- \n
- Mohammed M. Kurdi Al-Dulaimi, Sahilah Abd. Mutalib and Ma`aruf Abd.Ghani<\/strong><\/li>\n<\/ul>\n
Key words:<\/strong> Vibrio vulnificus<\/em>, cockles, isolation, characterization, Malaysia.<\/p>\n
ABSTRACT:<\/h3>\n
Vibrio<\/em> vulnificus<\/em> infections is worldwide public health problems associated with illnesses resulting from consumption of raw or partially cooked seafood worldwide. The aim of this study was to investigate the presence and identification of V. vulnificus<\/em> 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<\/em> and collected from four local supermarkets sites of Selangor hypermarkets, V. vulnificus<\/em> was present in at about 32% (25\/78) of the 78 seafood cockle samples were positive to this bacterium. Colonies morphological observation and biochemical characterization for those isolates showed 60% (15\/78) of isolates were classified as biotype 1 and 40% (10\/78) belong to biotype 2.<\/p>\n
INTRODUCTION:<\/h2>\n
Vibrio vulnificus<\/em> 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<\/a>) infections of V. vulnificus<\/em> 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 Thong 2013).<\/p>\n
Three major syndromes of clinical illness caused by pathogenic 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, oysters and clams (Bisharat et al., 1999). Probability of septicemia and necrotizing fasciitis increased in the patients with liver cirrhosis and diabetes mellitus that reduce host resistance to bacterial infections owing to the immunocom-promised status (Ito et al., 2012).<\/p>\n
V. vulnificus<\/em> 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<\/em> 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<\/em> 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 few human infections caused by biotype 2 isolates have been reported worldwide. This study aims to isolateion and identifiy characterization of the V. vulnificus<\/em> among cockle from Selangor, Malaysia.<\/p>\n
MATERIALS AND METHODS<\/h2>\n
Samples collection and preparing<\/h3>\n
For 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 medium) medium and incubated for 18-24 hours at 37\u00baC.<\/p>\n
Isolation and morphological characterization<\/h3>\n
All samples were analyzed for potentially pathogenic Vibrios. All samples enriched in <\/a>APW[DS1]<\/a> and transferred a loop full of enrichment broth on to Thiosulphate citrate bile salt sucrose agar (TCBS)(Difco Laboratories, USA) and CHROMagar Vibrio (CV)(CHROMagar, France) and incubated for 24 h at 37\u00baC. After incubation colonies suspected to be V. vulnficus<\/em> were picked up from the <\/a>TCBS[DS2]<\/a> agar and CV plates and cultured on Tryptic Soy Agar (TSA) to obtain pure colonies<\/a>.[DS3]<\/a> (Elhadi 2012).V. vulnificus<\/em> produce green colonies on TCBS and blue green (Turquoise) on Chromagar Vibrio, these colonies were picked up to nutrient agar slant and stored at 4\u00b0C for morphological and biochemical identification.<\/p>\n
<\/a>Biochemical characterization<\/h3>\n
The isolates were identified at the species level with the use of biochemical tests that may differentiated among closer species of vibrio<\/em> that gave similar morphological characteristics on selective media, in this study we use some biochemical tests that distinguish among V. vulnificus<\/em> and other vibrio species such as V. parahemolyticus<\/em>. [DS4]<\/a><\/p>\n
Indole Production<\/h3>\n
The 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<\/em> culture and incubated for 24- 48 h at 37\u00baC, <\/a>adding 1 ml of Kovac`s indicator(P-dimethyl amnobenzaldehyde in amyl) , positive reaction immediately forming red color ring in the broth. [DS5]<\/a><\/p>\n
Oxidase test<\/h3>\n
<\/a>:<\/strong>[DS6]<\/a><\/p>\n
Bacterial 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.<\/p>\n
Tolerance to NaCl<\/h3>\n
<\/a>:<\/strong>[DS7]<\/a><\/p>\n
Tolerance to NaCl was determined by the addition of NaCl to 1 % peptone broth with percentage of 0, 3, 6 and 10 % <\/a>(w\/v) [DS8]<\/a>and cultures were examined for growth after 2 days at 37o<\/sup>C.<\/p>\n
RESULTS<\/h2>\n
Isolation of V. vulnificus<\/em><\/a>:<\/em><\/strong> [DS9]<\/a><\/h3>\n
A total of 78 cockle <\/a>samples 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 August 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<\/em> (table 1),<\/em> The highest numbers of V. vulnificus<\/em> were isolated from pasar kajang and gaint bangi , the lowest numbers of V. vunlificus<\/em> were isolated from gaint kajang (Fig.1).[DS10]<\/a><\/p>\n
Table 1<\/strong> <\/a>percentage[DS11]<\/a> of presence of V. vulnificus<\/em> in cockle samples.<\/em><\/p>\n
![\"\"](\"https:\/\/s3-eu-west-1.amazonaws.com\/aaimagestore\/essays\/0949409.001.png\"\/)
Figure 1<\/strong> percentage of positive samples.<\/p>\n![\"\"](\"https:\/\/s3-eu-west-1.amazonaws.com\/aaimagestore\/essays\/0949409.002.png\"\/)
V. vulnificus<\/em> 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<\/em> 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<\/sup> Vibrio medium are accurately (99%) detect V. vulnificus<\/em> isolates and differentiate it from other vibrio species.<\/p>\n![\"\"](\"https:\/\/s3-eu-west-1.amazonaws.com\/aaimagestore\/essays\/0949409.004.png\"\/)
![\"\"](\"https:\/\/s3-eu-west-1.amazonaws.com\/aaimagestore\/essays\/0949409.003.png\"\/)
Fig 2 V vulnificus<\/em> isolates on TCBS (A) and Chromagar Vibrio (B).<\/p>\n