Vibrio alginolyticus is a Gram-negative halophilic bacterium, widely spread geographically in marine and estuarine waters [1]. Vibrio alginolyticus is recognized as a causative agent of gastroenteritis, wound infection and septicemia inhuman, and skin ulcer in marine animals [2].

Vibriospecies express a variety of hemolysis toxins. Some of these toxins are very similar, however, not necessarily identical [3]. Many studies have reported Vibrio alginolyticus strains carrying virulence genes derived from other Vibriospecies. In the United States (USA), during a 2004 investigation of a Vibrio parahaemolyticus outbreak, researchers isolated Vibrioalginolyticus that possessed and expressed a trh gene with 98% homology to the trh2 gene of Vibrio parahaemolyticus [4]. In Morocco, researchers isolated Vibrio alginolyticus carrying a trh gene [5]. Vibrio alginolyticus can be a reservoir of many virulence genes of other Vibrio species in the marine environment [6]. The tdh and trh are considered the two major pathogenic virulence factors of Vibrio parahaemolyticus [7]. In order to differentiate between pathogenic and non-pathogenic strains, Vibrio parahaemolyticus were examined for the presence of tdh and/or trh genes [7]. In this study, the virulence genes of Vibrio alginolyticus and Vibrio parahaemolyticus in shrimps were examined, targeting the specific toxR genes considered to be the global regulator of Vibrio species.

Sampling

Five samples of shrimp were purchased locally from hypermarket located in Selangor, Malaysia. All samples were transferred to sterile plastic bags for transportation and were processed and tested on the same day.

Most probable number method (MPN)

A quantity of 10 g of shrimp was mixed with 90 mL of Alkaline Peptone Water (APW) in a sterilized stomacher bag, and homogenized for one minute using a stomacher (Inter-science, France), as described in the U.S. Food and Drug Administration, Bacterial Analytical Manual (BAM) [8]. The sample was determined by the three-tube MPN method with slight modifications as described previously [9]. Briefly, a 10-fold serial dilution was applied by adding 1 ml of the homogenate to 9 ml of APW. A 1 ml of dilution was transferred into sterile centrifuge tubes; each tube contained 9 ml of APW with final dilution samples of 1:10 to 1:10000 in triplicates. The tubes were incubated at 37°C for 18-24 h, followed by plating of each dilution on the CHROMTM Vibrioagar. The total number of Vibriowas determined by PCR method.

DNA preparation

The DNA extraction was carried out using the boiling cell method [10]. Briefly, one milliliter of inoculated APW was incubated at 37°C for 18-24 h, and centrifuged at 10,000 RPM for 3 minutes. The supernatant was discarded, and 200 μl of sterile distilled water was added to the pellet and mixed gently using a vortex mixer. The resulting mixture was then boiled in a dry bath for 15 minutes followed by a centrifuge run at 10,000 RPM for one minute. The supernatant was used for PCR.

Multiplex PCR assay

The sets of primers (Sigma, USA) used are shown in Table 1; the primer sets have been reported to be effective in detecting Vibrio parahaemolyticus toxin genes [11,12]. Vibrio parahaemolyticus (ATCC17802) was used as a reference strain in the current study [13]. A total PCR mixture reaction of 25 μL was used following the protocol.

Table 1: Oligonucleotide primers used in PCR reaction and 16S rRNA sequencing.

The reaction mixture was applied with the following cycles: predenaturation at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 30 sec, then annealing at 60°C for 45 sec, and extension at 68°C for 1 min. The final extension was at 72°C for 3 min. The PCR products were separated by agarose gel electrophoresis stained with ethidium bromide, and were visualized and photographed with a UV trans illuminator (SYNGENE, USA). A 100 bp DNA ladder was used as a marker (PROMEGA, USA).

Identification of Vibrio alginolyticus

The amplified PCR products were sequenced by using full-length 16S ribosomal RNA sequences in order to identify the isolated Vibrio strain. The primers used are as shown in Table 1. Sequencing was carried out by First Base Asia Laboratory, Selangor, Malaysia.

Isolation and enumeration of total Vibrio

A total of 2 (40%) out of 3 samples were found to be positive. The thermostable Direct Hemolysin toxin (TDH) is encoded by the tdh gene whereas the TDH-Related Hemolysin toxin (TRH) is encoded by the trh gene. One sample of the Vibrio alginolyticus carried the tdh/ trh toxin, and the two samples carried toxR toxin gene. The colour of the colonies on CHROM TM Vibrio agar was colour less to creamy and light mauve, indicating the existence of a mixed culture. In the first plating, more than 90% of the colonies were colour less to creamy colour, indicating the presence of Vibrio alginolyticus. However, a few colonies were of light mauve colour, indicating the presence of Vibrio parahaemolyticus. Purification was carried out by sub culturing twice. Furthermore, the enrichment was tested on an alternative agar to confirm the results, through plating on TCBS agar at 37C for 18 h. The colonies were largely yellow in colour, indicating the presence of Vibrio alginolyticus. The two samples were isolated and saved in glycerol under -20°C, for further experiment.

Multiplex PCR

The agarose gel electrophoresis of PCR products determined the presence of trh, tdh and toxR genes at bands 484 bp, 251 bp and 368 bp respectively (Figure 1). The results clearly indicate the presence of virulence toxins (trh and tdh) and a regulator toxin (toxR). The PCR method was repeated five times to optimize the reaction conditions. According to the culture morphology and PCR results, it was assumed that Vibrio alginolyticus possessed the toxin genes of Vibrio parahaemolyticus. In order to identify the Vibrio species, we tested a DNA template for both samples by using 16S rRNA 1.5Kb full length sequencing. The sequencing reaction was carried out by the First Base Laboratory, Malaysia. Universal Primers (First Base Laboratories Sdn Bhd) were used as shown in Table 1. At the same time, the PCR products were tested with DNA sequencing to identify the toxin genes. The results revealed a 98% homology to the tdh gene, a 99% homology to the toxR gene, and a 98% homology to the trh gene of Vibrio parahaemolyticus. The trh toxin gene which was isolated showed 98% homology to Vibriostrain Vp 93A-5807 (accession number DQ359748). The Vibrio alginolyticus reported possessed homologues of the virulence gene trh1 [5].

Figure 1: Agarose gel electrophoresis of PCR products, lane M=DNA ladder, lane 1=negative control, lane 2=positive control (Vp. ATCC17802), lane 9, 10, 12, 13,14, 15, 17, 18=negative results, lane 3, 4, 5, 6, 7, 8, 11, 16=toxR positive at band 368bp, lane 6, 7=tdh & trh positive at bands 251bp and 484bp, respectively.

Identification of Vibrio alginolyticus

According to NCBI blast sequence analysis, the Vibriostrain was confirmed and identified by 16S rRNA sequences to be 99% homologically identical to the Vibrio alginolyticus strain ATCC17749 16S ribosomal RNA gene, partial sequence as shown in Figure 2.

Figure 2: Phylogenetic tree-neighbor joining of 16S rRNA sequencing result, ID number (lcl32699) was generated automatically by the NCBI blast tree method.

This study was carried out with the main goal of quantifying pathogenic and non-pathogenic Vibriotoxin factors in fresh shrimps in Selangor, Malaysia. Three toxin genes were determined, which were isolated from live shrimp samples (directly from the tank). When plated on CHROMTM Vibrioagar and incubated overnight at 37°C, it was found that 90% of colonies were white in colour, related to the presence of Vibrio alginolyticus, and a few colonies were light mauve in colour which indicated the presence of Vibrio parahaemolyticus. The results of multiplex PCR confirmed the presence of toxin genes. Vibrio alginolyticus and Vibrio parahaemolyticus are considered as causative organisms of gastrointestinal disease and are both responsible for substantial financial damage in the aquaculture industry [14,2]. Vibrio alginolyticus was found to have a high similarity to Vibrio species [2]. There are many reports that Vibriospecies possess and express Vibrio parahaemolyticus toxin genes, such as V. hollisae which has been reported to be capable of producing TDH toxin [9]. In addition, Vibrio alginolyticus might act as a reservoir of virulence factors from other Vibriospecies [15]. In relation to this hypothesis, researchers involved in the investigations of an outbreak in the USA in 2004 isolated Vibrio alginolyticus expression trh 2 toxin gene [5]. Under starvation, Vibrio alginolyticus and Vibrio parahaemolyticus were reported to exhibit morphological changes and changes in expression of their toxins [16]. Reports of isolating Vibrio alginolyticus carrying trh toxic gene have been increasing in frequency. The first few reports that identified the trh gene in Vibrio alginolyticus occurred in Alaska [5] and Tunisia [17]. In addition, a strain of Vibrio alginolyticus carrying the trh gene has also been reported in Morocco [18] is this all upper case or it starts with an upper case-please standardize!. In this study, Vibrio alginolyticus possessed and expressed the main three pathogenic and non-pathogenic genes (trh, tdh and toxR) of Vibrio parahaemolyticus, isolated from their shrimp sample and this supports our hypothesis mentioned above. However, the detection of these virulence genes, or one of them, in a mixed culture does not always imply that pathogenic Vibrio parahaemolyticus is present [5]. In conclusion, increasing number of reports from various locations around the world on Vibrio alginolyticus expression and possession of virulence genes suggest that this organism might be a reservoir for these genes. To our knowledge, this is the first report indicating that Vibrio alginolyticus possessed and expressed tdh and trh toxin genes of Vibrio parahaemolyticus in an environmental sample. This phenomenon needs further investigation by researchers [19]. Nucleotide sequence accession numbers. The gene toxin sequences have been deposited in GenBank under accession numbers as following:

• toxR gene sequence accession number KP146109

• trh gene sequence accession number KP146110

• tdh gene sequence accession number KP146112

• toxR gene sequence accession number KP146111

The toxR gene, with accession number KP146111, isolated from other samples of Vibrio alginolyticus, was positive to toxR gene toxin and negative to tdh and trh gene toxins [20,21].

This study was funded by the Government of Libya, and laboratory research was funded by the E-Science fund from the Ministry of Science, Technology and Innovation, Malaysia (E-Science 5450683) and the RP 026/2012F grant (under sub-program food security and safety), Asia Africa Development, University of Malaya, Kuala Lumpur and, in part, by the Kakenhi Grant-in-Aid for Scientific Research (KAKENHI 24249038), Japan Society for the Promotion of Sciences and Grant-in-aid of the Ministry of Health, Labour and Welfare, Japan.