Distribution of Putative Virulence Genes in Aeromonas hydrophila and Aeromonas salmonicida Isolated from Marketed Fish Samples

MATERIALS AND METHODS

Isolation and Identification of Bacterial Isolates
Presumptive Aeromonas sp. cultures were isolated from fish samples gathered from various vendors of fish markets situated in Vadapalani, Chennai, India during August 2006 to February 2007. The edible flesh portion of the fishes (10 g) were macerated and transferred into 10 mL sterile distilled water and kept it for shaking incubation at 37°C for 15 min. After incubation, 1 mL of suspension was transferred to Alkaline Peptone Water (APW) and incubated at 37°C for 18 h. Bacterial colonies were grown on Starch-Ampicillin (SA) agar plates (Palumbo et al., 1985) and Gram-negative, oxidase positive and catalase-positive isolates were plated out onto selective Rimler Shotts (RS) agar medium (HiMedia, India) and yellow-to honey-coloured colonies were taken for confirmation analysis by using Kaper’s multitest medium. Appearance of alkaline surface and acid butt after 24 h at 37°C demonstrated the presence of A. hydrophila, whereas black butt exhibited the presence of A. salmonicida. The presumptive identification was performed by oxidase reaction, fermentation of glucose, presence of lysine, arginine decarboxylation and absence of ornithine decarboxylase, proposed by Cowan and Steel (1970).

Detection of Virulence Genes in A. salmonicida and A. hydrophila Primers
Unique primers were designed for the amplification of the genes coding for the cytotoxic virulence factor of Aeromonas isolated from fish, shrimps and water sources. All the available partial and full-length gene sequences for a given virulence factor was determined according to Sen and Rodgers (2004). hlyA gene primer F1 (5’-GGC CGG TGG CCC GAA GAT GCA GG-3’) and R1 (5’-GGC GGC GCC GGA CGA GAC GGG-3’) to amplify a 597 bp; aerA gene primer F1(5’-GCC TGA GCG AGA AGG T-3’) and R1 (5’-CAG TCC CAC CCA CTT C-3’) to amplify a 416 bp. Bin-Kingombe et al. (1999) also suggested the above primers.

PCR Analysis
Reactions were performed in 25 μL volumes in 0-2 mL optical-grade PCR tubes (Tarsons, India). Each 25 μL of reaction mix contained 1 μM of each primer, 12.5 μL of Taq PCR Master mix (2X) containing, MgCl₂, Taq DNA polymerase and dNTPs (Genei, India). The amount of template used was 80 ng in 5 μL volume. Cycling conditions consisted of an initial single cycle at 95°C for 5 min, followed by 25 cycles of melting for 25 sec at 9524°C, annealing for 30 s at 55°C, elongation for 1 min at 72°C and a final single cycle at 70°C for 5 min. PCR was performed in PCR thermalcycler (MJ Research Model PTC 150 MiniCycler, Watertown, Mass, USA). Each DNA extract was first evaluated with a single primer set. The assay was then made multiplex by including the second primer set. Before performing PCR on the water isolates, each primer set was tested with MTCC control strains to confirm the production of an amplicon of predicted size.

Post-PCR Analysis
The PCR products were detected by subjecting a sample from each reaction tube to 2% agarose gel electrophoresis stained with ethidium bromide (1.6 mg mL^-1) at 11 V cm 1 for 90 min in TBE buffer (0.089 M Tris, 0.089 M boric acid, 0.002 M EDTA (pH 8). Representative PCR amplicons from each primer set were purified by QIAquick PCR purification kit (Qiagen). The sequences of the amplicons were determined in order to confirm the successful amplification of the target genes. Sizes of the amplified products were determined by comparison with DNA ladder (3000-100 bp, Genei, India).

RESULTS

Incidence of A. salmonicida and A. hydrophila
In this study, occurrence of A. salmonicida and A. hydrophila in fish were procured from marketed fish specimen. A total of 32 fish samples, 46.87% of fishes were contaminated with A. salmonicida and A. hydrophila (Table 1). It was the significant level of incidence in the sampling site.

Table 1:	Incidence of A. salmonicida and A. hydrophila from fish samples

Table 2:	Biochemical identification of A. salmonicida and A. hydrophila
+: Positive; -: Negative; F: Fermentative; A: Acid; G: Gas

About 31.2 and 15.6% of fishes were found to be A. salmonicida and A. hydrophila, respectively. The fish isolates were identified by using selective identification tests and biochemical tests. The results were presented in Table 2. A. salmonicida and A. hydrophila has been differentiated easily by Kaper’s multitest medium.

Identification of Virulence Gene of A. hydrophila
The results of the present study haemolysin was encoded by the hlyA and aerA genes which were observed in the fish isolates of A. hydrophila and A. salmonicida. However, this study targeted and aligned the gene of interest was hlyA and aerA genes from fish isolates. Most of the A. hydrophila found conserved region for hlyA gene. From these conserved sequences, the respective primers were designed and directed the amplification of 597 bp fragment. Of the 15 samples, presence of hlyA gene in 73.3% of the haemolysin producers (Fig. 1). However, hlyA gene was not available in whereas strain As 5, As 8, Ah 2 and Ah 5. These might have not produce the haemolysin on blood agar plate and 416 bp responsible for the production of aerolysin (Fig. 2). In all other samples has to be homology among the strains except the strain As 8 and Ah 2. This was demonstrated by a tendency for isolates carrying variable hlyA and aerA genotypes (hlyA⁺ aerA⁺, hlyA¯ aerA⁺, hlyA^- aerA¯ and hlyA⁺ aerA¯) available in all the isolates.

Fig. 1:	Haemolysin (hlyA) gene of A. salmonicida and A. hydrophila isolated from fish; Lane 1-15: Strain As 1-10, Lane 11-15: Strains Ah 1-5, Lane M. DNA Marker (3000-100 bp)

Fig. 2:	Aerolysin (aerA) gene of A. salmonicida and A. hydrophila from fish; Lane 1-15: Strain As 1-10, Lane 11-15: Strains As 1-5, Lane M: DNA Marker (3000-100 bp)

DISCUSSION

Incidence of A. salmonicida and A. hydrophila in Fish
Fishery products, which are of great importance for human nutrition worldwide and provide clear health benefits (Darlington and Stone, 2001), can also act as a source of foodborne pathogens Overall, the majority of reported seafood-associated outbreaks are caused by toxins (biotoxins and histamine) and viruses (noroviruses and hepatitis A virus) but fish and shellfish may also be a vehicle for pathogenic bacteria naturally occurring in aquatic environments, referred to as indigenous, or derived from polluted waters and/or from post-capture contamination (Huss et al., 2003). In the present investigation, a significant level of incidence of A. salmonicida and A. hydrophila were recorded in the fish samples. During the period of sampling we have observed higher (46.8%) contaminations in the marketed fish this may due to improper handling of fish, transportation of seafoods from the catching area. About 31.2 and 15.6% of fishes were found to be A. salmonicida and A. hydrophila, respectively.

The incidence of A. salmonicida and A. hydrophila in fish sold in retail outlets of various parts of the world has been well documented, but distribution of this pathogen in various fishes from retail markets was not well elaborated so far. The result of the present study expressed that the contamination of seafoods with Aeromonas in vadapalani market was found to be considerably higher than the previous reports. Although the source of the organisms may be ambient environment, secondary contamination due to catching, transportation, handling and etc. may also contribute for its distribution. Moreover, days of storage and processing of seafood in retail outlets may cause variations in the level of incidence of A. hydrophila. This may be the possible reason for the increased prevalence of Aeromonas observed in fish markets though all the fishes available in the market has the same source of origin. Because of obvious differences in sampling period, geographical location, origin of the samples and methodology for analysis it is difficult to compare the level of incidence.

Virulence Gene of A. salmonicida and A. hydrophila
Nucleic acid amplification methods targeting virulence genes are used for detection of pathogenic bacteria and to differentiate pathogenic from non-pathogenic strains (Bin-Kingombe et al., 1999; Sen and Rodgers, 2004). Wang et al. (2003) developed a multiplex PCR method for detection of hemolysin and aerolysin genes in A. hydrophila and A. sobria and used it to screen 121 clinical and 7 reference strains for ahh1, asa1 and aerA genes. Several authors detected the aerolysin gene coding for the β-hemolysin in A. salmonicida and A. hydrophila (Pollard et al., 1990). Castro-Escarpulli et al. (2003) reported that 96% of strains were positive for aerolysin/haemolysin genes and the strains were also found to the hemolytic. Various putative virulence fishes have been described to A. hydrophila to explain the gene responsible for their pathogenicity. A number of studies have shown that haemolytic factors are involved in the virulence of aeromonads. Two haemolytic toxins have been described in A. hydrophila: the AHH1 haemolysin (Hirono and Aoki, 1991) and aerolysin (Howard et al., 1987). These haemolytic toxins have only 18% homology and are distinct (Hirono and Aoki, 1991). Aerolysin is the best studied haemolysins (Howard et al., 1987), but Aeromonas strains can produce more than one haemolytic toxin with virulence properties (Chopra et al., 1991; Wong et al., 1998). Most of the A. hydrophila found conserved region for hlyA and aerA gene responsible for the production of haemolysin. From these conserved sequences, the respective primers (hlyA and aerA) were designed and directed the amplification of 597 and 416 bp fragments.

The aerA gene was wide spread in A. veronii biotype sobria and A. hydrophila but less so in A. caviae. In contrast, the studies of Pollard et al. (1990), showed that the aerA was only detected in hemolytic, cytotoxic and entereotoxic strains of A. hydrophila but not in A. veronii biotype sobria and A. caviae. Since previous studies (Wong et al., 1998), have suggested that the combined effect of aerolysin (aerA) and Vibrio cholerae. HlyA-like haemolysin (hlyA) contributes to virulence is A. hydrophila a different approach for the identification of potentially pathogenic Aeromonas isolates is the PCR detection of the genes for the haemolysins (aerA and hlyA) (Pollard et al., 1990; Heuzenroeder et al., 1999).

In this study haemolysin (hlyA) and aerolysin (aerA) were encoded by the hlyA and aerA gene, respectively, included multifactorial activities. However, this study targeted and aligned the gene of interest was hlyA and aerA genes from A. hydrophila isolated from fishes. However, this study targeted and aligned the gene of interest was hlyA genes from A. hydrophila and A. salmonicida isolated from fishes. Aerolysin is synthesized as a high molecular weight precursor, which crosses the inner bacterial membrane as a preprotoxin containing a signal sequence which is removed cotranslationally (Howard and Buckley, 1985). Most of the A. hydrophila found conserved region for hly A gene. From these conserved sequences, the respective primers (hlyA) were designed and directed the amplification of 597 bp fragment. The range of virulence of aeromonads is thought to result from the variety of genotypes (hlyA⁺ aerA⁺, hlyA^¯ aerA⁺, hlyA¯ aerA^- and hlyA⁺ aerA¯) present in the environment. Both phenotypic and genotypic heterogeneity are common among aeromonads.

ACKNOWLEDGMENT

The author is very thankful to Life Tech Research Centre, Vadapalani, Chennai-600 026 India for providing the necessary facilities.