Cockle Isolates of Vibrio Vulnificus | Research Experiment

Isolation and identification characterization among cockle isolates of Vibrio vulnificus isolated from Selangor, Malaysia Coastal area

• Mohammed M. Kurdi Al-Dulaimi, Sahilah Abd. Mutalib and Ma`aruf Abd.Ghani

Key words: Vibrio vulnificus, cockles, isolation, characterization, Malaysia.

ABSTRACT:

Vibrio vulnificus 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 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 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.

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INTRODUCTION:

Vibrio 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 Thong 2013).

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).

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 few 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 METHODS

Samples collection and preparing

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 37ºC.

Isolation and morphological characterization

All samples were analyzed for potentially pathogenic Vibrios. All samples enriched in APW[DS1] 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 37ºC. After incubation colonies suspected to be V. vulnficus were picked up from the TCBS[DS2] agar and CV plates and cultured on Tryptic Soy Agar (TSA) to obtain pure colonies.[DS3] (Elhadi 2012).V. vulnificus 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°C for morphological and biochemical identification.

Biochemical characterization

The isolates were identified at the species level with the use of biochemical tests that may differentiated among closer species of vibrio that gave similar morphological characteristics on selective media, in this study we use some biochemical tests that distinguish among V. vulnificus and other vibrio species such as V. parahemolyticus. [DS4]

Indole Production

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 culture and incubated for 24- 48 h at 37ºC, adding 1 ml of Kovac`s indicator(P-dimethyl amnobenzaldehyde in amyl) , positive reaction immediately forming red color ring in the broth. [DS5]

Oxidase test

:[DS6]

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.

Tolerance to NaCl

:[DS7]

Tolerance to NaCl was determined by the addition of NaCl to 1 % peptone broth with percentage of 0, 3, 6 and 10 % (w/v) [DS8]and cultures were examined for growth after 2 days at 37^oC.

RESULTS

Isolation of V. vulnificus: [DS9]

A total of 78 cockle 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 (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).[DS10]

Table 1 percentage[DS11] of presence of V. vulnificus in cockle samples.

Figure 1 percentage of positive samples.

Morphological and biochemical identification:

V. 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 detection 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 Chromagar^TM Vibrio medium are accurately (99%) detect V. vulnificus isolates and differentiate it from other vibrio species.

Fig 2 V vulnificus isolates on TCBS (A) and Chromagar Vibrio (B).

Biochemical characterization:

Distinguish 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.

DISCUSSION

Isolation and morphological identification of V. vulnificus:

The 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 identification:

On 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 were 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 most 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 13°C to 40°C, optimal temperature for isolates in this study was 37°C that is an optimal temperature for pathogenic bacteria.

REFERENCES

Horseman, M.A. and Surani, S. (2011). A comprehensive review of Vibrio vulnificus: an important cause of severe sepsis and skin and soft-tissue infection. Int. J. of Infect. Dis., 15(3):157-166.

Bisharat, N., Agmon, V., Finkelstein, R., Raz, R., Ben-Dror, G., Lerner, L., et al.(1999) .Clinical, epidemiological, and microbiological features of Vibrio vulnificus biotype 3causing outbreaks of wound infection and bacteraemia in Israel. The Lancet 354(9188):1421–1424.

Biosca EG, Amaro C, Larsen JL, Pedersen K (1997) Phenotypic and genotypic characterization of Vibrio vulnificus: proposal for the substitution of the subspecific taxon biotype for serovar. Appl Environ Microbiol 63: 1460–1466.

Tison, D.L., Nishibuchi, M., Greenwood, J.D., Seidler, R.J.(1982). Vibrio vulnificus biogroup2: new biogroup pathogenic for eels. Applied and Environmental Microbiology 44,640–646.

Biosca, E. G., and Amaro C.(1996).Toxic and enzymatic activities of Vibrio vulnificus biotype 2 with respect to host specificity. Appl. Environ. Microbiol. 62:2331–2337.

Paydar M, Thong K.L.(2013). Prevalence and genetic characterization of Vibrio vulnificus in raw seafood and seawater in Malaysia. J Food Prot.76(10):1797-1800.

Linkous D.A. and Oliver J.D.( 1999). Pathogenesis of Vibrio vulnificus, FEMS Microbiol. Letters. 174 :207-214.

Hlady WG , Klontz KC(1996). The Epidemiology of Vibrio Infections in Florida, 1981-1993, J. of Infec. Dis. 173(5):1176-1183.

Elhadi N, Radu S, Chen CH, Nishibuchi M.(2004) Prevalence of potentially pathogenic Vibrio species in the seafood marketed in Malaysia. Journal of Food Protection; 67(7): 1469-75.

Chuang, Y.C., Yuan, C.Y., Liu, C.Y., Lan, C.K. & Huang, A.H. (1992). Vibrio vulnificus infection in Taiwan: report of 28 cases and review of clinical manifestations and treatment. Clinical Infectious Diseases, 15: 271–276.

Elhadi, N. (2012). Antibiotic Resistance and Plasmid Profiling of Clinically Significant Vibrio vulnificus Isolated from Coastal Water in Eastern Province of Saudi Arabia. British Journal of Pharmacology and Toxicology 3(2): 93-97.

Radu, S., Elhadi, N. Hassan, Z. Rusul, G. Lihan,S. Fifadara, N. Yuherman and E. Purwati, 1998.Characterization of Vibrio vulnificus isolated from cockles (Anadara granosa): Antimicrobial resistance,plasmid profiles and random amplification of polymorphic DNA analysis. FEMS. Microbiol.Lett.,165: 139-143.

Ito, H., Shibayama A., Abe M., Antoku S., Nawata H., Isonishi M., Fujita M., Kato S. (2012). Vibrio vulnificus septicemia and necrotizing fasciitis in the patients with liver cirrhosis and diabetes mellitus. J. of Diabetes Mellitus :2(1)122-125.

Dalsgaard, I., Høi, L., Siebeling, R. J. & Dalsgaard, A. (1999). Indole-positive Vibrio vulnificus isolated from disease outbreaks on a Danish eel-farm. Dis Aquat Organ 35, 187–194.

Amaro, C., Biosca, E. G., Esteve, C., Fouz, B. & Toranzo, A. E. (1992). 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, 29–35.

Tamplin M, Rodrick G.E., Blake N.J., Cuba T.(1982). Isolation and characterization of Vibrio vulnificus from two Florida estuaries. Appl. Environ. Microbiol. 44:1466–1470.

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