Cockle Isolates of Vibrio Vulnificus | Research Experiment

Cockle Isolates of vibrio Vulnificus Research Experiment isolation and appellation characterization among entwine isolates of Vibrio vulnificus isolated from Selangor, Malaysia Coastal areaMohammed M. Kurdi Al-Dulaimi, Sahilah Abd. Mutalib and Maaruf Abd.GhaniKey words Vibrio vulnificus, cockles, isolation, characterization, Malaysia.ABSTRACTVibrio vulnificus infections is worldwide public health problems associated with illnesses resulting from consumption of raw or parti eithery cooked seafood worldwide. The aim of this take away was to investigate the forepart and identification of V. vulnificus in cockle collected from local wet (2) and supermarkets (2) from Selangor, Malaysia from July 2013 to February 2014. A total of 78 (n=78) cockle samples were examined for the presence of V. vulnificus and collected from four local supermarkets sites of Selangor hypermarkets, V. vulnificus was present in at about 32% (25/78) of the 78 seafood cockle samples were unequivocal to this ba cterium. Colonies geomorphological observation and biochemical characterization for those isolates showed 60% (15/78) of isolates were separate as biotype 1 and 40% (10/78) belong to biotype 2.INTRODUCTIONVibrio vulnificus is a motile, asporgenic, halophilic gram-negative bacterium that found worldwide in estuarine and coastal warm waters that frequently contaminates seafood like oysters , cockles , shrimps and other seafood (Horseman and Surani, 2011) infections of V. vulnificus are reported in many different countries ,USA, Europe, Korea, Taiwan , Malaysia and Saudi Arabia(Tamplin et al. 1982 Chuang et al.1992 Dalsgaard et al. 1999 Hlady and Klontz 1996 Elhadi et al.,2004 Elhadi 2012 Paydar and eyelash 2013).Three major syndromes of clinical illness ca apply by unhealthful vibrio septicemia, gastroenteritis and wound infections. The majority of these infections is foodborne and associated with consumption of raw or undercooked variety of seafood, including shrimp, fish, oyster s and pillage (Bisharat et al., 1999). Probability of septicemia and necrotizing fasciitis increased in the patients with coloured cirrhosis and diabetes mellitus that reduce host resistance to bacterial infections owing to the immunocom-promised status (Ito et al., 2012).V. vulnificus is a bacterial species that is virulent for humans and fish ,In terms of virulence, it has been classified based on phenotypical and serological characteristics(Biosca et al.,1997 Tison et al., 1982), therefore V.vulnificus isolates classified into three biotypes, biotypes 1 and 3 are classified as a human isolates and biotype 2 is classified fish and eel isolates , The virulence mechanism of biotype 2 V. vulnificus strains in eels remains unclear, although some virulence factors have been proposed. The extracellular products of biotype 2 strains exhibit hydrolytic/toxic activities and lethality for the eel similar to those produced by the biotype 1 strain (Biosca and Amaro 1996). However, a hardl y a(prenominal) human infections caused by biotype 2 isolates have been reported worldwide. This study aims to isolateion and identifiy characterization of the V. vulnificus among cockle from Selangor, Malaysia.MATERIALS AND METHODSSamples collection and preparingFor this In the present study, cockle samples (n=78) were purchased from wet markets (2) and hypermarkets (2) in Selangor-Malaysia from July 2013 to February 2014 .The cockles were washed in distilled water and scrubbed free of dirt and shucked with a sterile scalpel and cut to small pieces with sterile scissor, 25 g of muscle and intravalvar fluid were collected homogenized into sterile stomacher bags containing 225 ml of Akaline Salt Peptone Water (ASPW) (Oxoid, England) with 3% (w/v) NaCl. Samples were homogenized with using stomacher (Stomacher Lab-Blender 400) for 2 minutes. enriched in alkaline peptone salt water and The solution was diluted and then spread onto ______(what spiritualist) medium and incubated for 18-24 hours at 37C.Isolation and morphological characterizationAll samples were analyzed for potentially pathogenic Vibrios. All samples enriched in APWDS1 and transferred a loop full of enrichment broth on to Thiosulphate citrate saddle sore salt sucrose nutrient agar (TCBS)(Difco Laboratories, USA) and CHROMagar Vibrio (CV)(CHROMagar, France) and incubated for 24 h at 37C. After incubation colonies suspected to be V. vulnficus were picked up from the TCBSDS2 agar and CV plates and coatingd on Tryptic Soy Agar (TSA) to obtain pure colonies.DS3 (Elhadi 2012).V. vulnificus produce green colonies on TCBS and spunky green (Turquoise) on Chromagar Vibrio, these colonies were picked up to nutrient agar slant and stored at 4C for morphological and biochemical identification.Biochemical characterizationThe isolates were identify at the species level with the use of biochemical tastes that may tell apart among closer species of vibrio that gave similar morphological characteristics on selec tive media, in this study we use some biochemical tests that distinguish among V. vulnificus and other vibrio species such(prenominal) as V. parahemolyticus. DS4Indole ProductionThe ability of bacteria to splitting of tryptophan to indole tested , test tubes contain 9 ml of tryptone water inoculated with loop full of V. vulnificus culture and incubated for 24- 48 h at 37C, adding 1 ml of Kovacs indicator(P-dimethyl amnobenzaldehyde in amyl) , positive reaction immediately forming red color ring in the broth. DS5Oxidase testDS6Bacterial colonies were transferred with a sterile glass rod to filter paper moistened with oxidase reagent. Appearance of a dark purple color within few seconds was considered a positive reaction.Tolerance to NaClDS7Tolerance to NaCl was determined by the addition of NaCl to 1 % peptone broth with percentage of 0, 3, 6 and 10 % (w/v) DS8and cultures were examined for process after 2 days at 37oC.RESULTSIsolation of V. vulnificus DS9A total of 78 cockle sample s were obtained from four different location of kajang.30, 28, 10 and 10 cockle samples were collected from pasar kajang, gaint bangi , gaint kajang and pasar bangi respectively, from terrific 2013 until January 2014, 11 sample (36%) of pasar Kajang was positive, 9 samples (32%) of Gaint Bangi was positive, 3 samples (30%) of Gaint Kajang was positive and 2 (20%) of Pasar Bangi was positive for V. vulnificus (table 1), The highest numbers of V. vulnificus were isolated from pasar kajang and gaint bangi , the lowest numbers of V. vunlificus were isolated from gaint kajang (Fig.1).DS10Table 1 percentageDS11 of presence of V. vulnificus in cockle samples.Figure 1 percentage of positive samples.Morphological and biochemical identificationV. vulnificus in this study were identified as biotype I because all the isolates were positive for indole production. CHROMagar Vibrio (CHROMagar Paris,France) was used in this study in conjunction with TCBS agar because of its ability to isolate and identifyV. vulnificus compared to the TCBS agar. CHROMagar Vibrio uses chromogenic technology to allow for the isolation and perception of V. vulnificus resulting in development dark blue colonies which can be distinguished from other Vibrio species as shown in Fig. 2. V. vulnificus were isolated from 25 of the 78 samples analyzed, and were identified with picked up single colonies on selective media for morphological identification, V.vulnificus isolates appeared as green colonies on TCBS medium and appeared as blue green colonies on Chromagar Vibrio medium Fig 2, green color on TCBS refer to inability of this bacteria to produce acids from sucrose, colonies on ChromagarTM Vibrio medium are accurately (99%) detect V. vulnificus isolates and fork it from other vibrio species.Fig 2 V vulnificus isolates on TCBS (A) and Chromagar Vibrio (B).Biochemical characterizationDistinguish between the different vibrios on the basis of colony morphology not always possible, the identification of Vibrio spp. is problematic because of phenotypic similarity of some species, there for we should be do biochemical tests to confirm the results obtained from morphological identification. V. vulnificus is divided into three biotypes according to their different biochemical and biological properties (Linkous and Oliver 1999).Twenty-five strains identified as V. vulnificus were submitted to biochemical tests. All isolates used in this study were positive for oxidase test (100%), all isolates grow on solid selective media (TCBS and CV agar). Salt tolerance show that 100% of isolates grow in 3% NaCl whiles no growth in 0 % of NaCl (table 2) because this bacterium is halophilic and it is abundantly present in estuarine ecosystems throughout the world.Table 2 biochemical tests of V. vulnificus isolates.DISCUSSIONIsolation and morphological identification of V. vulnificusThe main objective of this study was to detect the incidence of pathogenic V. vulnificus in cockle samples collected from five sites in Malaysia, 25 of 78 cockle samples were positive for V. vulnificus (table 1), high percentage of cockles positive for V. vulnificus attributed to filter-feeding mollusks such as oysters, clams and mussels have high concentrations of the bacteria in their guts and other tissues (Strom and Paranjpye 2000). A chromogenic medium used for differentiation of V. vulnificus which contains bile salts and high NaCl and pH. This medium selects mainly three vibrio species (V. vulnificus, V. cholera and V. parahemolyticus) easily to differentiate among these species depend upon colony color whereas V. vulnificus isolates gave distinctive blue green color, various biochemical tests were used for more identification (Elhadi 2012).Biochemical identificationOn the basis of differences in biochemical property V. vulnificus includes two biotypes (Tison et al. 1982). In this study, V. vulnificus biotype 1 and 2 were isolated. Isolates from human infections and environmental sources w ere described as indole positive and belong to biotype 1, whereas strains recovered from diseased eels were indole-negative and classified as biotype 2. (Amaro et al., 1992 Biosca et al., 1996 Radu et al., 1998).Ability to produce indole from trypton show 60% of isolates positive classified as biotype 1, and 40% was negative to indole production classified as biotype 2 (table 2), that indole production was the single biochemical trait which distinguished biotype 1 and biotype 2 (Tison et al. 1982). All isolates in this study were positive for oxidase, majority of V. vulnificus have cytochrome c oxidase enzyme, V. vulnificus is a halophilic marine bacterium tolerant for NaCl from 1% to 10% whereas closely of V. vulnificus isolates tolerant up to 6% NaCl (table 2) but no growth in 10% NaCl and 0% of NaCl (Dalsgaard et al., 1999 Elhadi, 2012). V. vulnificus grow in wide range of temperature from 13C to 40C, optimal temperature for isolates in this study was 37C that is an optimal temp erature for pathogenic bacteria.REFERENCESHorseman, M.A. and Surani, S. (2011). 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Comparative study of phenotypic and virulence properties in V. vulnificus biotype 1 and 2 obtained from a European eel farm experiencing mortalities. Dis Aquat Organ 13, 2935.Tamplin M, Rodrick G.E., Blake N.J., Cuba T.(1982). Isolation and characterization of Vibrio vulnificus from two Florida estuaries. Appl. Environ. Microbiol. 4414661470.DS1Write in ful, and short capital garner with bracket after thatDS2Write in fullDS3Shouldnt has dot pleaseDS4Omit this partDS5Not clear please clarifyDS6removeDS7removeDS8 please addDS9removeDS10make sure the valu tele with your move upDS11Shouldn write in bold, please write properly and precise. Too simple is not describsableDS12write in fullDS13remove this column