Halophilic vibriosjp

• 1. Dr.R.Jayaprada

2. Taxonomy Family vibrionaceae now presently composed of 6 genera. Vibrio, Photobacterium , Salinivibrio, Enterovibrio, Grimontia and Aliivibrio. Pathogenic species for humans were found in 3 genera , including Vibrio (10 species), Photobacterium and Grimontia (one species each). 3. Halophilic Vibrios Definition: Species of vibrios that are unable to grow in media without added salt (NaCl) [They require (6-10% NaCl) to grow]. Halophilic means salt loving. e.g. V.parahaemolyticus, V.alginolyticus, V.vulnificus, V.fluvialis, V. hollisae . 4. Differentiation of halophilic vibrio from non-halophilic vibrios 1. Growth on Cystine Lactose Electrolyte Deficient (CLED) medium (pH 7.5): Non-halophilic vibrios grow on this medium but halophilic vibrios dont grow. 2. Growth in peptone water with different concentrations of NaCl: A loopful of bacterial suspension is inoculated into a series of peptone water with NaCl concentration of 0%, 0.5%, 3%, 6%, 8% and 10%. Halophilic vibrios dont grow at 0% NaCl concentration, but show increasing growth from 0.5% to 3% or more. Non-halophilic vibrios dont grow in NaCl concentration at or beyond 6%. 5. Vibrio parahaemolyticus Vibrio parahaemolyticus is a gram-negative halophilic bacterium that is indigenous to coastal marine and estuaries (where rivers flow into the sea) throughout the world (1). It is a common cause of acute gastroenteritis characterized by diarrhea, vomiting and abdominal cramps and the source of some cases of septicemia(2). It also causes traveller's diarrhea, wound infection, ear infection and secondary septicemia in humans (Pavia et. al., 1989). Typically, human infections with this organism result from the consumption of raw or undercooked seafood especially with molluscan shellfish, such as clams, oysters, mussels, and scallops(3). 1.Twedt, R. M. 1989. Vibrio parahaemolyticus, p. 552554. In M. P. Doyle (ed.), Foodborne bacterial pathogens. Marcel Dekker Inc., New York, N.Y. 2.Blake PA, Weaver RE, Hollis DG. Diseases of humans (other than cholera) caused by vibrios. Annu Rev Microbiol. 1980;34:341-67. Review. 3.Rippey SR. Infectious diseases associated with molluscan shellfish consumption. Clin Microbiol Rev. 1994 Oct;7(4):419-25. Review. 6. Scientific Classification Kingdom: Bacteria Phylum: Proteobacteria Class: Gamma Proteobacteria Order: Vibrionales Family: Vibrionaceae Genus: Vibrio Species: V. parahaemolyticus 7. History This organism was first identified as a causative agent of food borne gastroenteritis after a large outbreak (272 illnesses and 20 deaths) associated with consumption of half-dried sardines called shirasu, in Japan in 1951 (Fujino et. al., 1953)(4). Fujino (1951) reported the discovery of this pathogenic bacterium at the 25th Annual Meeting of the Japanese Association for Infectious Diseases in 1951. 4.Fujino T, Okuno Y, Nakada D, et al. On the bacteriological examination of shirasu-food poisoning. Med J Osaka Univ. 1953;4:299-304. 8. History In India V. parahaemolyticus was first isolated from a case of gastroenteritis by Chatterjee et. al. (1970) and about 10% of the cases of gastroenteritis in patients admitted to the Infectious Disease Hospital in Kolkata are due to V. parahaemolyticus (Deb et. al., 1975) (5). The United States Centers for Disease Control and Prevention (CDC) estimates that of the approximately 7,880 Vibrio illnesses each year in the United States, approximately 2,800 are estimated to be associated with Vibrio parahaemolyticus and raw oyster consumption. 5.S Nelapati, K Nelapati, B K Chinnam. Vibrio parahaemolyticus- An emerging foodborne pathogen-A Review. Vet. World, 2012, Vol.5(1): 48-62. 9. Morphology & Characteristics In morphology, it resembles the cholera vibrio,except that it is capsulated, shows bipolar staining and has a tendency to pleomorphism, especially when grown on 3% salt agar and in old cultures. Unlike other vibrios, it produces peritrichous flagella when grown on solid media. Polar flagella are formed in liquid cultures. It grows only in media containing NaCl. It can tolerate salt concentration up to 8% but not 10 %. The optimum salt concentration is 2-4 %. On TCBS agar, the colonies are green with an opaque, raised centre and flat translucent periphery. String test is positive. 10. Morphology & Characteristics It is oxidase, catalase, nitrate, indole and citrate positive. Glucose, maltose, mannitol, mannose and arabinose are fermented producing acid only. Lactose, sucrose, salicin, xylose, adonitol, inositol and sorbitol are not fermented. It is killed at 60 C in 15 minutes. It does not grow at 4 C but can survive refrigeration and freezing. Drying destroys it. It dies in distilled water or vinegar in a few minutes. 11. Epidemiology Surveillance for V. parahaemolyticus infection was initiated in January 1994 in Calcutta, India. A group of strains belonging to serovar O3:K6 and possessing the tdh gene but not the trh gene appeared suddenly in February 1996 and was shown to be responsible for the high incidence of V. parahaemolyticus infection since then in Calcutta (6) A large number of the clinical cases seen in the years after 1996 were associated with a unique clone of V. parahaemolyticus serotype O3:K6 (7). It was determined that many of the O3:K6 strains isolated since 1996 contain a filamentous phage, f237. f237contains a unique open reading frame, ORF8. Two other V. parahaemolyticus serotypes that were subsequently isolated, O4:K68 and O1:KUT. O4:K68 and O1:KUT also contains the f237 phage and therefore the ORF8 gene (7). 6.Okuda J, Ishibashi M, Hayakawa E, Nishino T, Takeda Y, Mukhopadhyay AK, Garg S, Bhattacharya SK, Nair GB, Nishibuchi M. Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta, India, and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan. J Clin Microbiol. 1997 Dec;35(12):3150-5. 7.Matsumoto C, Okuda J, Ishibashi M, Iwanaga M, Garg P, Rammamurthy T, Wong HC, Depaola A, Kim YB, Albert MJ, Nishibuchi M. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses. J Clin Microbiol. 2000 Feb;38(2):578-85. 12. Contd It has been demonstrated that serovars O3:K6, O4:K68, and O1: KUT are closely related, and molecular biology studies have suggested that O4:K68 and O1:KUT diverged from O3:K6 by genetic alteration of the O and K antigens. Together, these serotypes have been designated the pandemic group (8). It has been suggested that ORF8 could play a role in the increased virulence of the pandemic group strains. ORF8 could be a useful genetic marker for identification of these strains (9, 10). 8. Okura, M., R. Osawa, A. Iguchi, E. Arakawa, J. Terjima, and H. Watanabe. 2003. Genotypic analyses of Vibrio parahaemolyticus and development of a pandemic group-specific multiplex PCR assay. J. Clin. Microbiol. 41:4676 4682. 9. Myers, M. L., G. Panicker, and A. K. Bej. 2003. PCR detection of a newly emerged pandemic Vibrio parahaemolyticus O3:K6 pathogen in pure cultures and seeded waters from the Gulf of Mexico. Appl. Environ. Microbiol. 69:21942200. 10. Nasu, H., T. Iida, T. Sugahara, Y. Yamaichi, K.-S. Park, K. Yokoyama, K. Makino, H. Shinagawa, and T. Honda. 2000. A filamentous phage associated with recent pandemic Vibrio parahaemolyticus O3:K6 strains. J. Clin. Microbiol. 38:21562161. 13. Incidence & Geographic distribution V. parahaemolyticus-associated gastroenteritis has been reported from North America, Central America, Europe, Asia, and Africa. In Asia, V. parahaemolyticus is an established enteric pathogen in Japan, where consumption of uncooked seafood is common. V. parahaemolyticus has been reported to cause about 5070% of the cases of bacterial food poisoning in Japan (Miwatani & Takeda, 1976; Obata et al., 2001). Apart from Japan, various Asian countries, such as Thailand (Echeverria et al., 1983; Sriratanaban & Reinprayoon, 1982), India (Chatterjee et al., 1978; Bag et al., 1999), Bangladesh (Hughes et al., 1978; Bhuiyan et al., 2002), Laos (Matsumoto et al., 2000), Vietnam (Neumann et al., 1972; Van and Tuan, 1974), Tanzania (Mhalu et al., 1982), Hong Kong (Ho & Wong, 1985), Indonesia (Matsumoto et al., 2000), Philippines (Adkins et al., 1987), and Kuwait (Matsumoto et al., 2000), have reported the isolation of V. parahaemolyticus from patients suffering from diarrhea. 14. Incidence & Geographic distribution V. parahaemolyticus has also been isolated from diarrheal cases in Russia (Boiko et al., 1994), China (Jiang, 1991;Wu et al., 1998), Korea (Matsumoto et al., 2000), and Taiwan (Wu et al, 1996; Wong et al., 2000). In USA, V. parahaemolyticus-mediated outbreak was first identified in 1971 during which 320 people suffered from gastroenteritis in Maryland due to food poisoning (Dadisman et al.,1972; Molenda et al., 1972). This was the first confirmed outbreak of food poisoning due to V. parahaemolyticus outside Japan which is due to consumption of raw oysters. 15. Pathogenesis The incubation period of V. parahaemolyticus associated gastroenteritis varies between 4 and 96 h (Miwatani & Takeda, 1976), the mean incubation period being 15 h (Centers for Disease Control and Prevention, 1998). The experimental dosages required for initiation of gastroenteritis range from 2x105 to 3x107 CFU. Pathogenicity was closely correlated with kanagawa phenomenon(KP+) due to release of kanagawa hemolysin or the thermostable direct hemolysin ((Takeda, 1983; Nishibuchi et. al., 1989). Clinical isolates of KP negative V. parahaemolyticus from traveler produced hemolysin, named TDH-related hemolysin (TRH), which is also an important virulence factor. Clinical isolates of V.parahemolyticus may produce either TDH/ TRH encoded by tdh and trh genes respectively(Nishibuchi and Kapar, 1995). 16. Kanagawa phenomenon Clinical strains of V. parahaemolyticus were observed to produce hemolysis on special blood agar medium (Kato et al., 1965). In 1968, Wagatsuma developed a special medium for measuring the hemolytic character of V. parahaemolyticus called the Wagatsuma agar. It is a high-salt (7%) blood agar (defibrinated human or rabbit blood) medium containing d-mannitol as the carbohydrate source. Hemolysis observed on Wagatsuma agar medium, referred to as the Kanagawa phenomenon (KP). Kanagawa phenomenon (KP) has diagnostic as well as pathogenic significance for V. parahaemolyticus. 17. Kanagawa phenomenon KP is known to occur due to the expression of TDH that is more frequently detected in clinical strains of V. parahaemolyticus. Only 12% of the environmental strains of V. parahaemolyticus express the hemolytic protein and therefore most non-clinical isolates of V. parahaemolyticus are KP-negative (Miyamoto et al., 1969). 18. Virulence factors of V.parahemolyticus 1.Thermostable direct hemolysin (TDH): TDH was the first recognized virulence factor for V. parahaemolyticus and has been used as an important marker for identifying virulent strains (Okuda et. al., 1997a; Cook et. al., 2002). The hemolysin is a homodimer protein with a molecular mass of 46 kDa, each peptide being composed of 165 amino acids with one disulfide bond near the carboxyl terminus. (Tsunasawa et al., 1987; Honda & Iida, 1993). TDH is encoded by the tdh gene located in the chromosome, and all KP-positive V. parahaemolyticus strains contain two tdh gene copies, tdh1 and tdh2, that are 97% homologous (Nishibuchi & Kaper, 1990, 11). Vp-TDH exhibits - hemolytic activity on Wagatsuma medium, which has been termed the Kanagawa phenomenon (KP). Purified Vp-TDH is heat-stable, even at 100 C for 10 min and has hemolytic, cytotoxic, enterotoxic, mouse lethality and cardiotoxic activities. 11.Honda T, Ni Y, Miwatani T, Adachi T, Kim J. The thermostable direct hemolysin of Vibrio parahaemolyticus is a pore-forming toxin. Can J Microbiol. 1992 Nov;38(11):1175-80. 19. Thermostable direct hemolysin (TDH) Vp-TDH damages the erythrocyte membrane by acting as a pore-forming toxin, with the pores estimated at 2 nm in diameter. Vp-TDH-induced hemolysis occurs in three sequential steps : 1) binding to the erythrocyte membrane, 2) formation of a transmembrane pore, and 3) disruption of the cell membrane. Primary target of TDH is the intestinal epithelial cell. Raimondi et. al. (1995) demonstrated (using rabbit as animal model) that the TDH is one of the enterotoxin whose action mediated by intracellular calcium. TDH increases Cl-secretion in human colonic epithelial cells, through mechanisms involving cell binding and Ca2 + influx, followed by elevation of [Ca2+]in association with protein kinase C phosphorylation. TDH has cardiotoxic and cytotoxic effects (Sakazaki et. al., 1974). 20. Thermostable direct hemolysin- related hemolysin (TRH) TDH-like hemolysin or named Vp-TRH produced by a KP-negative clinical isolate of V. parahaemolyticus. Vp- TRH stimulates fluid secretion in the rabbit ileal loop test. It has an amino acid sequence that is approximately 67% homologous with Vp- TDH. Vp-TRH is heat labile at 60 C for 10 min. TRH is encoded by the trh gene, two copies (trh1 and trh2) of which are found to be chromosomally located in the V. parahaemolyticus genome. The two trh loci share 84% sequence identity (Kishishita et al., 1992). 21. Vp-TDH and Vp-TRH Both Vp-TDH and Vp-TRH induce chloride secretion in human colonic epithelial cells (12,13,14). In cultured human colonic epithelial cells, TRH increases Cl(-) secretion, followed by elevation of intracellular calcium. 12.Takahashi A, Kenjyo N, Imur K, Myonsun Y, Honda T : Cl-secretion in colonic epithelial cells induced by the Vibrio parahaemolyticus hemolytic toxin related to thermostable direct hemo-lysin. Infect Immun 68 : 5435-5438, 2000. 13.Park KS, Ono T, Rokuda M, Jang MH, Iida T, Honda T : Cytotoxicity and enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio parahaemolyticus. Microbiol Immunol 48 : 313-318, 2004. 14.Shimohata T, Takahashi A. Diarrhea induced by infection of Vibrio parahaemolyticus. J Med Invest. 2010 Aug;57(3-4):179-82. Review. 22. Thermo labile haemolysin (TLH) Another thermo-labile haemolysin (TLH) which is encoded by tlh gene is considered as a species-specific marker(15). This gene has been detected in all clinical and environmental V. parahaemolyticus strains. 15.Bej AK, Patterson DP, Brasher CW, Vickery MCL, Jones DD, Kaysner CA. Detection of total and hemolysin- producing Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of tlh, tdh and trh. J. Microbiol Methods. 1999;36:215-25. 23. Other putative virulence factors A serine protease (protease A) has been purified directly from the supernatant of V. parahaemolyticus and identified as a potential virulence factor (Lee et al., 2002). The protease A was a monomeric protein having a molecular mass of 43 kDa and an isoelectric point of 5.0. Purified protease-induced tissue hemorrhage and caused death of experimental mice when injected intravenously and intraperitoneally (Lee et al., 2002). 24. T3SS Apart fromTDH or TRH, there are factors that contribute to the pathogenesis of V. parahaemolyticus which include T3SS. Analysis of the genome sequence of V. parahaemolyticus strain IMD2210633 revealed two type III secretion systems (T3SS) on chromosomes 1 (T3SS1) and 2 (T3SS2). T3SS is an apparatus used by several Gram-negative pathogenic bacteria to secrete and translocate virulence factor proteins into the cytosol of eukaryotic cells. V. parahaemolyticus T3SS1 is analogous to the Ysc secretion system in Yersinia, and the V. parahaemolyticus T3SS2 is analogous to the Inv-Mxi-Spa secretion system in Salmonella and Shigella(16). 16.Park KS, Ono T, Rokuda M, Jang MH, Okada K, Iida T, Honda T : Functional characterization of two type III secretion systems of Vibrio parahaemolyticus. Infect Immun 72 : 6659- 6665, 2004. 25. T3SS The Ysc secretion system is typically related to cytotoxicity and the Inv-Mxi- Spa secretion system is associated with host cell invasion. T3SS1 has been found in all isolated strains and is related to the cytotoxicity observed in HeLa cells. T3SS2 is found only in Kanagawa phenomenon (KP)-positive strains and produces enterotoxicity which was assayed using rabbit ileal loop test. Two known T3SS variants: observations have shown T3SS2 correlating with tdh+/trh- strains, while T3SS2 correlates with tdh-/trh+ strains(17). 17.Noriea, Nicholas; CN Johnson, KJ Griffitt, DJ Grimes (September 2010). "Distribution of type III secretion systems in 'Vibrio parahaemolyticus from the northern Gulf of Mexico". Journal of Applied Microbiology 109 (3): 953962. 26. Clinical disease V. parahaemolyticus causes three major syndromes of clinical illness in humans (Daniels et al., 2000a; Morris & Black, 1985). 1.Gastroenteritis, - Most common manifestation characterized by watery or bloody diarrhea, vomiting, abdominal cramps, headache, fever, general lassitude, nausea, chills, and tenesmus. Stool of patients with diarrhea at the acute stage are of a characteristic meat washed/ reddish watery consistency 2. Wound infections and 3.Septicemia 27. Modes of transmission Ingestion of improperly processed or undercooked seafood that include marine fish, crustacean shellfish (prawns, lobsters, crabs), and molluscan shellfish is the most common mode of transmission. Person-to-person transmission of infection may occur from contact with an ill individual, either directly or via secondary contamination of household water, food, or fomites. V. parahaemolyticus has been detected from flies in all months of the year in Calcutta. Mean percentage of isolation was: housefly, 14.1; blue bottle fly, 18.5; external washing, 10.1; viscera, 22.6; flies of sweet shops, 9.7; and those from fishmarket, 22.7 (Chatterjee et al., 1978). Flies revealed higher (4.4%) isolation of Kanagawa positive V. parahaemolyticus strains than those of water and fish specimens (0.8%) studied. 28. Laboratory detection of V.parahaemolyticus Detection and identification and enumeration of V. parahaemolyticus (ISO method 8914, 1990; FDA, 1998), include an inoculationof the test portion into the selective enrichment medium APW (alkaline peptone water) and incubation at optimum temperatures, 2.Streaking onto the selective solid medium thiosulphate citrate bile salt agar (TCBS). 3. Presumptive colonies, sub-cultured on TSA (trypticase soya agar), are subjected to microscopic and biochemical analysis, such as Gram staining, motility, oxidase, arginine dihydrolase, lysine decarboxylase, ortho-nitrophenyl-galactopyranoside (ONPG), acidgas from glucose, saccharose, cellobiose, and halophilic characteristics according to Bergeys Manual of Systematic Bacteriology (Baumann and Schubert 1984). 29. Laboratory detection of V.parahaemolyticus 4. FDA procedure includes the evaluation of V. parahaemolyticus pathogenicity by detection of the Kanagawa phenomenon using Wagatsuma agar, containing high-salt blood to detect the haemolytic activity of V. parahaemolyticus thermostable direct haemolysin (TDH)-positive strains only. 5. Vibrio species may be serotyped according to both somatic O and capsular polysaccharide K antigens. 30. Identification of V.parahaemolyticus Microscopic Appearance: Gram stain : Gram negative rods straight or curved or comma-shaped of size 0.5-0.9 x 1.5-2.5 , forming no capsule and spores. This characteristic appearance is not always observed when the organism is Gram-stained from solid media. Primary Isolation Media : Blood agar incubated in air at 35-37C for 18-24 hr TCBS agar incubated in air at 35-37C for 18-24 hr 31. Identification of V. parahaemolyticus Colony Appearance : On blood agar colonies are 2-3 mm in diameter. On TCBS agar, it grows with green colored colonies due to absence of sucrose fermentation. On Macconkey agar it produces non-lactose fermenting colonies. Strains isolated from clinical specimens produce beta hemolysis on Wagatsuma Blood Agar (human blood) while strains isolated from environment dont. This is known as Kanagawa phenomenon. It grows well in peptone water with 8% NaCl but not at 10%. 32. Biochemical characteristics of V. parahaemolyticus Oxidase----------------------------------------------- + N03-NO2 + 1% NaCl------------------------------- + Indole + 1% NaCl----------------------------------- + Voges-Proskauer + 1% NaCl --------------------- -- Urease ---------------------------------------------- --/+ Lysine decarboxylase + 1% NaCl---------------- + Ornithine decarboxylase + 1% NaCl----------- + Arginine dihydrolase + 1% NaCI---------------- -- Fermentation of Sucrose------------------------------------------------ -- Lactose------------------------------------------------ -- L-Arabinose ----------------------------------------- + Gas from glucose---------------------------------- -- Growth in nutrient broth 0% NaCl---------------------------------------------- -- 3% NaCl---------------------------------------------- + 6% NaCI----------------------------------------------- + 8% NaCl----------------------------------------------- + 10% NaCl--------------------------------------------- -- Susceptibility to 0/129 ( 2,4-diamino-6,7-diisopropylpteridine phosphate) 10g------------------------------------------------- Resistant 150 g----------------------------------------------- Sensitive Growth on TCBS---------------------------------------- Green colonies (18). 18.Janda JM, Powers C, Bryant RG, Abbott SL. Current Perspectives on the Epidemiology and Pathogenesis of Clinically Significant Vibrio spp. Clin Microbiol Rev. 1988;1:245-267. 33. Serotyping of V.parahaemolyticus V. parahaemolyticus is classified on the basis of its somatic O antigen and capsular K antigen; the flagellar H antigens are identical in all strains of this species (Sakazaki, 1992). From the 13 known O types and more than 60 K types (Joseph et al., 1982; Hisatsune et al., 1993), 75 combinations of O:K serovars of V. parahaemolyticus are possible. ELISA-based system is commercially available for the detection of V. parahaemolyticus thermo stable direct haemolysin (Honda et al. 1985). O:K serovar of each test strain was determined by agglutination tests with specific antisera (Toshiba Kagaku Co.) or through the courtesy of H. Zen- Yoji. 34. O K 1 1 5 20 25 26 32 38 41 56 58 60 64 69 2 3 28 3 4 5 6 7 29 31 33 37 43 45 48 54 56 57 58 59 65 72 4 4 8 9 10 11 12 13 34 42 49 53 55 63 67 73 5 15 17 30 47 60 61 68 6 18 46 7 19 8 20 21 22 39 70 74 9 23 44 10 24 52 61 66 71 11 19 36 40 50 51 61 The serotyping kit of V. parahaemolyticus is commercially available from TOSHIBA KAGAKU KOGYO Co. Ltd., Tokyo, Japan. Current O (somatic antigen) and K (capsular antigen) serotype scheme of V. parahaemolyticus. 35. Molecular typing 1.16S rRNA gene analysis. 2. Polymerase chain reaction method, based on the use of combined species-specific primers directed against the variable regions of 16S rRNA. Multiplex PCR was established for detection of virulence genes tdh, trh and tlh. A molecular test based on the detection of the tdh and/or trh genes, encoding for thermo stable direct haemolysin and thermo stable-related hemolysin has been applied to identify V. parahaemolyticus (Joseph et al. 1982; Johnson et al. 1984; Tada et al. 1992). Lee et al. (1995) have developed a molecular approach based on the amplification of a DNA fragment, that is highly conservative in all strains of V. parahaemolyticus. 36. Molecular typing PCR procedures targeting the gyrB gene, encoding the B subunit of DNA gyrase essential for DNA replication, and the regulatory toxR gene, were used for the specific detection of V. parahaemolyticus (Miller et al. 1987; Lin et al. 1993; Reich and Schoolnik 1994; Kim et al. 1999). V. parahaemolyticus can be further confirmed by the toxR PCR; the toxR gene is species specific and can distinguish V. parahaemolyticus from other members of the genus Vibrio (Kim et al., 1999). FDA procedure includes the use of a gene probe targeting tdh gene for detecting of V. parahaemolyticus (Morris et al. 1987; Yamamoto et al. 1990). 37. Molecular typing DNA colony hybridization detects hemolysin genes. Presence or absence of the tdh, trh1, and trh2 genes in each test strain was determined by the DNA colony hybridization method using specific DNA probes. Arbitrarily primed PCR (AP-PCR) , Random amplified polymorphic DNA (RAPD), AFLP (Amplified restriction fragment length polymorphism), Ribotyping, PFGE, MLST, MALDI-TOF mass spectrometry. toxRS sequence determination: The toxRS region of the test DNA was amplified by a PCR method, and the nucleotide sequence of the amplicon was determined. Group-specific PCR : A PCR method to specifically detect the toxRS sequence of the new O3:K6 clone was established by a modification of the method of Wu et al(19,20). 19.J. Okuda, M. Ishibashi, E. Hayakawa, T. Nishino, Y. Takeda, A. K. Mukhopadhyay, S. Garg, S. K. Bhattacharya, G. B. Nair, and M. Nishibuchi, J. Clin. Microbiol. 35:31503155, 1997). 20.Wu, D. Y., L. Ugozzoli, B. K. Pal, and R. B. Wallace. 1989. Allele-specific enzymatic amplification of b-globin genomic DNA for diagnosis of sickle cell anemia. Proc. Natl. Acad. Sci. USA 86:27572760. 38. Markers of Pandemicity Group specific PCR(GS-PCR : Matsumoto et al. (2000) first reported the use of group-specific PCR (GS-PCR) for rapid identification of the pandemic clone. GS-PCR is based on the nucleotide differences at the ToxRS region identified between the pre-1995 and post-1995 group of O3:K6 strains. All post-1995 O3:K6 strains analyzed were 100% homologous at the nucleotide level of the ToxRS region and differed from pre-1995 isolates by 1114 bp (Matsumoto et al., 2000). The pandemic properties of serotypes O1:KUT, O4:K68, and O1:K25 were first identified by GS-PCR analysis. 39. Group-specific PCR for identification of pandemic strains of V. parahaemolyticus. PCR products were loaded on1%agarose gels and the amplicons visualized by ethidium bromide staining. Lane 1: 100 bp molecular weight standard (Gibco-BRL, USA); lane 2: negative control (non- pandemic strain AQ3810, an O3:K6 1983 isolate from Singapore); lanes 3 and 4: O3:K6 pandemic strains (VP294, 1999/Calcutta, and VP362, 2000/Calcutta, respectively); lane 5: O4:K68 pandemic strain (VP293, 1999/Calcutta); lane 6: O1:KUT pandemic strain (VP356, 2000/Calcutta); and lane 7: O1:K25 pandemic strain (VP343, 2000/Calcutta). 40. Markers of Pandemicity ORF8 assay: Presence of a unique f237 filamentous phage has been proposed to be a genetic marker for the identification of pandemic strains. The f237 filamentous phage was initially shown to be present only in the new O3:K6 strains isolated after 1995 from different geographical regions (Nasu et al., 2000). The importance of the phage significance is due to the presence of an open reading frame (designated ORF8) encoding a protein that had no homology with any proteins in the database. GS-PCR assay and the ORF8 assay are now routinely used as rapid diagnostic markers for distinction of pandemic strains from the non-pandemic V. parahaemolyticus. 41. Detection of the ORF8 from the bacteriophage f237 by PCR. PCR products were loaded on 1% agarose gels and the amplicons visualized by ethidium bromide staining. Lane 1: negative control (non-pandemic strain AQ3810, an O3:K6 1983 isolate from Singapore); lanes 2 and 3: O3:K6 pandemic strains (VP294, 1999/Calcutta, and VP362, 2000/Calcutta, respectively); lane 4: O4:K68 pandemic strain (VP293, 1999/Calcutta); lane 5: O1:KUT pandemic strain (VP356, 2000/Calcutta); lane 6: O1:K25 pandemic strain (VP343, 2000/Calcutta); and lane 7: 100 bp molecular weight standard (Gibco- BRL, USA). 42. Antibiotic susceptibility testing (CLSI)document M45-A Tests of susceptibility to the following antibiotics were performed by using the method of Bauer et al. (1): ampicillin (10 ,g), cephalin (30 p.g), chloramphenicol (30 , g), colymicin (10 , g), erythromycin (15 , g), gentamicin (10 , g), kanamycin (30 , g), lincomycin (2 , g), methicillin (5 , g), nafcillin (1 , g), nalidixic acid (30 , g), neomycin (30 , g), penicillin (10 IU), polymyxin B (300 IU), streptomycin (10 , g), and tetracycline (30 , g) (21). 21.Molitoris E, Joseph SW, Krichevsky MI, Sindhuhardja W, Colwell RR. Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia. Appl Environ Microbiol. 1985 Dec;50(6):1388-94. 43. Pathogenicity was tested by KP, RIL, and i.p. inoculation of mice Ligated RIL. New Zealand White rabbits of either sex and weighing approximately 1.0 kg were anesthetized by intravenous injection of 45 mg of sodium pentobarbitol per kg of body weight. The ileum was externalized and ligated, and 2.0 ml of whole culture in brain heart infusion broth was injected into each loop. Test loops were separated by control intervals which were injected with sterile brain heart infusion broth. The ileum was returned, and the incision was closed. After 18 to 20 h, the rabbits were sacrificed, and the loops were examined for fluid accumulation. Response was measured as the ratio of milliliter of fluid accumulation to centimeter of loop length. 44. Pathogenicity was tested by KP, RIL, and i.p. inoculation of mice Mouse i.p. inoculations. For each strain, 0.5 ml of whole culture was inoculated i.p. into two 18- to 21-day-old white mice, NIHINMRI CU strain. The mice were kept in holding cages at room temperature and were examined hourly for 10 h. They were subsequently examined daily for 10 days(21). 21.Molitoris E, Joseph SW, Krichevsky MI, Sindhuhardja W, Colwell RR. Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia. Appl Environ Microbiol. 1985 Dec;50(6):1388-94. 45. Treatment Rehydration is usually the only treatment needed. There is no evidence that antibiotic treatment decreases the severity or the length of the illness. In severe or prolonged illnesses, antibiotics such as tetracycline or ciprofloxacin can be used. Choice of antibiotics should be based on antimicrobial susceptibilities of the organism. 46. V.vulnificus V.vulnificus is the most important pathogenic vibrio because of its invasiveness and the high fatality rates associated with infection. The first indication of the existence of a new Vibrio species pathogenic for humans (originally called the L+, or lactose-positive Vibrio or Beneckea vulnifica) was in 1976 (22). It was first identified and described by the CDC in 1976 (22). The first documented case of disease caused by the bacterium was in 1979 by Blake et al of the US Centers for Disease Control. Vulnificus means wounding. Vibrio vulnificus is a typical marine vibrio - a slightly curved bacterium, motile by means of a single polar flagellum. 22.Hollis DG, Weaver RE, Baker CN, Thornsberry C. Halophilic Vibrio sp. Isolated from blood cultures. J Clin Microbiol. 1976;3:425. 47. Scientific Classification Kingdom: Bacteria Phylum: Proteobacteria Class: Gammaproteobacteria Order: Vibrionales Family: Vibrionaceae Genus: Vibrio Species: V. vulnificus 48. HISTORY The first description of a fatal vulnificus infection has been attributed to Hippocrates in the 5th century B.C. (1). He described the case of Criton of Thasos, an island in the Agean. This patient developed violent pain in his foot; followed by shivering and fever rapidly soon became delirious, and the following day, the foot was noted to be swollen and red, with small black blisters. Then he developed diarrhea and he died on the second day. Because Criton was an island resident, perhaps he ingested raw shellfish or food contaminated by seawater. 49. Introduction Vibrio vulnificus is a Gram-negative, motile curved bacterium found in marine and estuarine environments. It has been isolated from seawater, sediments, plankton and shellfish (oysters, clams and crabs) located in the Gulf of Mexico, the Atlantic Coast as far north as Cape Cod, and the entire U.S. West Coast. Vibrio vulnificus is an emerging pathogen of humans. It causes wound infections, gastroenteritis, or a syndrome known as primary septicemia. 50. Epidemiology On the basis of lipopolysaccharide (LPS) antigens, the species can be organized into three biotypes. Biotype 1, which accounts for almost all human infections; Biotype 2, which consists primarily of eel pathogens; and Biotype 3, an apparent hybrid of biotypes 1 and 2 that has been recently isolated from fish (tilapia) handlers in Israel. Biotype 1 was originally divided into 5 antigenic subgroups, other subgroups are known to exist. Biotype 1 is almost invariably associated with human disease, and one particular LPS type (1/5) is significantly more prevalent among clinical strains. This suggests that the presence of this LPS type itself causes increased virulence. 51. Biotype 2 This biotype of V. vulnificus, first reported by Tison et al. (23), differs from biotype 1 (the originally described strains) in several biochemical reactions (e.g. being indole negative) and in having a different Lipopolysaccharide type. Biotype 2 consists of a homogeneous type of LPS. 23.Tison DL, Nishibuchi M, Greenwood JD, Seidler RJ. Vibrio vulnificus biogroup 2: new biogroup pathogenic for eels. Appl Environ Microbiol 1982; 44: 640 646. 52. Biotype 3 First isolated in 1999, biotype-3 strains of V. vulnificus differ from biotypes 1 and 2 in several biochemical traits (e.g. lack of salicin and cellobiose fermentation) (24,25). Biotype 3 causes cellulitis, necrotizing fasciitis, and osteomyelitis. 24.Bisharat N, Agmon V, Finkelstein R, et al. Clinical, epidemiological, and microbiological features of Vibrio vulnificus biogroup 3 causing outbreaks of wound infection and bacteraemia in Israel. Israel Vibrio study Group. Lancet 1999; 3554: 14211424. 25.Colodner R, Raz R, Meir I, et al. Identification of the emerging pathogen Vibrio vulnificus Biotype 3 by commercially available phenotypic methods. J Clin Microbiol 2004; 42: 41374140. 53. Risk factors Severity of V vulnificus infections (and probably the infectious dose required for infection) depends on both bacterial and host factors. V. vulnificus produces a number of enzymes (hyaluronidase, mucinase, DNAase, lipase, and protease) that may facilitate pathogenesis. In addition, the presence of a capsule appears to be associated with invasive forms of V vulnificus. (encapsulated forms are more commonly found among clinical isolates than among environmental isolates.(26,27). 26.Hor LI, Chang TT, Wang ST. Survival of Vibrio vulnificus in whole blood from patients with chronic liver diseases: association with phagocytosis by neutrophils and serum ferritin levels. J Infect Dis. 1999;179:275-278. 27.Bullen JJ, Spalding PB, Ward CG, et al. Hemochromatosis, iron, and septicemia caused by Vibrio vulnificus. Arch Intern Med. 1991;151:1606-1609. 54. Risk factors V.vulnificus is sensitive to the degree of iron bound by transferrin in a given host, because it uses transferrin-bound iron for growth.(28,29). Persons with elevated transferrin-bound iron saturation (greater than 70%) or elevated ferritin levels, which includes persons with hemochromatosis, thalassemia, or liver disease, immunosuppression and hypochlorhydria are at increased risk for invasive infections. 28.Brennt CE, Wright AC, Dutta SK, et al. Growth of Vibrio vulnificus in serum from alcoholics: association with high transferrin iron saturation. J Infect Dis. 1991;164:1030-1032. 29.Centers for Disease Control and Prevention. Vibrio vulnificus infections associated with raw oyster consumption -- Florida, 1981-1992. MMWR. 1993;42:405-407. 55. Septicemia Risk Factors Liver disease Cancer Cirrhosis Immunosuppressive drugs Hemochromatosis Immunodeficiency syndromes Alcoholism Hemolytic anemia-eg. Thalassemia. major Chronic renal disease Hypochlorhydria 56. Modes of transmission Oysters, the most common vehicle for V. vulnificus infections, are typically found in estuaries and bays. Wound infections, which are often necrotizing, are usually acquired when an open wound is exposed to warm seawater or seafood drippings contaminated with V. vulnificus. Consumption of Raw or undercooked shellfish especially oysters is another source of infection. 57. Clinical disease Infections with V. vulnificus 1. Wound infection-Invasion is characterized by the occurrence of blister-like skin lesions or bullae, and rapidly-spreading necrosis resembling necrotizing fasciitis. 2.Primary septicaemias following ingestion of raw seafood include pneumonia(30), osteomyelitis,(31)spontaneous bacterial peritonitis,(32), eye infections,(33) and meningitis (34), endometritis or fallopian tube infection. 3.Gastroenteritis which usually develops within 16 hours of eating the contaminated food. 30.Kelly MT, Avery DM. Lactose-positive Vibrio in seawater: a cause of pneumonia and septicemia in a drowning victim. J Clin Microbiol. 1980;11: 278-280. 31.Vartian CV, Septimus EJ. Osteomyelitis caused by Vibrio vulnificus. J Infect Dis. 1990;161:363. 32.Holcombe DJ. Vibrio vulnificus peritonitis. A unique case. J La State Med Soc. 1991;143:27-28. 33.DiGaetano M, Ball SF, Straus JG. Vibrio vulnificus corneal ulcer. Case reports. Arch Ophthalmol. 1989;107:323-324. 34.Katz BZ. Vibrio vulnificus meningitis in a boy with thalassemia after eating raw oysters. Pediatrics. 1988;82:784-786. 58. Wound infections caused by V.vulnificus Characteristic skin lesions associated with Vibrio vulnificus infection on the leg in a 75-year-old patient with liver cirrhosis in whom septic shock and bacteremia developed. B) V. vulnificus bacteremia developed one day after a fish bone injury on the fourth finger of the left hand (arrow) in a 45-year-old patient with uremia. C. Gram-negative curved bacilli isolated from a blood sample of the 45-year-old patient with uremia. Photos from Hsueh, et al. Vibrio vulnificus in Taiwan. CDC Emerging Infectious Diseases Volume 10, Number 8, August 2004. 59. Symptoms and signs associated with Vibrio. vulnificus wound infections Symptom/sign % Cellulitis at wound site 88 Bullae 88 Fever (>37.8 C) 65 Chills 29 Mental status changes# 18 Ecchymosis 18 Hypotension$ 12 Diarrhoea 6 Vomiting 6 * Adapted from Klontz et al.(35) (n=168). # Obtundation, disorientation, or lethargy. $ Systolic blood pressure less than 90 mmHg. 35.Klontz KC, Lieb S, Schreiber M, Janowski HT, Baldy LM, Gunn RA. Syndromes of Vibrio vulnificus infections. Clinical and epidemiological features in Florida cases, 19811987. Ann Intern Med 1988; 109: 318323. 60. VIRULENCE AND PATHOGENESIS Virulence Factors include * Capsule * LPS * Extracellular proteins & cell associated factors Capsule:The acidic mucopolysaccharide capsule is thought to be the major virulence factor. Uronic acid component of the capsule is itself a strong stimulant to the release of tissue necrosis factor by macrophages. LPS: Cell wall of V. vulnificus contains lipopolysaccharide (36). In vivo studies suggest this material is active and may account for the hypotension seen in septicemia. 36.Elmore SP, Watts JA, Simpson LM, et al. Reversal of hypotension induced by Vibrio vulnificus lipopolysaccharide in the rat by inhibition of nitric oxide synthase. Microbiol Pathogenesis 1992;13:391-397. 61. VIRULENCE AND PATHOGENESIS Extracellular proteins & cell associated factors: Pili, hyaluronidase, collagenase,metalloprotease,elastase,cytolysin(Hemolysin), phospholipase, mucinase. The metalloprotease increases vascular permeability by releasing histamine from mast cells and/or by activating the Hageman factor-plasma kallikreinkinin system (37). 37.Powell JL, Wright AC, Harris AM, et al. The role of Vibrio vulnificus virulence factors in the in vitro stimulation ofTNFa. In: proceedings of the Annual Meeting of the American Society of Microbiology (Las Vegas, Nevada) ASM p. 78, 1994. 62. Identification of V.vulnificus V. vulnificus infection is diagnosed by routine stool, wound, or blood culture. Microscopic Appearance: Gram stain : Gram negative rods straight or curved or comma-shaped of size 0.5-0.9 x 1.5-2.5 , forming no capsule and spores. This characteristic appearance is not always observed when the organism is Gram-stained from solid media. Primary Isolation Media : Blood agar incubated in air at 35-37C for 18-24 hr TCBS agar incubated in air at 35-37C for 18-24 hr Colony Appearance : On blood agar colonies are 2-3 mm in diameter. On TCBS agar, it grows with green/ yellow colored colonies. On MacConkey agar, V.vulnificus produces lactose fermenting colonies. 63. Biochemical characteristics of V.vulnificus Oxidase ----------------------------------------Positive N03-NO2 + 1% NaCl------------------------- Positive Indole + 1% NaCl------------------------------ Positive Voges-Proskauer + 1% NaCl-----------------Negative Urease-------------------------------------------- -- Lysine decarboxylase + 1% NaCl------------ + Ornithine decarboxylase + 1% NaCl-------- +/-- Arginine dihydrolase + 1% NaCI-------------- -- Fermentation of Sucrose-------------------------------------------- --/+ Lactose -------------------------------------------- + L-Arabinose--------------------------------------- -- Gas from glucose-------------------------------- -- Growth in nutrient broth---------------------- -- 0% NaCl-------------------------------------------- -- 3% NaCl-------------------------------------------- + 6% NaCI-------------------------------------------- +/-- 8% NaCl-------------------------------------------- -- 10% NaCl------------------------------------------- -- Susceptibility to 0/129 10g------------------------------------------------- Sensitive 150 g----------------------------------------------- Sensitive Growth on TCBS------------------------------------ Green/Yellow colonies(17). 18.Janda JM, Powers C, Bryant RG, Abbott SL. Current Perspectives on the Epidemiology and Pathogenesis of Clinically Significant Vibrio spp. Clin Microbiol Rev. 1988;1:245-267. 64. Serotyping & Molecular typing of V. Vulnificus The identity of V. vulnificus strains may be confirmed by immunological test, such as enzyme-linked immunosorbent assay (ELISA) with monoclonal antibody species-specific for an intracellular antigen. Molecular typing : Polymerase chain reaction: It is based on the use of combined species-specific primers directed against the variable regions of 16S rRNA, was developed for the specific detection of V. vulnificus strains (Kim and Jeong 2001). A rapid, specific enumeration technique for V. vulnificus, using a nonradioactive probe, is currently being evaluated (Wright et al. 1993; FDA, 1998). FDA procedure includes the use of a gene probe for the identification of V. vulnificus, targeting cytotoxin, haemolysin (Morris et al. 1987; Yamamoto et al. 1990). 65. Treatment Surgical debridement. Antimicrobial agents most effective against V vulnificus infections include tetracycline,(38,39) fluoroquinolones (for example, ciprofloxacin), third-generation cephalosporins (for example, ceftazidime), and aminoglycosides (for example, gentamicin). The most current antimicrobial recommendation includes treatment with ceftazidime (2 g IV tid) and doxycycline (100 mg PO or IV bid) (40) or doxycycline in combination with ciprofloxacin or an aminoglycoside. Early administration of antimicrobial therapy may reduce the morbidity and mortality associated with V vulnificus infections. 38.Bowdre JH, Hull JH, Cocchetto DM. Antibiotic efficacy against Vibrio vulnificus in the mouse: superiority of tetracycline. J Pharmacol Exp Ther. 1983;225:595- 598. 39. Fang FC. Use of tetracycline for treatment of Vibrio vulnificus infections. Clin Infect Dis. 1992; 15:1071. 40.Gilbert DN, Moellering RC, Sande MA. Sanford Guide to Antimicrobial Therapy. 29th ed. Hyde Park, Vt: Antimicrobial Therapy, Inc; 1999. 66. Prevention Pasteurization of oysters has been shown to reduce or eliminate V. vulnificus without altering the taste or consistency of oysters. Capsular polysaccharide is the major determinant of virulence. Experimental vaccines utilizing capsular polysaccharide, conjugated to tetanus toxoid or homologous extracellular proteins are capable of eliciting an immune response in mice (41). Passive immunoprophylaxis or immumotherapy may be feasible using conjugate induced IV immune globulin (42). 41.Devi S, Hayat U, Kreger A, et al. Protection conferred by capsular conjugate vaccines of Vibrio vulnificus in a marine model. In: Proceedings of the Annual Meeting of the American Society of Microbiology (Las Vegas, Nevada) ASM p. 150, 1994. 42. Devi S, Hayat U, Powell J, et al. Passive immunization of mice with antiserum to capsular polysaccharide-tetanus toxoid conjugate confers protection against Vibrio vulnificus septicemia. In the Proceedings of the Interscience Conference on Antimicrobials and Chemotherapy (Orlando, Florida) ICAAC, p. 189, 1994. 67. Characteristics of V.Vulnificus & V.parahemolyticus Traits V.Vulnificus* V.parahemolyticus Oxidase Positive Positive TSI Agar medium Slant Stab Gas H2 S N A -- -- N A -- -- LIM medium Lysine Decarboxylation Indole production Motility + + + + + + Ornithine decarboxylation + + Arginine hydrolysis -- -- Growth in peptone medium 0% NaCl 1 3 8 10 -- + + -- -- -- + + + -- VP -- -- 68. Contd Traits V.Vulnificus V.parahemolyticus Utilization of Arabinose Mannitol Inositol -- d -- + + -- ONPG hydrolysis + -- *Biotype1, A : Acid production, N: No acid production, d: Varies in strains, ONPG: o-nitrophenyl-- D- galactopyranoside 43.Shinoda S. [Pathogenic factors of vibrios with special emphasis on Vibrio vulnificus]. Yakugaku Zasshi. 2005 Jul;125(7):531-47. Review. 69. V.alginolyticus V alginolyticus is a halophilic Vibrio first recognized as being pathogenic in humans in 1973. Wound infections account for 71% of V alginolyticus infections(44). Ear infections ( Otitis media-most common) are also seen with this organism. Gastroenteritis was thought to be a rare presentation accounting for 12% of infections (44). Other clinical syndromes reported in association with V alginolyticus infection include chronic diarrhea in a patient with AIDS,(45) conjunctivitis, (46) and post-traumatic intracranial infection (47). 44.Hlady WG, Klontz KC. The epidemiology of Vibrio infections in Florida, 1981- 1993. J Infect Dis. 1996;173:1176-1183. 45.Caccemese SM, Rastegar DA. Chronic diarrhea associated with Vibrio alginolyticus in an immunocompromised patient. Clin Infect Dis. 1999;29:946- 947. 46. Lessner AM, Webb RM, Rabin B. Vibrio alginolyticus conjunctivitis. Arch Ophthalmol. 1985; 103:229-230. 47. Opal SM, Saxon JR. Intracranial infection of Vibrio alginolyticus following injury in salt water. J Clin Microbiol. 1986;23:373-374. 70. V . alginolyticus Colonies were larger (from 6-9mm) irregular in shape, transparent or opaque, with a wavy margin on blood agar. On TCBS agar, colonies are yellow in colour. It has a higher salt tolerance, is VP positive and ferments sucrose. Growth in the presence of 10% NaCl, acetoin production, and fermentation of arabinose are important features which distinguish V. alginolyticus from V. parahaemolyticus. Resistance to tetracycline and chloramphenicol has been reported in a few isolates of V alginolyticus, but all strains appear to be sensitive to ciprofloxacin (48). 48. French GL. Antibiotics for marine Vibrios. Lancet. 1990;336:568-569. 71. Association of pathogenic Vibrio species with three major clinical syndromes Vibrio Species Primary septicemia Gastroenteritis Wound infection V.parahemolyticus + +++ ++ V.alginolyticus +++ * +++ V.vulnificus + ++ +++ +++, most common association; ++, possible association; +, rare association. *, The association remains to be firmly established. Adapted from West (1989). 49.Tantillo GM, Fontanarosa M, Di Pinto A, Musti M. Updated perspectives on emerging vibrios associated with human infections. Lett Appl Microbiol. 2004;39(2):117-26. Review. 72. V. fluvialis V fluvialis is a halophilic Vibrio first identified in 1975 in a patient with diarrhea in Bahrain.(50) It is biochemically similar to Aeromonas hydrophila but can be differentiated from this organism by its ability to grow well on media containing 6% to 7% sodium chloride. The largest series of V fluvialis infections involved 500 patients in Bangladesh, half of whom were young children.(51) In that series, patients presented with diarrhea (100%, 75% bloody), vomiting (97%), abdominal pain (75%), dehydration (67%), and fever (35%). V fluvialis rarely causes wound infections or primary septicemia.(44,52) 44.Hlady WG, Klontz KC. The epidemiology of Vibrio infections in Florida, 1981- 1993. J Infect Dis. 1996;173:1176-1183. 50. Furniss AL, Lee JV, Donovan TJ. Group F, a new Vibrio? Lancet. 1977;10:73-94. 51. Huq MI, Alam KMJ, Brenner DJ, et al. Isolation of Vibrio-like group, EF-6, from patients with diarrhea. J Clin Microbiol. 1980;11:621-624. 52. Varghese MR, Farr RW, Wax MK, et al. Vibrio fluvialis wound infection associated with medicinal leech therapy. Clin Infect Dis. 1996;22:709-710. 73. Vibrio hollisae V hollisae, a halophilic Vibrio like species first described in 1982, most commonly causes gastroenteritis. Now it is named as Grimontia hollisae. V hollisae is difficult to isolate, since it grows poorly on selective TCBS media and it needs to be isolated from colonies on a blood agar plate. V hollisae septicemia and wound infections have been reported but are rare.(44,53) 44.Hlady WG, Klontz KC. The epidemiology of Vibrio infections in Florida, 1981- 1993. J Infect Dis. 1996;173:1176- 1183. 53.Rank EL, Smith IB, Langer M. Bacteremia caused by Vibrio hollisae. J Clin Microbiol. 1988; 26:375-376. 74. Photobacterium damsela P damsela (formerly Vibrio damsela) is a halophilic gram-negative bacillus similar to V .vulnificus that strictly causes soft tissue infections following exposure of wounds to brackish water or injury by saltwater animals.(54) P damsela infections can be fulminant and are frequently fatal even in immunocompetent hosts. Of the 16 cases of P damsela infection reported between 1982 and 1996, 4 were fatal.(55) 54. Barber GR, Swygert JS. Necrotizing fasciitis due to Photobacterium damsela in a man lashed by a stingray. N Engl J Med. 2000;342:824. 55. Fraser SL, Purcell BK, Delgado B, et al. Rapidly fatal infection due to Photobacterium (Vibrio) damsela. Clin Infect Dis. 1997;25:935-936.