Chapter 20: Vibrio spp.

Updated: 07/18/2007

Potential Food Safety Hazard

Vibrio spp.
V. cholerae
V. parahaemolyticus
V. vulnificus
Other Vibrio species

Control Measures
Guidelines

FDA Guidelines
ICMSF Recommended Microbial Limits

Growth
Heat Resistance
Analytical Procedures

Compendium of Analytical Methods (HC)
Food Sampling and Preparation of Sample Homogenate (USFDA)
Definition of Terms; Collection of Samples; Supplement to all Methods in the HC Compendium (HC)
V cholerae, V. parahaemolyticus, V. vulnificus, and other Vibrio species
Detection of enterotoxigenic Vibrio cholerae in foods by the polymerase chain reaction (USFDA)
Detection of halophilic Vibrio species in seafood (HC)
The isolation and identification of Vibrio cholerae 01 and non-01 from foods (HC)
The isolation and enumeration of Vibrio vulnificus from fish and seafoods (HC)
Other analytical procedures

Commercial Test Products
References

Potential Food Safety Hazard

Contents

Vibrio spp.

Contents

The genus Vibrio includes Gram-negative, oxidase-positive (except two species), rod or curved rod-shaped facultative anaerobes. Many Vibrio spp. are pathogenic to humans and have been implicated in food-borne disease. Vibrio spp. other than V. cholerae and V. mimicus do not grow in media that lack added sodium chloride, and are referred to as "halophilic" (Elliot et al., 1998)

Association of Vibrio spp. with different clinical syndromes^a,b.

	Clinical Syndrome
Species	Gastroenteritis	Wound Infection	Ear Infection	Primary Septicemia	Secondary Septicemia
V. cholerae O1	+++	+
V. cholerae non-O1	+++	++	+	+	+
V. mimicus	++		+
V. fluvialis	++
V. parahaemolyticus	+++	+	+		+
V. alginolyticus	(+)	++	++	+
V. cincinnatiensis				+
V. hollisae	++			+
V. vulnificus	+	++		++	++
V. furnissii	(+)
V. damsela		++
V. metschnikovii	(+)			(+)
V. carchariae		+

^a+++ = frequently reported, ++ = less common (6-100 reports); + = rare (1-5 reports), and (+) = association is unclear.
^bTable taken from Pavia et al. (1989).

Vibrio cholerae

Contents

V. cholerae was first described as the cause of cholera by Pacini in 1854. Pathogenic V. cholerae produces a heat-sensitive enterotoxin that causes the characteristic cholera symptoms, including "rice water stool." The species comprises several somatic (O) antigen groups, including O-group-1, which is associated with classical and El Tor biotypes. V. cholerae Ol may have several serotypes, including Inaba, Ogawa, and Hikojima. V. cholerae non-O1 (referred to in older literature as nonagglutinable or NAG vibrios) also can cause gastrointestinal disease, though typically less severe than that caused by V. cholerae O1 (Yamamoto et al., 1983). Serotype O139 is an exception, and produces classic cholera symptoms. This serotype was first identified in 1992 (CWG, 1933) as the cause of a new epidemic of cholera in India and Bangladesh. Non-O1 V. cholerae is found more readily in estuarin! e waters and seafood in the United States than is the Ol serogroup; however, the 0139 serogroup has not yet been found here. Because this species can grow in media lacking sodium chloride, it is not considered a halophilic Vibrio, although traces of sodium ion are required for growth. The standard FDA method for recovery of V. cholerae is qualitative (presence/absence). Testing V. cholerae O1 and non-O1 isolates for production of cholera toxin is recommended.

Some diarrheal and otitis isolates, once thought to be atypical V. cholerae non-O1 (sucrose-negative), are now recognized as a separate species, V. mimicus (Davis et al., 1981; Shantera et al., 1983). Members of the species may produce cholera-like enterotoxins. V. mimicus can be identified by biochemical procedures used for the identification of V. cholerae (Elliot et al., 1998).

Vibrio parahaemolyticus

Contents

V. parahaemolyticus is a halophilic bacterium found naturally in estuarine waters and animals. It was first described as the cause of gastroenteritis in Japan (Fujino et al., 1951) and was first found in the United States by Baross and Liston (1968) in the estuarine waters of Puget Sound. It has a worldwide distribution in estuarine and coastal environments and has been isolated from many species of fish, shellfish, and crustaceans. V. parahaemolyticus has been implicated in numerous outbreaks of seafood-borne gastroenteritis in the United States. Between 1971 and 1978, crab, oyster, shrimp, and lobster were implicated in 14 outbreaks, which may have resulted from the consumption of raw or insufficiently heated seafood or properly cooked seafood contaminated after cooking. The FDA method of enumeration uses an MPN format (Elliott et al., 1998).

Vibrio vulnificus

Contents

V. vulnificus is a halophilic bacterium found in the estuarine environment and is similar phenotypically to V. parahaemolyticus (Oliver, 1989). The species was first described as "lactose-positive" because most strains ferment lactose and are o-nitrophenyl-b -D-galactosidase (ONPG) positive. It causes food-borne and wound disease, either of which may progress to rapidly fatal septicemia in individuals with liver disease (cirrhosis) or other underlying illnesses such as diabetes. Raw oysters are the major source of food-borne disease caused by V. vulnificus. The FDA method of enumeration uses an MPN series confirmed by biochemical testing or an immunological test, such as the ELISA, with monoclonal antibody to a species-specific intracellular antigen (Elliott et al., 1998).

Other Vibrio species

Contents

Other halophilic Vibrio spp., including V. fluvialis, V. hollisae, V. alginolyticus, V. furnissii, and V. metschnikovii, have been associated with gastroenteritis and are present in estuarine environments along with other pathogenic and nonpathogenic species of Vibrio. V. cincinnatiensis, V. damsela, and V. carchariae have not been associated with gastroenteritis, but on rare occasions are pathogenic to humans (Table 20-1). V. anguillarum, V. damsela, and V. carchariae are pathogenic to fish. Biochemical testing is required for taxonomic speciation (Elliott et al., 1998).

Control Measures

Contents

Hazards from Vibrio can be prevented by cooking seafood thoroughly and by preventing cross-contamination once the seafood is cooked. Freezing is ineffective in killing the bacteria (Ward et al., 1997).

If V. parahaemolyticus has produced the heat-stable Kanagawa hemolysin, some cooking procedures may not destroy the hemolysin (Bradshaw et al., 1984).

The risk of V. vulnificus infection can also be reduced by rapidly refrigerating oysters from the Gulf Coast during warm-weather months. Individuals in the "high risk" groups should not consume raw molluscan shellfish (Ward et al., 1997).

Guidelines

Contents

FDA Guidelines

Contents

FDA guidelines for Vibrio in fish.

ICMSF Recommended Microbial Limits

Contents

Recommended microbiological limits for V. parahaemolyticus in fish (ICMSF, 1986).

Product	n¹	c²	Bacteria/gram or/cm²
Product	n¹	c²	m³	M⁴
Fresh and frozen fish and cold-smoked fish	5	2	10²	10³
Frozen raw crustaceans	5	1	10²	10³
Frozen cooked crustaceans	5	1	10²	10³
Cooked, chilled, and frozen crabmeat	10	1	10²	10³
Fresh and frozen bivalve molluscs	10	1	10²	10³

¹Number of representative sample units.
²Maximum number of acceptable sample units with bacterial counts between m and M.
³Maximum recommended bacterial counts for good quality products.
⁴Maximum recommended bacterial counts for marginally acceptable quality products.

Plate counts below "m" are considered good quality. Plate counts between "m" and "M" are considered marginally acceptable quality, but can be accepted if the number of samples does not exceed "c." Plate counts at or above "M" are considered unacceptable quality (ICMSF, 1986).

Growth

Contents

Limiting conditions for V. cholerae, V. parahaemolyticus, and V. vulnificus growth.

Heat Resistance

Contents

Heat resistance of V. cholerae.

Temp.		D-Value	Medium	Reference
(ºC)	(ºF)	(min.)
48.9	120	9.17	Shrimp homogenate	Hinton and Grodner, 1985
49	120.2	8.15	Crabmeat	Shultz et al., 1984
54	129.2	5.02	Crabmeat	Shultz et al., 1984
54.4	129.9	0.43	Shrimp homogenate	Hinton and Grodner, 1985
60	140	2.65	Crabmeat	Shultz et al., 1984
60	140	0.39	Shrimp homogenate	Hinton and Grodner, 1985
65.5	149.9	0.32	Shrimp homogenate	Hinton and Grodner, 1985
66	150.8	1.60	Crabmeat	Shultz et al., 1984
66	150.8	1.22	Crayfish homogenate	Grodner and Hinton, 1985
71	159.8	0.30	Crabmeat	Shultz et al., 1984
71	159.8	0.30	Crayfish homogenate	Grodner and Hinton, 1985
71.1	160	0.31	Shrimp homogenate	Hinton and Grodner, 1985
76.7	170.1	0.30	Shrimp homogenate	Hinton and Grodner, 1985
77	170.6	0.27	Crayfish homogenate	Grodner and Hinton, 1985
82	179.6	0.27	Crayfish homogenate	Grodner and Hinton, 1985
82.2	180	0.28	Shrimp homogenate	Hinton and Grodner, 1985

Heat resistance of V. parahaemolyticus.

Temp.		D-Value	Medium	Reference
(ºC)	(ºF)	(min.)
47	116.6	65.1	7.5% NaCl	Beuchat and Worthington, 1976
49	120.0	0.82	Clam homogenate	Delmore and Chrisley, 1979
51	123.8	0.66	Clam homogenate	Delmore and Chrisley, 1979
53	127.4	0.40	Clam homogenate	Delmore and Chrisley, 1979
55	131	0.29	Clam homogenate	Delmore and Chrisley, 1979

Heat resistance of V. vulnificus.

Temp.		D-Value	Medium	Reference
(ºC)	(ºF)	(min.)
47	116.6	2.40	Buffered saline	Cook and Ruple, 1992
50	122	1.15	Buffered saline	Cook and Ruple, 1992

Analytical Procedures

Contents

Compendium of Analytical Methods (HC)
Food sampling and preparation of sample homogenate (USFDA)	Contents

Definition of Terms (HC Appendix A); Collection of samples (HC Appendix B); Supplement to All Methods in the HC Compendium: General Microbiological Guidance (HC Appendix I) (Supplement to HC Appendix I: A. General Microbiological Guidance on Pre-warming of Broths in All Qualitative Methods in the Compendium)

Contents

V. cholerae, V. parahaemolyticus, V. vulnificus, and other Vibrio species (USFDA)

Contents

Detection of enterotoxigenic Vibrio cholerae in foods by the polymerase chain reaction (USFDA)

Contents

Detection of Halophilic Vibrio Species in Seafood (HC MFLP-37)	Contents
The isolation and identification of Vibrio cholerae 01 and non-01 from foods (HC MFLP-72) (pdf file)	Contents

The isolation and enumeration of Vibrio vulnificus from fish and seafoods (HC MFLP-73) (pdf file)

Contents

Other analytical procedures

Contents

Vibrio cholerae in oysters: Elevated temperature enrichment method (AOAC, 1995a).
Vibrio vulnificus: Gas chromatographic identification method by microbial fatty acid profile (AOAC, 1995b).
V. vulnificus in oysters: DNA probe (DePaola et al., 1997).

Commercial Test Products

Contents

Commercial test products for V. cholerae.

Test Kit	Analytical Technique	Approx. Total Test Time¹	Supplier
CHECK 3 Vibrio sp.	Chemical, visual detection	4-18 h	Contamination Sciences LLC Contact: Robert Steinhauser 4230 East Towne Blvd., Suite 191 Madison, WI 53704 Phone: 608/825-6125 E-mail: bsteinha@contam-sci.com Web: www.contam-sci.com
Chromogenic Vibrio [Presumptive differentiation of V. parahaemolyticus and V. vulnificus]	Chromogenic media	48 h	Biomedix Contact: Claver Bundac 1105 #F North Golden Springs Dr. Diamond Bar, CA 91765 Phone: 800/674-8648 #4282; 909/396-0244 E-mail: cb4biomedx@aol.com
ISO-GRID Method [For Vibrio parahaemolyticus count using VSP agar]	Membrane filtration with selective and differential culture medium using sucrose fermentation	24 h	QA Life Sciences, Inc. 6645 Nancy Ridge Dr. San Diego, CA 92121 Phone: 800/788-4446; 858/622-0560 E-mail: bugsy@qalife.com
VET-RPLA TD920 [Used to identify V. cholerae enterotoxin]	Reversed passive latex agglutination	24 h (bacterial culture)	Oxoid, Inc. Contact: Jim Bell 217 Colonnade Rd. Nepean, Ontario K2E 7K3 Canada Phone: 613/226-1318 E-mail: jbell@oxoid.ca

¹Includes enrichment

References

Contents

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AOAC. 1995a. Vibrio cholerae in oysters: Elevated temperature enrichment method. Sec. 17.11.01, Method 988.20. In Official Methods of Analysis of AOAC International, 16th ed., P.A. Cunniff (Ed.), p. 106B-108. AOAC International, Gaithersburg, MD.

AOAC. 1995b. Vibrio vulnificus: Gas chromatographic identification method by microbial fatty acid profile. Sec. 17.11.02, Method 994.06. In Official Methods of Analysis of AOAC International, 16th ed., P.A. Cunniff (Ed.), p. 108-108B. AOAC International, Gaithersburg, MD.

Baross, J., and J. Liston. 1968. Isolation of Vibrio parahaemolyticus from the Northwest Pacific. Nature 217:1263-1264.

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Beuchat, L.R. 1974. Combined effects of water activity, solute, and temperature on the growth of Vibrio parahaemolyticus. Appl. Microbiol. 27:1075-1080.

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Davis, B.R., G.R. Fanning, J.M. Madden, A.G. Steigerwalt, H.B. Bradford, Jr., H.L. Smith, Jr., and D.J. Brenner. 1981. Characterization of biochemically atypical Vibrio cholerae strains and designation of a new pathogenic species, Vibrio mimicus. J. Clin. Microbiol. 14:631-639.

Delmore, R.P., Jr. and Chrisley, F.D. 1979. Thermal resistance of Vibrio parahaemolyticus in clam homogenate. J. Food Protect. 42(2):131-134.

Depaola, A., Kaysner, C.A., and McPhearson, R.M. 1987. Elevated temperature method for recovery of Vibrio cholerae from oysters (Crassostrea gigas). Appl. Environ. Microbiol. 53:1181-1182.

DePaola, A., Motes, M.L., Cook, D.W., Veazey, J., Garthwright, W.E., and Blodgett, R. 1997. Evaluation of an alkaline phosphatase-labeled DNA probe for enumeration of Vibrio vulnificus in Gulf Coast oysters. J. Microbiol. Methods 29(2):115-120.

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Ewing, W.H., K.M. Tomfohrde, and P.J. Naudo. 1979. Isolation and identification of Vibrio cholerae and certain related vibrios: an outline of methods. Species 2:10-22.

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Fujino, T., Y. Okuno, D. Nakada, A. Aoyama, K. Fukai, T. Mukai, and T. Ueho. 1951. On the bacteriological examination of Shirasu food poisoning. J. Jpn. Assoc. Infect. Dis. 35:11-12.

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Grimes, D.J., J. Stemmler, H. Hada, E.B. May, D. Maneval, F.M. Hetrick, R.T. Jones, M. Stoskopf, and R. R. Colwell. 1984. Vibrio species associated with mortality of sharks held in captivity. Microbial Ecol. 10:271-282.

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Honda, T., Y. Ni, and T. Miwatani. 1988. Purification and characterization of a hemolysin produced by a clinical isolate of Kanagawa phenomenon-negative Vibrio parahaemolyticus and related to the thermostable direct hemolysin. Infect. Immun. 56:961-965.

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Jackson, H. 1974. Temperature relationships of Vibrio parahaemolyticus. In International Symposium on Vibrio parahaemolyticus. T. Fujino, G. Sakaguchi, R. Sakazaki, and Y. Takeda (Eds.), p. 139-145. Saikon Publ. Co., Ltd., Tokyo. Cited in Jarvis, N.D. 1987. Curing of Fishery Products. Teaparty Books, Kingston, MA.

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MacDonell, M.T., F.L. Singleton, and M.A. Hood. 1982. Diluent decomposition of use of API 20E in characterizing marine and estuarine bacteria. Appl. Environ. Microbiol. 44:423-427.

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Sloan, E.M., C.J. Hagen, G.A. Lancette, J.T. Peeler, and J.N. Sofos. 1992. Comparison of five selective enrichment broths and two selective agars for recovery of Vibrio vulnificus from oysters. J. Food. Prot. 55:356-359.

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