Vibrio is a comma shape Gram-negative bacteria and several species of this bacteria can cause foodborne infection which is usually associated with eating undercooked sea food, Vibrio spp. are facultative anaerobes that shows positive result for oxidase test and are non-spore formers. All species of Vibrio are motile and have polar flagella with sheaths. Recently phylogenies have been constructed based upon the group of genes (multilocus sequence analysis). While these common bacteria persist as a natural component of the marine microbial flora, a small percentage of environmental isolates carry the genetic determinants for human pathogenesis (Nishibuchi and Kaper, 1995; Chakraborty et al., 2000; Rivera et al., 2001). Currently, Vibrio infections are the major cause of seafood-borne bacterial gastroenteritis in the United States, Vibrio cholerae (non-infection, usually associated with the consumption of raw or undercooked seafood, typically found in saltwater -O1 and non-O139), V. parahaemolyticus and V. vulnificus responsible for the majority of those infections (Mead et al., 1999). Among the 4,754 Vibrio infections reported to the Centers for Disease Control and Prevention (CDC) from 1997-2006, 3,544 (75%) of those infections were foodborne in origin and 1,210 (25%) of those infections were non-foodborne in origin (Dechet et al., 2008). Vibrio originally defined on the basis of biochemical characteristics, but recent studies relied on DNA related data, amino acid sequence divergence of enzymes such as glutamine synthetase and superoxide dismutase, and cellular fatty acid profiles. Results from the studies show a relationship between the genus Vibrio and the family enterobacteriaceae.
1.1 Vibrio disease spectrum
Biochemical and serological tests has been developed to distinguish phenotypically similar Vibrios and to identify important serotypes of Vibrios species that cause major public health hazards. By far the most common feature observed in individuals presenting with Vibrio infections were usually gastroenteritis, which is caused due to consumption of raw sea foods. Individuals who were identified with such a food pattern in their milieu, irrespective of their presenting symptoms, should be considered likely candidates for having a Vibrio infection. Other significant conditions that should be considered potentially analytical of Vibrio infections include foreign travel (e.g., Mexico), recent immigration, accidental trauma during contact with sea water or marine associated products (e.g., shellfish), or gastroenteritis of cholera like ("rice water stools") nature. Although these situations are not in and of themselves pathognomonic for Vibrio spp., they are highly suggestive and help to define a specific population upon which additional diagnostic tests for the presence of Vibrios should be performed. In one recent study by Bonner et al.1983 on Vibrio infections at their institution over a 10-year period, 87% (20 of 23) of the patients interviewed indicated a recent history of contact with the marine environment or associated products. Laboratory workers uses commercially prepared Thiosulphate-citrate-bile salts-sucrose agar (TCBS), which enable the specific isolation of Vibrios from contaminated samples. It is a selective and differential media contain high concentration of sodium thiosulphate and sodium citrate which inhibits the growth of enterobacteriaceae and to provide optimum growth and metabolic activity of halophilic Vibrio species. This media contain bile salts which inhibits the growth of gram positive bacteria. And the sucrose present in this media serve as the fermentable carbohydrate, with the help of the pH indicators, allows for the differentiation of those Vibrios spp. which utilize sucrose, results in production of yellow-brown colonies.
1.2 Properties of TCBS medium
It has high pH in the range of 8.5 to 9.5 that suppresses the growth of intestinal flora other than Vibrio spp. 1% NaCl enables the growth and metabolic activity of halophilic Vibrio sp. Bile salt inhibits growth of Gram positive bacteria. Sodium thiosulfate detects the production of H2S. Sucrose serves as fermentable CHO, differeniates that Vibrio sp. which utilizes sucrose and V. cholerae and its biotype Eltor ferment sucrose, results in a pH shift and production of Yellow brown colonies (Fig. 1).
Fig.1: A generalized view of Vibrio spp. growing on TCBS plate
Virulence factor both cell associated and extracellular may have multiple biological function or properties by making the study of such molecules even more difficult. Besides adherence properties, cell surface factors regulate resistance to complement mediated lyses and other naturally occurring host defenses. Few cell association and extracellular virulence factor for pathogenesis, specific markers to identify pathogenic strains of a given species are general location.
1.3 Environmental studies
Vibrio spp. have been regarded as a member of a group of organisms whose major habitats are aquatic ecosystems. The concentration of Vibrios varies directly with temperature with higher numbers occurring in water from 17 to 35??C containing salinity 5 to 25 %. In any aquatic source, the concentration of the Vibrio may vary for relatively short period of time according to the amount of local rainfall and amount of freshwater runoff. Most pathogenic Vibrios exhibit wide strains to strain variance in virulence, isolated from the environment.
V. cholerae produces the abundant diarrhea characteristic of cholera by means of a potent enterotoxin, cholera toxin (CT). The A subunit of CT, encoded by ctxA, stimulates adenylate cyclase in intestinal epithelial cells, which results in net secretion of fluid into the intestinal lumen. Vibrio spp. is among the most commonly isolated bacteria in marine and estuarine waters. They significantly affect nutrient cycling in these habitats and often comprise a major portion of the members of the genus are pathogenic either for humans or marine animals. Food contamination with antibiotic-resistant bacteria is a major threat to public health, as the antibiotic resistance determinants can be transferred to other bacteria of human clinical significance.
In assessing the public health significance, two critical properties of V. cholerae are taken into account. These include the production of CT, which is responsible for the severe diarrhea, and the possession of the O1 or O139 antigen, which acts as a marker of epidemic potential, since the actual determinant of such potential is not clearly known. However, molecular analysis has revealed that in addition to genes encoding CT, all strains capable of causing cholera invariably carry genes for a colonization factor known as toxin-coregulated pilus (TCP) and a regulatory protein, ToxR, which coregulates the expression of CT and TCP (Fig. 2). Thus, cholera pathogenesis relies on the synergistic effect of a number of pathogenic factors produced by toxigenic V. cholerae and the structure of TCP Pathogenicity Island and the CTX genetic element are as a horizontally transferred gene clusters for the origination of new pathogenic clones of vibrio spp.
Fig.2: Expression of CT and TCP are coregulated by the ToxR regulatory system
Clinical isolates of V. parahaemolyticus most often produce either the thermo-stable direct haemolysin (TDH) or TDH related haemolysin (TRH) encoded by tdh and trh genes, respectively. More than 90% of clinical V. parahaemolyticus isolates possess tdh (DePaola et al., 2000). In contrast, the tdh and trh genes were rarely detected in the environmental strains of V. parahaemolyticus. The incidence of pathogenic V. parahaemolyticus has been reported to be less than 1-2% among environmental strains, but studies using molecular techniques indicate higher prevalence of pathogenic strains. It seems possible that acquisition of TCP Pathogenicity Island and the CTX genetic element has allowed specific strains of Vibrio spp. to become adapted to the human intestinal environment (Fig. 3). Colonization of brush borders in the small intestine, a crucial component of the infection strategy, is assumed to be mediated by a rigid pilus colonization factor, TCP and it is under the same genetic control as CT.
Fig.3: ToxR regulatory genes influence TcpA synthesis and pilin synthesis
The coordinate regulation of virulence genes through the ToxR regulon demonstrates that the organism has developed a mechanism of sampling and responding to its environment. Different regulatory systems in Vibrio spp. apparently allow the bacterium to vary the expression of its genes to optimize survival in different environments, which include the human intestine and the estuarine environment. The survival of Vibrio spp. may be dependent on several factors, such as the occurrence of certain physicochemical conditions, a specific association of the bacteria with aquatic plants or animals, and the existence of specific ecological associations involving several components of the aquatic environment. It has been postulated that the Vibrio spp. are converted into viable but non-culturable (VNC) form, under stress condition. And this forms are not be recovered by standard culture techniques and that such VNC forms are able to cause infection and can revert to the culturable from. The public health and ecological importance of this VNC form depends on whether these forms can be converted back to live infectious bacteria.
1.4 Antibiotic susceptibility and resistance
The Vibrio spp. which is clinically significant usually grows well on Mueller-Hinton agar (MHA) for antibiotic susceptibility testing, without the addition of NaCl. Addition of NaCl is not recommended as it may cause the alteration in the activity of some antibiotics (Hollis et al., 1976).
Morris et al. (1985) has been reported that most strains of Vibrio spp. are susceptible to tetracycline, chloramphenicol, and aminoglycosides. Most strains were susceptible to tetracycline, chloramphenicol, gentamicin, and nalidixic acid, whereas susceptibility to sulfonamides was variable. V. parahaemolyticus, V. alginolyticus, and V. furnissii were resistant to ampicillin, carbenicillin, and cephalothin. Similar findings have been reported by other investigators (Joseph et al., 1978; Morris et al., 1985).
In the United States, although plasmid-mediated resistance does not seem to be a problem with V. cholerae strains,there have been outbreaks in Africa and Asia with strains resistant to tetracycline, ampicillin, and trimethoprim-sulfamethoxazole have been reported ( Lee et al.,1985; Mhalu et al.,1979).
Fig 4: Bacterial mechanisms of antibiotic resistance
The basic three mechanism of antibiotic resistance in bacteria that are encoded by plasmids, which are potentially transmissible to other bacteria (Fig. 4).
(i) Efflux pumps are located in the bacterial membrane that has high- affinity reverse transport systems. This system transports the antibiotic out of the cell. e.g. tetracycline.
(ii) A specific enzyme modifies the antibiotic in such a way that it loses its activity. e.g. streptomycin.
(iii) Some enzyme is produced that degrade the antibiotic, thereby inactivating it. e.g. the pencillinases are a group of beta-lactamase enzymes that cleave the beta lactam ring of the pencillin molecule.
1.5 Heavy metal resistance
Heavy metals are essential micronutrients for bacterial growth and enzymatic activities in small amounts; but they are toxic at elevated concentrations results in binding with other cellular components to form complex compounds (Lopez-Maury et al., 2002).
Trace metals are significant contaminants in many aquatic systems, partly from anthropogenic sources such as industrial and mining inputs. Metal-resistant microorganisms may be useful as indicators of potential toxicity to other forms of life and are important in studies of mechanisms, determinants and genetic transfer of microbial metal-resistance (De Rore et al., 1994). The study of interaction between heavy metal and bacteria will help in bioremediation process .Mechanisms of metal resistance in microbes include precipitation of metals as phosphates, carbonates and/or sulfides; volatilization via methylation or ethylation; physical exclusion of electronegative components in membranes and extracellular polymeric substances (EPS); energy-dependent metal efflux systems; and intracellular sequestration with low molecular weight, cysteine-rich proteins (Gadd, 1990; Silver, 1996). Anne Spain and Elizabeth Alm, (2003) attributed increase of antibiotics resistance genes to presence of antibiotics reisstance genes in bacterial strains to presence of heavy metals in the environment, hence posturing threat to human health and environment in general.
The entry of heavy metals into the bacterial cell is done by the regulation systems of the divalent cations or oxyanions. Indeed, heavy metals are not distinguishable for these transmembrane proteins, from the other divalent cations or oxyanions (for example: SO42-, HPO42-, Fe2+, Mg2+, Mn2+, etc'). Bacteria have developed two kinds of resistance mechanisms to cure this passive entry. The first of them is fast, non-specific and is based on the gradient of concentration or on proto-motive force. The second mechanism is more specific, inducible and is activated in the presence of a metal and generally requires ATP hydrolysis
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