates. H um an volunteer and

ani ma l experiments generally tend t o subs tant iate this view, altho ugh the significance of

the few cases caused by KP- stra ins remains partially unexp lained. T he suspicion is that

these strains produce hemolysin but in very small quantities. This has been bo rne o ut, in

fact, by using a serological meth od

of

detection, w hich reveals that, indee d, hemolysin is

produced by some presumed KP- strains.13' Th e suggestion has been m ad e that V.

parahuemolyricus produces a n cnterotoxin.'I6 Th e possibility th at enter oto xin is

produced and contributes to the disease process to some extent, regardless of KP

reaction, has yet

to

be clearly es tabl i~hed. ' '~

B.

Toxin

Assessment

1 . Mouse Inocularion

Stu die s using inoculation in adu lt and suckling mice generally confirm th at large

num bers of vibrios are required to cause lethality. Fujino* observed the lethal toxicity of

V. parahaemolyricus

to m ice an d guinea pigs. Zen-Yoji e t 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. Co nc ent rate d cell-free filtrates introduced

via foot inoculation showed lethal toxicity, whereas all lysates provoked only slightly

edem atous responses. A ttempts to detect a n E. coli-like heat-stable toxin (ST) in suckling

mice were inconclusive. Recently, Jo hn so n and Calia,'" using co nce ntra ted filtrates,

demonstrated weak ST-like responses in the same system.

W hole cells of

V.parahaernolyricus

administered per

os,

SC, r I P resulted in mouse

lethality caused by septicemia.' Sa ka zak i et al." fo un d th at 0.5 mP f subgroups I and 2,

now

V.

arahaemolyricus and

.V.

lginolyricus, injected IP int o mice resulted in lethality

within 24 t o 48 hr. Lethality is dose dep ende nt. Tw o differen t KP + clinical strains



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95

olume 10, Issue

I

inoculated into NMRI mice yielded LDso values a t concentrations 2 10' but were

nonle thal a t lesser concentrations.'" Inoc ulation s of cell-free filtrates

of

over 200

separate clinical isolates, even at IOX concentration, did not cause death of

NMRI

mice,221 urther emphasizing the a pp are nt requirem ent f or large num bers 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 m ore frequen tly in the RIL

system, than are

KP-

strains.2'4225~z2*ell filtrates are not capable of eliciting a fluid

accumulation in

RIL,

lthough 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 t o be substantiated when hea ting at 100C for 30

min eliminated activity of tenfold co ncen trated cell-free filtrates. R ecently, Jo hn so n and

Calia233 ave shown t hat concen tration of filtrates produces solutions containing >20

NaCI and reported tha t media containing N aC l2 4 95 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 in

RIL,

when NaCl

concentrations were

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96


KP- strains, the effect

w a s

more rapid a nd 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 adhe re to HeLa

cells and adhered to H F I cells a t 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 carb ohy dra te on the ou ter m embran e of the bacterial cell wall apparently

necessary for the Using clinical str ain s of KP+ and KP- V.

parahaemolyticus, Sochard and Joseph were unable to differentiate between either

gro up similarly exposed to HeLa cells.2a Gingras and Ho wa rdz4 lso were unable to

detect significant differences in vitro, by radioassay. in the adherence of KP+ and KP-

strai ns of

V.

parahoemolyticus to human intestinal cells.

Culture filtrates of V. porahaemolyricus isolates from human gastrointestinal

infections, when exposed to Y-1 drena l cells, d o not cause morphologic change^.^^^-^@ In

contra st , H ond a et al .27 ave isolated a heat-labile fac tor from the culture fi l trates of

K P+ strains which causes morphological changes of Chinese ham ster ovary (C H O ) cells,

the premise being that this factor is an entero toxic substan ce separable from the cardio-

toxic hemolysin.

Oishi et al.2 45 ave recently described the presence of exohem agglutinins ( H A ) from

marine vibrio strains, which had tax ono mic properties similar to

V. parahaemolyticus.

HA showed remarkably high hemagglutinating titers for a wide spectrum of

erythrocytes. The more widely reactive H A were not easily inhibited, whereas the HA in

the g rou p with narrow er spectra, were specifically and co mpletely inhibited by L-fucose

an d D -arabinose. T he physiologic significance of HA is not clear, bu t they may eventually

emerge as an imp ortant feature of adherence, an essential step in pathogenicity.

Clinical exam ination of a few patients in India, Bangladesh,246 nd the U.S.,247who

were suffering from

V. parahaemolyricus

dysentery-like diarrhea, suggested that

invasion of the bowel may occur, i.e., blood a nd mucu s in the stoo l, polymorp honuclear

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 sin ga direct fluorescent an tibo dy

method, studied invasiveness as a step in pathogenesis during host organ-bacterial

interaction. All strains tested, K P+ an d KP-, penetrated in to the lam ina pro pria of the

ileum and were eventually isolated f ro m the spleen. How ever, not all were ab le to cause

fluid dilatation

of

RIL.

KP- stra ins caused only occasional positive responses.

Interestingly. V. parahaemolyricus do es no t ex hibit a positive Se re ny test, i.e.,

penetration of the corneal epithelium

of

guinea Attempts to demon strate

invasiveness by V. parahaemolyricus an d oth er closely related vibrios with the He La cell

techniques described by Me hlman et reinforced the impression tha t, by this

experimen tal m ethod, a s well,

V.

parahaemolyticus penetrat ion

or

invasion of tissue is

still a questionable phenomenon.240

Joseph et aL2 found that a

KP-

strain injected into th e i leum of suckling rabb its

caused focal necrosis and appeared to adhere in damaged areas, but no t on apparen tly un-

effected tissue, leading t o speculatioh th at tissue dam age , adherence , an d invasion were

interrelated, possibly with toxicity. Fu rthe r examin ation showed that a dhere nce was

possibly mediated by lateral appen dage s (flagella), and played i n im po rtan t role in the

stimulation of fluid accumulation in R I L and lethality to mice by the whole, intact

Similar investigations reported simultaneously by Shinoda et al .2s

sub stan tiate d these results.

D.

Permeability Factor

Th e permeabili ty fac tor (P F ) or skin toxin, foun d to be capa ble of pro ducin g increased

vascular perm eability in the skin of exp eri

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