5 Foodborne Pathogenic Vibrios T. Ramamurthy and G. Balakrish Nair 1. BACKGROUND In the past few decades, many foodborne outbreaks associated with consumption of contaminated foods have occurred in both developing and developed countries. Factors that influence the epidemiology of the foodborne diseases include: level of hygiene in handling foods, globalization of the supply and distribution of raw foods, aquaculture practices, farming, introduction of pathogens into new geographical areas, changes in the virulence and environmental resistance of pathogens, decrease in immunity among certain population, and changes in the eating habits. Members belonging to the genus Vibrio of the family Vibrionaceae have acquired increasing significance because of its association with mild-to-severe human diseases. There is a species preponderance of vibrios and their geographic distribution of infec- tion. For example, incidences of some vibrios are related to poor sanitation and hygiene in developing countries, whereas other species are associated with frequent foodborne infections in developed countries. Most of the vibrios reside in aquatic realms and asso- ciated fauna and flora as autochthonous microflora. Toxigenic Vibrio cholerae is the most important, because this species causes cholera, a devastating disease of global prominence. The other important species is Vibrio parahaemolyticus. The recently emerged pandemic strains of this species caused major concerns in both the developed and developing countries. Studies conducted at molecular level have clearly shown that the pandemic strains are of recent origin and are undergoing rapid changes that have not been recorded earlier. Other vibrios of medical importance are Vibrio vulnificus, Vibrio mimicus, Vibrio alginolyticus, Vibrio fluvialis, Vibrio hollisae, and Vibrio damsela. However, the significance of V. furnissii and V. metschnikovii is not known owing to inadequate number of investigations carried out on these pathogens. With the exception of cholera, Vibrio diseases are not designated as reportable. This chapter reviews the health hazards associated with foods carrying pathogenic vibrios, detection techniques, virulence factors, and the effect of different decontamination methods in eliminating the vibrios. 2. CLASSIFICATION The family Vibrionaceae consists of seven genera. The genus Vibrio currently has 48 species; among them 11 are of clinical importance. Recently, the species V. damsela is placed under the genus Photobacterium. Species representing the family Vibrionaceae From: Infectious Disease: Foodborne Diseases Edited by: S. Simjee © Humana Press Inc., Totowa, NJ 115 116 Ramamurthy and Nair Fig. 1. Classification and names of taxa within the family Vibrionaceae. For each species, the type strain number is given in parentheses. The other six genus in this family includes Photobacterium, Allomonas, Listonella, Enhydrobacter, Salinivibrio, and Enterovibrio. The species V. damselae is now placed under the genus Photobacterium and the nomenclature is Photobacterrium damselae. are given in Fig. 1. Members of the genus Vibrio are defined as Gram-negative, asporogenous rods that are straight or have a single, rigid curve. They are motile; most have a single polar flagellum, when grown in liquid medium. Sodium ions at varying concentrations stimulate the growth of all species, which are a basic growth requirement for most of the vibrios. Most produce oxidase and catalase, and ferment glucose without producing gas. The G+C content of the genus Vibrio is 38–51% compared to the Photobacterium (40–44%), Aeromonas (57–63%), and Plesiomonas (51%). 3. ISOLATION AND IDENTIFICATION For isolation of vibrios, initial enrichment in alkaline peptone water (pH 8.0) is useful as this medium supports the growth of viable cells. An altered alkaline broth is recommended for specific enrichment of V. fluvialis from seafood, and environmental sample (1). Majority of the clinically important vibrios can be isolated using common media like blood agar, MacConkey agar, taurocholate–tellurite–gelatin agar, and thiosulfate–citrate–bile salt–sucrose agar (TCBS). The selective nature of TCBS may be inhibitory to some of the vibrios such as V. hollisae, V. damsela, V. metschnikovii, and V. cincinnatiensis. Use of 1–3% NaCl to the food diluent is necessary to maintain the viability of the halophilic vibrios. Vibrios 117 V. parahaemolyticus is known to exist in a viable but nonculturable state especially in foods when incubated at low temperatures and in routine cultural methods. It is difficult to culture cells at this state. The resuscitation of nonculturable cells of V. parahaemolyticus is achievable after spreading onto an agar medium supplemented with H2O2-degrading compounds, such as catalase or sodium pyruvate (2). Conventionally, vibrios are identified using biochemical characteristics and specific growth conditions (Table 1). However, it is recommended to use standard strains for quality control to ensure the results. V. cholerae and V. mimicus are nonhalophils (grows in the absence of NaCl) and are closely related. Because the production of arginine dihydrolase by V. fluvialis and V. hollisae is often confused with the identification of Aermonas spp., salt tolerance test is recommended for confirmation. The vibriostatic compound 2,4-diamino-6,7-diisopropylpteridine (O/129) at concentration of 10 and 150 Rg/mL has been used to discriminate between Aeromonas and Vibrio species. However, because many recent strains of V. cholerae have been found resistant to O/129 compound (3), these results should be interpreted with caution and used in conjunction with other biochemical and salt tolerance tests. Many diagnostic kits are now commercially available for the identification of vibrios of clinical importance (Table 2). An antibody enzyme immuno assay for the detection of V. cholerae O1 was developed using the rabbit antiserum, G-D-galactosidase-labeled goat antirabbit immunoglobulin G as the tracer (4). With the detection limit of 4500 cells, this assay can be used for screening V. cholerae directly from food homogenates. V. cincinnatiensis, a new species, was identified in 1986 from the blood and cerebro- spinal fluid of a patient. On the basis of 5S rRNA comparative sequence analysis, the organism appears to share a recent common ancestry with V. gazogenes (98% homo- logy) and close ancestry with V. mimicus, V. fluvialis, and V. metschnikovii (5). Strains belonged to the aerobic biogroup 2 of V. fluvialis are now designated as V. furnissii (6). Based on the production of gas from the fermentation of carbohydrates, V. furnissii can be differentiated from V. fluvialis. The distinct features of V. furnissii compared to other halophilic vibrios include positive reaction for L-arginine, L-arabinose, maltose, and D-mannitol and negative reaction for L-lysine, L-ornithine, lactose, and Voges– Proskauer (6). V. cholerae serogroup O1 includes two biotypes—classical and El Tor—each including Inaba and Ogawa, and rarely Hikojima serotypes. Currently, the El Tor biotype is predominant. Vibrios that are bichemically indistinguishable, but do not agglutinate with V. cholerae serogroup O1 antiserum (non-O1 strains, formerly known as nonagglutinable vibrios [NAGs] or noncholera vibrios [NCVs] are now included in the species V. cholerae). Before 1992, non-O1 strains were recognized to cause sporadic infections and not associated with large epidemics. However, in late 1992, large-scale epidemics of cholera-like infection were reported in India and Bangladesh (7,8). The causative organism was identified as a new serogroup V. cholerae O139 (synonym: Bengal), which also elaborates cholera toxin (CT). The clinical and epidemiological picture of illness caused by the O139 serogroup is typical of cholera, and the cases should be reported as cholera (9). The reporting of V. cholerae non-O1 infections, other than O139, as cholera is inaccurate and leads to confusion. Apart from O1 and O139 serogroups, 206 O-serogroups are recognized in the current V. cholerae serotyping scheme. Table 1 Biochemical and Growth Characteristics of Clinically Important Vibrios Test responsea V. V. V.parahaemoly- V. V. V. V. V. V. V. Test cholerae mimicus ticus alginolyticus vulnificus damsela fluvialis hollisae metschnikovii cincinatiensis Growth in TCBS Yellow Green Green Yellow Green/ Green Yellow Green/ Yellow Yellow agar Yellow NG mCPC agar Purple NG NG Purple ND ND NG NG NG ND CC agar Purple NG NG Purple ND ND NG NG NG ND AGS K/a K/A K/A K/K ND ND K/K K/a K/K ND Oxidase ++ + + ++++ + Indoleb ++ + ± + + + + ± + + + 118 Voges– Proskauerb b ++ + + ++++ + NO3 to NO2 Simmons’ ++ ± + ± + citrate ONPG ++ ± + ±± Urea ± hydrolysis Gelatin +± + + ± ± ± hydrolysis Motility ++ + + ++ ± ±± Polymyxin B + +± ± ±++ + + inhibition Arginine ++ ± dihydrolaseb Lysin ++ + + +± + ± decarboxylaseb Ornithine ++ + ± ± decarboxylaseb Acid production from: D-Glucose ++ + + +++++ + L-Arabinose ± ++ + D-Arabitol ± Cellobiose + + + Lactose + ± ± Maltose ++ + + +++ ++ D-Mannitol ++ + + + + ++ Salicin + + Sucrose + + + + ++ Utilization of: L-leucine ++ ND L-putrescine ++± ND Ethanol +±+ ND 119 D-glucuronate + + ± + + ND Growth in NaCl: 0% ++ 3% ++ + + +++++ + 6% ±± + + ±+±++ + 8% ++ ± 10% ±+ Growth at 42°C ++ + + ND ND ± ND ± ND Sensitivity to: 10 Rg of O/129 S S R R S S R R S R 150 Rg of O/129 S S S S S S S S S S Abbreviations: TCBS, thisulfate–citrate–bile salts–sucrose agar; mCPC, modified polymyxin