Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species 663 highest numbers of both 01 and non-01 strains occurred in August and November.38 Neither the fecal coliform nor the total coliform index was an adequate indicator of the presence of V cholerae, but the former was more useful than the latter Along the Santa Cruz coast of, California, the highest numbers of non-01 strains occurred during the summer months and were associated with high coliform counts.70 Both 01 and non-01 strains have been recovered from aquatic birds in Colorado,96 and both types have been shown to be endemic in the Texas gulf as evidenced by antibody titers in human subjects.59 V cholerae 01 El Tor synthesizes a 82-kDa preprotoxin and secretes it into culture media, where it is further processed into a 65-kDa active cytolysin.139 Non-01 strains produce a cytotoxin and a hemolysin with a molecular weight of 60 kDa, which is immunologically related to the hemolysin of the El Tor strain The OmpU outer membrane protein has been shown to be an adherence factor of V cholerae, which may facilitate adherence to small intestines Monoclonal antibodies raised to OmpU protected HeLa, HEp-2, Caco-2, and Henle 407 epithelial cells from invasion by viable organisms.118 From a patient with travelers’ diarrhea a strain of 01 was isolated from which the STa (NAG-STa) gene was cloned.95 The NAG-STa was chromosomal, and the toxin had a molecular weight of 8815 The NAG-STa shared 50% and 46% homology to E coli STh and STp, respectively.95 NAG-ST is methanol soluble, active in the infant mouse model, and similar to the ST of Citrobacter freundii.126 Monoclonal antibodies to NAG-ST cross-react with the ST of Yersinia enterocolitica Further, V mimicus ST and Y enterocolitica ST are neutralized by monoclonal antibodies to NAG-ST but not E coli STh or STp.126 In a study of the survival of V cholerae El Tor serotype Inaba in several foods, it was found that in meats with an inoculum of × 103 /g, cells remained viable for up to 90 days at −5◦ C and for up to 300 days at −25◦ C.36 The organism was not detected in milk after 34 days at −5◦ C and 150 days at −25◦ C with an inoculum of × 104 /ml In milk at 7◦ C, it survived 32 days on average but only 18–20 days in other foods The virulence mechanisms of V cholerae are discussed in Chapter 22 Vibrio vulnificus This organism is found in seawater and some seafoods It is isolated more often from oysters and clams than from crustacean shellfish products It has been isolated from seawater from the coast of Miami, Florida, to Cape Cod, Massachusetts, with most (84%) isolated from clams Upon injection into mice, 82% of tested strains were lethal V vulnificus, along with other vibrios, have been recovered from mussels, clams, and oysters in Hong Kong at rates between 6% and 9%.34 In a study of the estuarine waters of eastern North Carolina, V vulnificus was isolated only when the water temperatures were between 15◦ C and 22◦ C.103 Following the warm summer of 1994 in Denmark during which 11 clinical cases of V vulnificus were reported, a study was undertaken to determine the prevalence of the organism in Danish waters.56 Upon testing suspect colonies with a DNA probe, from 0.8 to 19 colony-forming units (cfu) per liter were found in water between June and mid-September, and 0.04 to >11 cfu/g in sediment samples from July to mid-November A strong correlation was found between the presence of V vulnificus and water temperature The organism was found in of 17 mussels examined from of 13 locations, and also from wild fish Biotype constituted 99.6% of 706 V vulnificus isolates.56 Like V alginolyticus (see below), V vulnificus causes soft-tissue infections and primary septicemia in humans, especially in the immunocompromised and those with cirrhosis The fatality rate for those with septicemia is more than 50%, and more than 90% among those who become hypotensive.139 These organisms are highly invasive, and they produce a cytotoxin with a molecular weight of about 56 kDa that is toxic to CHO cells and lytic to erythrocytes However, the cytolysin appears not to be a critical virulence factor.137 664 Modern Food Microbiology Also, a hemolysin is produced with a molecular weight of about 36 kDa.138 It also produces a zinc metalloprotease of the thermolysin family that induces a hemorrhagic reaction in skin by digesting type IV collagen, a key structure of the basement membrane.85 The structural genes of V vulnificus and the El Tor strain of V cholerae 01 share areas of similarity, suggesting a common origin.140 V vulnificus induces fluid accumulation in the RITARD ligated rabbit loop (see Chapter 12), suggesting the presence of an enterotoxin.119 V vulnificus strains from the same oysters have been shown to display wide genomic diversity, suggesting that infections may be caused by mixed populations of cells or that only a few of the different strains are virulent.20 V vulnificus bacteriophages are discussed in Chapter 20 relative to their association with host cells and their possible use as indicators Infections are rather common in many countries, most occur between May and October, and most patients are men over 40 years of age V vulnificus is a significant pathogen in individuals with higher than normal levels of iron (e.g., in hepatitis and chronic cirrhosis) even though its virulence is not explained entirely by its capacity to sequester iron For the period 1981–1992, 125 V vulnificus cases were reported to the Florida Department of Health, and 25 persons (35%) died.33 Raw oysters are the leading food source for this bacterium, and it is believed to be responsible for ∼95% of all seafoodassociated deaths in the United States In 1996, V vulnificus was the cause of 16 cases and deaths in Los Angeles associated with the consumption of raw oysters.27 The oysters were traced back to Galveston Bay, Texas, and Eloi Bay, Louisiana The addition of hot sauce to raw oysters has been shown to be ineffective in killing V vulnificus123 but diacetyl at a concentration of 0.05% decreased the numbers of V vulnificus cells in oysters.124 Vibrio alginolyticus and V hollisae V alginolyticus is a normal inhabitant of seawater and it has been found to cause soft-tissue and ear infections in humans Human pathogenicity was first confirmed in 1973 but first suspected in 1969.132 Wound infections occur on body extremities, with most patients being men with a history of exposure to seawater In coastal waters of the state of Washington, higher numbers of this organism were found in invertebrates and sediment samples than in open water, where the numbers were quite low.5 Numbers found in oysters correlated with the temperature of overlying waters, with the highest numbers associated with warmer waters In improperly stored oysters in Brazil, seven species of Vibrio were isolated at the rates shown82 : V alginolyticus (81%), V parahaemolyticus (77%), V cholerae non-01 (31%), V fluvialis (27%), V furnissii (19%), and V mimicus and V vulnificus (12% each) First described in 1982, V hollisae causes foodborne gastroenteritis, and for the period 1967–1990, 15 cases were recorded.106 In contrast, only one case of human illness traced to shellfish consumption was reported for V alginolyticus over the same period V hollisae produces an enterotoxin with a molecular weight of ∼33 kDa, and it is hemolytic on human and rabbit red blood cells.74 Unlike V parahaemolyticus, an isolate of V hollisae from coastal fish produced a TDH-related hemolysin.92 Using HeLa, Henle 407, and HCT-8 cell monolayers, V hollisae has been shown to invade via microfilaments and microtubules.84 The latter suggests that this organism may possess multiple modes of infection YERSINIOSIS (Yersinia ersinia enterocolitica enterocolitica) In the genus Yersinia, which belongs to the family Enterobacteriaceae, 11 species and biovars are recognized, including Y pestis, the cause of plague The species of primary interest in foods is Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species 665 Table 28–3 Species of Yersinia Associated with Y enterocolitica in the Environment and in Foods, and Minimum Biochemical Differences Between Them Species Y enterocolitica Y kristensenii Y frederiksenii Y intermedia Y bercovieri Y mollaretti VP∗ Sucrose Rhamnose Raffinose Melibiose + − + + − − + − + + + + − − + + − − − − − + − − − − − + − − VP = Voges–Proskauer reaction; + = positive reaction; − = negative reaction Y enterocolitica First isolated in New York State in 1933 by Coleman,54 this Gram-negative rod is somewhat unique in that it is motile below 30◦ C but not at 37◦ C It produces colonies of 1.0 mm or less on nutrient agar, is oxidase negative, ferments glucose with little or no gas, lacks phenylalanine deaminase, is urease positive, and is unique as a pathogen in being psychrotrophic It is often present in the environment with at least three other of the yersiniae noted in Table 28–3 Growth Requirements Growth of Y enterocolitica has been observed over the temperature range −2◦ C to 45◦ C, with an optimum between 22◦ C and 29◦ C For biochemical reactions, 29◦ C appears to be the optimum The upper limit for growth of some strains is 40◦ C, and not all grow below 4–5◦ C Growth at 0–2◦ C in milk after 20 days has been observed Growth at 0–1◦ C on pork and chicken has been observed,77 and three strains were found to grow on raw beef held for 10 days at 0–1◦ C.51 In milk at 4◦ C, Y enterocolitica grew and attained up to 107 cells/ml in days and competed well with the background biota.2 The addition of NaCl to growth media raises the minimum growth temperature In brain heart infusion (BHI) broth containing 7% NaCl, growth did not occur at 3◦ C or 25◦ C after 10 days At a pH of 7.2, growth of one strain was observed at 3◦ C and very slight growth at pH 9.0 at the same temperature; no growth occurred at pH 4.6 and 9.6.121 Although 7% NaCl was inhibitory at 3◦ C, growth occurred at 5% NaCl With no salt, growth was observed at 3◦ C over the pH range 4.6–9.0.121,125 Clinical strains were less affected by these parameters than were environmental isolates With respect to minimum growth pH, the following values were found for six strains of Y enterocolitica with the pH adjusted with HCl and incubated for 21 days: 4.42–4.80 at 4◦ C, 4.36–4.83 at 7◦ C, 4.26–4.50 at 10◦ C, and 4.18–4.36 at 20◦ C.19 When organic acids were used to adjust pH, the order of their effectiveness was acetic > lactic > citric On the other hand, the order of effectiveness of organic acids in tryptic soy broth was propionic ≥ lactic ≥ acetic > citric ≥ phosphoric ≥ HCl.17 A chemically defined growth medium has been devised, and it consists of four amino acids (lmethionine, l-glutamic acid, glycine, and l-histidine), inorganic salts, buffers, and potassium gluconate as carbon source.3 Y enterocolitica is destroyed in 1–3 minutes at 60◦ C.50 It is rather resistant to freezing, with numbers decreasing only slightly in chicken after 90 days at -18◦ C.77 The calculated D62.8◦ C for 21 strains in milk ranged from 0.7 to 17.8 sec, and none survived pasteurization.42 666 Modern Food Microbiology Distribution Y enterocolitica and the related species noted in Table 28–3 are widely distributed in the terrestrial environment and in lake, well, and stream waters, which are sources of the organisms to warm-blooded animals It is more animal adapted and is found more often among human isolates than the other species in Table 28–3 Of 149 strains of human origin, 81%, 12%, 5.4%, and 2% were, respectively, Y enterocolitica, Yersinia intermedia, Yersinia frederiksenii, and Yersinia kristensenii.114 Y intermedia and Y frederiksenii are found mainly in fresh waters, fish, and foods, and only occasionally are isolated from humans Y kristensenii is found mainly in soils and other environmental samples as well as in foods but rarely isolated from humans.7 Like Y enterocolitica, this species produces a heat-stable enterotoxin Many of the Y enterocolitica-like isolates of Hanna et al.52 were rhamnose positive and, consequently, are classified as Y intermedia and/or Y frederiksenii, and all grow at 4◦ C Rhamnosepositive yersiniae are not known to cause infections in humans Animals from which Y enterocolitica has been isolated include cats, birds, dogs, beavers, guinea pigs, rats, camels, horses, chickens, raccoons, chinchillas, deer, cattle, swine, lambs, fish, and oysters It is widely believed that swine constitutes the single most common source of Y enterocolitica to humans Of 43 samples of pork obtained from a slaughterhouse and examined for Y enterocolitica, Y intermedia, Y kristensenii, and Y frederiksenii, were positive and all four species were found.53 Along with Klebsiella pneumoniae, Y enterocolitica was recovered from crabs collected near Kodiak Island, Alaska, and was shown to be pathogenic.4 In a study in the United States, 95 of 103 (92.2%) lots of market hogs carried at least one Y enterocolitica isolate, and 98.7% of the pathogenic isolates were serotype 0:5 and 3.7% were 0:3.46 In a study in Finland, 92% of 51 tongue and 25% of 255 ground meat samples contained Y enterocolitica.44 These investigators used a yadA gene-targeted polymerase chain reaction (PCR) along with a culture method and by the two methods >98% of the pork tongues were positive Biotype was the most common, as was serotype 0:3 (see below) A TaqMan assay was compared to two other methods for their respective recovery of Y enterocolitica from fresh and frozen ground pork, and the sensitivity of TaqMan was to log10 cfu/g or ml.138 Results could be obtained in hours after an 18-hour enrichment The thin agar layer Oxyrase (TALO) method could detect as few as log10 cfu/g of freeze-injured cells Using these two methods along with a standard selective medium, no Y enterocolitica could be found in 100 ground pork samples.138 In regard to human carriage, an examination was made of 4,841 stool specimens from seven cities in as many U.S states from November 1989 to January 1990, and the findings were as follows: 38%, 49%, 60%, and 98% contained, respectively, Y enterocolitica, shigellae, Campylobacter, and salmonellae.75 Of the Y enterocolitica isolates, 92% were serotype 0:3 Serovars and Biovars The most commonly occurring Y enterocolitica serovars (serotypes) in human infections are 0:3, 0:5,27, 0:8, and 0:9 Each of 49 isolates belonging to these serovars produced a positive HeLa cell response, whereas only of 39 other serovars were positive.88 Most pathogenic strains in the United States are 0:8 (biovars and 3), and except for occasional isolations in Canada, it is rarely reported from other continents In Canada, Africa, Europe, and Japan, serovar 0:3 (biovar 4) is the most common.130 The second most common in Europe and Africa is 0:9, which has been reported also from Japan Serovar 0:3 (biovar 4, phage type 9b) was practically the only type found in the province of Quebec, Canada, and it was predominant in Ontario.130 The next most common were 0:5,27 and 0:6,30 From human infections in Canada, 0:3 represented 85% of 256 isolates, whereas for nonhuman sources, Foodborne Gastroenteritis Caused by Vibrio, Yersinia, and Campylobacter Species 667 Table 28–4 The Four Most Common Biovars of Y enterocolitica Biovars Substrate/Product Lipase (Tween 80) Deoxyribonuclease Indole d-Xylose + − + + − − + + − − − + − + − − 0:5,27 represented 27% of 22 isolates.130 Six isolates of 0:8 recovered from porcine tongues were lethal to adult mice,130 and only 0:8 was found by Mors and Pai88 to be Sereny positive Employing HeLa cells, the following serovars were found to be infective: 0:1, 0:2, 0:3, 0:4, 0:5, 0:8, 0:9, and 0:21 Serovar 0:8 strains are not only virulent in humans, but they possess mouse lethality and invasiveness by the Sereny test The four most common biovars of Y enterocolitica are indicated in Table 28–4 It appears that only biovars 2, 3, and carry the virulence plasmid Virulence Factors Y enterocolitica produces a heat-stable enterotoxin (ST) that survives 100◦ C for 20 minutes It is not affected by proteases and lipases and has a molecular weight of 9,000–9,700 daltons, and biological activity is lost upon treatment with 2-mercaptoethanol.97,98 When subjected to iso-electric focusing, two active fractions with iso-electric points (pIs) of 3.29 (ST-1) and 3.00 (ST-2) have been found.97 Antiserum from guinea pigs immunized with the purified ST neutralized the activity of Y enterocolitica ST and E coli ST.97 Like E coli ST, it elicits positive responses in suckling mice and rabbit ileal loop assays and negative responses in the CHO and Y-1 adrenal cell assays (see Chapter 12) It is methanol soluble and stimulates guanylate cyclase and the cyclic adenosine monophosphate (cAMP) response in intestines but not adenylate cyclase.98,107 It is produced only at or below 30◦ C,100 and its production is favored in the pH range 7–8 Of 46 milk isolates, only produced ST in milk at 25◦ C and none at 4◦ C In a synthetic medium, enterotoxin production was favored by aeration but inhibited by high iron content.3 At 25◦ C, more than 24 hours were required for ST production in a complex medium, and the gene that encodes its synthesis appears to be chromosomal.1996 V vulnificus was the cause of 16 cases and deaths in Los Angeles associated with the consumption of raw oysters.27 The oysters were traced back to Galveston Bay, Texas, and Eloi Bay, Louisiana In a study of 232 human isolates, 94% produced enterotoxin, whereas only 32% of 44 from raw milk and 18% of 55 from other foods were enterotoxigenic.101 Of the serovars 0:3, 0:8, 0:5,27, 0:6,30, and 0:9, 97% of 196 were enterotoxigenic It has been found that most natural waters in the United States contain rhamnose-positive strains that are either serologically untypeable or react with multiple serovars.54 In another study, 43 strains of Y enterocolitica from children with gastroenteritis and 18 laboratory strains were examined for ST production, and all clinical and laboratory strains produced ST as assessed by the infant mouse assay, and all were negative in the Y-1 adrenal cell assay.100 Regarding the production of ST by species other than Y enterocolitica, none of 21, 8, and of Y intermedia, Y frederiksenii, and Y aldovae, respectively, was positive in one study of species from raw milk, whereas 62.5% of Y enterocolitica were ST positive.135 On the other hand, about one-third ... ranged from 0.7 to 17.8 sec, and none survived pasteurization.42 666 Modern Food Microbiology Distribution Y enterocolitica and the related species noted in Table 28–3 are widely distributed in... Yersinia intermedia, Yersinia frederiksenii, and Yersinia kristensenii.114 Y intermedia and Y frederiksenii are found mainly in fresh waters, fish, and foods, and only occasionally are isolated from... died.33 Raw oysters are the leading food source for this bacterium, and it is believed to be responsible for ∼95% of all seafoodassociated deaths in the United States In 1996, V vulnificus was