Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species 659
tryptic soy broth). After employing the latter medium at 29
◦ C and various solutes to control a w ,
minimum values were 0.937 (glycerol), 0.945 (KCl), 0.948 (NaCl), 0.957 (sucrose), 0.983 (glucose),
and 0.986 with propylene glycol.
9 The organism is heat sensitive, with D 47 ◦ C values ranging from
0.8 to 65.1 minutes having been reported.
10 With one strain, destruction of 500 cells/ml in shrimp
homogenates was achieved at 60
◦ C in 1 minute, but with 2 × 10
5 cells/ml, some survived 80
◦ C for 15
minutes.
133 Cells are most heat resistant when grown at high temperatures in the presence of about 7%
NaCl.
When the growth of V. parahaemolyticus was compared in estuarine water and a rich culture
medium, differences were observed in cell envelope proteins and lipopolysaccharide and in alkaline
phosphatase levels of K
+ and K
− strains.
99 Alkaline phosphatase was slightly higher in K
− strains
grown in water. Changes in cell envelope composition may be associated with the capacity of V. parahaemolyticus to enter a viable yet nonculturable state in waters, making its recovery from water more
difficult.
99
Virulence Properties
The most widely used in vitro test of potential virulence for V. parahaemolyticus is the Kanagawa
reaction, with most all virulent strains being positive (K
+ ) and most avirulent strains being negative
(K
− ). About 1% of sea isolates and about 100% of those from patients with gastroenteritis are K
+ .
107 K
+
strains produce a thermostable direct hemolysin (TDH), K
− strains produce a heat-labile hemolysin,
and some strains produce both. A thermostable-related hemolysin (TRH) has been shown to be an
important virulence factor for at least some V. parahaemolyticus strains. Of 214 clinical strains tested,
52% produced TDH only, and 24% produced both TDH and TRH.
115 Of 71 environmental strains,
7% gave weak reactions to a TRH probe, but none reacted with a TDH probe. The Kanagawa reaction
is determined generally by use of human red blood cells in Wagatsuma’s agar medium. In addition
to human red blood cells, those of the dog and rat are lysed, those of the rabbit and sheep give weak
reactions, and those of the horse are not lysed.
111
To determine the K reaction, the culture is surface plated, incubated at 37
◦ C for 18–24 hours, and
read for the presence of beta hemolysis. Of 2,720 V. parahaemolyticus isolates from diarrheal patients,
96% were K
+ , whereas only 1% of 650 fish isolates were K
+ . In general, isolates from water are K
− .
The TDH has a molecular weight of 42,000 daltons and is a cardiotrophic, cytotoxic protein that is
lethal to mice
57 and induces a positive response in the rabbit ileal loop assay (see Chapter 12). Its mean
mouse LD 50 by intraperitoneal (i.p.) injection is 1.5 µg, and the rabbit ileal loop dose is 200 µg.
143 The
hemolysin is under pH control and was found to be produced only when the pH is 5.5–5.6.
35 That K
+
hemolysin may aid cells in obtaining iron stems from the observation that lysed erythrocyte extracts
enhanced the virulence of the organism for mice.
66 The membrane receptors of TDH are gangliosides
G T 1 and G D1a , with the former binding hemolysin more firmly than the latter.
127 The resistance of
horse erythrocytes to the hemolysin apparently is due to the absence of these gangliosides.
127
A synthetic medium has been developed for the production of both the thermostable direct and
the heat-labile hemolysins, and serine and glutamic acid were found to be indispensable.
65 The heat
stability of TDH is such that it can remain in foods after its production. In Tris buffer at pH 7.0, D 120 ◦ C
and D 130 ◦ C values of 34 and 13 minutes, respectively, were found for semipurified toxin, whereas in
shrimp D 120 ◦ C and D 130 ◦ C values were 21.9 and 10.4 minutes, respectively.
18 Hemolysin was detected
when cell counts reached 10
6 /g, and its heat resistance was greater at pH 5.5–6.5 than at 7.0–8.0.
18
The TDH gene (tdh) is chromosomal, and it has been cloned in E. coli. When the tdh gene was
introduced into a K
− strain, it produced extracellular hemolysin.
94 The nucleotide sequence of the
tryptic soy broth). After employing the latter medium at 29
◦ C and various solutes to control a w ,
minimum values were 0.937 (glycerol), 0.945 (KCl), 0.948 (NaCl), 0.957 (sucrose), 0.983 (glucose),
and 0.986 with propylene glycol.
9 The organism is heat sensitive, with D 47 ◦ C values ranging from
0.8 to 65.1 minutes having been reported.
10 With one strain, destruction of 500 cells/ml in shrimp
homogenates was achieved at 60
◦ C in 1 minute, but with 2 × 10
5 cells/ml, some survived 80
◦ C for 15
minutes.
133 Cells are most heat resistant when grown at high temperatures in the presence of about 7%
NaCl.
When the growth of V. parahaemolyticus was compared in estuarine water and a rich culture
medium, differences were observed in cell envelope proteins and lipopolysaccharide and in alkaline
phosphatase levels of K
+ and K
− strains.
99 Alkaline phosphatase was slightly higher in K
− strains
grown in water. Changes in cell envelope composition may be associated with the capacity of V. parahaemolyticus to enter a viable yet nonculturable state in waters, making its recovery from water more
difficult.
99
Virulence Properties
The most widely used in vitro test of potential virulence for V. parahaemolyticus is the Kanagawa
reaction, with most all virulent strains being positive (K
+ ) and most avirulent strains being negative
(K
− ). About 1% of sea isolates and about 100% of those from patients with gastroenteritis are K
+ .
107 K
+
strains produce a thermostable direct hemolysin (TDH), K
− strains produce a heat-labile hemolysin,
and some strains produce both. A thermostable-related hemolysin (TRH) has been shown to be an
important virulence factor for at least some V. parahaemolyticus strains. Of 214 clinical strains tested,
52% produced TDH only, and 24% produced both TDH and TRH.
115 Of 71 environmental strains,
7% gave weak reactions to a TRH probe, but none reacted with a TDH probe. The Kanagawa reaction
is determined generally by use of human red blood cells in Wagatsuma’s agar medium. In addition
to human red blood cells, those of the dog and rat are lysed, those of the rabbit and sheep give weak
reactions, and those of the horse are not lysed.
111
To determine the K reaction, the culture is surface plated, incubated at 37
◦ C for 18–24 hours, and
read for the presence of beta hemolysis. Of 2,720 V. parahaemolyticus isolates from diarrheal patients,
96% were K
+ , whereas only 1% of 650 fish isolates were K
+ . In general, isolates from water are K
− .
The TDH has a molecular weight of 42,000 daltons and is a cardiotrophic, cytotoxic protein that is
lethal to mice
57 and induces a positive response in the rabbit ileal loop assay (see Chapter 12). Its mean
mouse LD 50 by intraperitoneal (i.p.) injection is 1.5 µg, and the rabbit ileal loop dose is 200 µg.
143 The
hemolysin is under pH control and was found to be produced only when the pH is 5.5–5.6.
35 That K
+
hemolysin may aid cells in obtaining iron stems from the observation that lysed erythrocyte extracts
enhanced the virulence of the organism for mice.
66 The membrane receptors of TDH are gangliosides
G T 1 and G D1a , with the former binding hemolysin more firmly than the latter.
127 The resistance of
horse erythrocytes to the hemolysin apparently is due to the absence of these gangliosides.
127
A synthetic medium has been developed for the production of both the thermostable direct and
the heat-labile hemolysins, and serine and glutamic acid were found to be indispensable.
65 The heat
stability of TDH is such that it can remain in foods after its production. In Tris buffer at pH 7.0, D 120 ◦ C
and D 130 ◦ C values of 34 and 13 minutes, respectively, were found for semipurified toxin, whereas in
shrimp D 120 ◦ C and D 130 ◦ C values were 21.9 and 10.4 minutes, respectively.
18 Hemolysin was detected
when cell counts reached 10
6 /g, and its heat resistance was greater at pH 5.5–6.5 than at 7.0–8.0.
18
The TDH gene (tdh) is chromosomal, and it has been cloned in E. coli. When the tdh gene was
introduced into a K
− strain, it produced extracellular hemolysin.
94 The nucleotide sequence of the