Occurrence of Vibrio , Salmonella and Staphylococcus aureus in retail fresh fish , mussel and shrimp

This study aimed to determine the presence of pathogenic Vibrio spp., S. aureus and Salmonella in 100 seafood samples purchased from retail outlets in Bursa city (Turkey). Of the samples examined including fish, mussel and shrimp, 67% were found to be contaminated with Vibrio. Presumed Vibrio spp. were identified by standard biochemical tests, and further confirmed by API 20E system. Identified Vibrio spp. were V. parahaemolyticus (28%), V. vulnificus (1%) and V. cholerae (1%), with the most prevalent being V. alginolyticus (37%). Six (6%) of the samples analysed were positive for S. aureus. However, no contamination of the samples with Salmonella was observed. Our results showed that seafood from retail outlets can be a likely vehicle for infections with Vibrio spp. and S. aureus. Seafood, pathogen bacteria, incidence, microbiological quality In addition to being a healthy food with nutritional value, seafood can also act as a source of foodborne pathogens (Hudecová et al. 2010; Bakr et al. 2011). Various outbreaks of bacterial disease associated with the consumption of seafood have been reported (Guerin et al. 2004; Friesema et al. 2012). From these seafood-borne bacteria, Vibrio spp. are Gram-negative rod-shaped and halophilic bacteria that generally widespread in the coastal and estuarine environments (Austin 2010). Among more than 20 Vibrio species known to be associated with human disease, V. cholerae, V. parahaemolyticus and V. vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health (Gopal et al. 2005; Cariani et al. 2012). Salmonella and S. aureus are the leading foodborne pathogens, causative agents of the most common enteric infections to humans (Lei et al. 2008; Kumar et al. 2009). These bacteria can enter the aquatic environment through wild animals, domestic stock, poor sanitation and inappropriate disposal of human and animal wastes (Amagliani et al. 2012). Various authors have reported the incidence of Salmonella (Woodring et al. 2012) and enterotoxigenic S. aureus (Popovic et al. 2010) in seafood. In this study, we investigated the prevalence of pathogenic Vibrio spp., Salmonella and S. aureus contamination in fish, mussel and shrimp from the retail level in order to assess health risks for consumers, and also determined the identification to species level of the Vibrio strains isolated from samples. Materials and Methods

In addition to being a healthy food with nutritional value, seafood can also act as a source of foodborne pathogens (Hudecová et al. 2010;Bakr et al. 2011).Various outbreaks of bacterial disease associated with the consumption of seafood have been reported (Guerin et al. 2004;Friesema et al. 2012).From these seafood-borne bacteria, Vibrio spp.are Gram-negative rod-shaped and halophilic bacteria that generally widespread in the coastal and estuarine environments (Austin 2010).Among more than 20 Vibrio species known to be associated with human disease, V. cholerae, V. parahaemolyticus and V. vulnificus are the pathogenic species of Vibrio that pose the greatest threat to human health (Gopal et al. 2005;Cariani et al. 2012).
Salmonella and S. aureus are the leading foodborne pathogens, causative agents of the most common enteric infections to humans (Lei et al. 2008;Kumar et al. 2009).These bacteria can enter the aquatic environment through wild animals, domestic stock, poor sanitation and inappropriate disposal of human and animal wastes (Amagliani et al. 2012).Various authors have reported the incidence of Salmonella (Woodring et al. 2012) and enterotoxigenic S. aureus (Popovic et al. 2010) in seafood.
In this study, we investigated the prevalence of pathogenic Vibrio spp., Salmonella and S. aureus contamination in fish, mussel and shrimp from the retail level in order to assess health risks for consumers, and also determined the identification to species level of the Vibrio strains isolated from samples.

Sample collection
During 2012, a total of 100 fresh seafood samples including 78 fish, 12 mussel and 10 shrimp were collected from several supermarkets, fish markets and neighbourhood bazaars in Bursa province, Turkey.The samples were placed into sterile bags and transported to the laboratory under refrigerated conditions for analysis.

Isolation and identification
The detection of pathogenic Vibrio species was achieved according to the standard methods of the U.S. Food and Drug Administration (FDA) (DePaola and Kaysner 2004).Approximately 25 g of each sample were homogenized in 225 ml Alkaline Peptone Water (APW) (Merck 1.01800) with 2% NaCl and incubated overnight at 35 ± 2 ºC.A loop of the pre-enriched culture was streaked onto Thiosulphate Citrate Bile Salts Sucrose (TCBS) agar (Merck 1.10263).After incubation at 35 ± 2 ºC for 18-24 h, presumptive colonies were firstly characterized by biochemical tests which included Gram staining, motility, oxidase production, 0129 vibriostat susceptibility (Oxoid DD0015), and then identified at the genus and species level using API 20E (BioMerieux, France) test kits.
For the detection of S. aureus, 10 g seafood samples were homogenized with 90 ml of a sterile 0.1% peptone water solution.Serial dilutions of the homogenate were made with sterile peptone water and plated in duplicates on Baird Parker agar (Merck 1.05406) supplemented with egg yolk-tellurite emulsion (Merck 1.03785).After incubation at 35 ºC for 48 h, the typical black with clear halo colonies of S. aureus were tested for coagulase activity using Dry Spot Staphytect Plus (Oxoid DR0100) (Harrigan 1998).

Results
This study was conducted to determine the incidence of Vibrio spp., S. aureus and Salmonella spp. in seafood (78 fish, 12 mussel and 10 shrimp) for sale in retail outlets.The results related to the incidence and identification at the species level of Vibrio spp.are summarized in Table 1.Sixty-seven (67%) out of 100 samples were found to be contaminated with Vibrio spp.The isolates were identified as V. vulnificus (one isolate), V. cholerae (one isolate), V. parahaemolyticus (28 isolates) and V. alginolyticus (37 isolates) using API 20E system.V. vulnificus was isolated from one fish sample while V. cholerae was detected in one shrimp sample.Among 28 V. parahaemolyticus isolates, 25 were found in fish samples and three in mussels.As seen in Table 2, 6 (6%) from 100 seafood samples were contaminated with S. aureus.Salmonella was not detected in the samples analysed in this study.

Discussion
Vibrio spp. was isolated in 67% of total count of the samples.By API 20E system, the isolates were identified as V. vulnificus, V. cholerae, V. parahaemolyticus and V. alginolyticus.The total rate of isolation of V. parahaemolyticus in the samples analysed was 28% (Table 1).In comparison to the results of our study, previous studies with seafood from several countries demonstrated higher prevalence of V. parahaemolyticus.In a study on seafood products in China (Chao et al. 2009), the incidence of this bacterium was found to be 56.6%.The rates of V. parahaemolyticus contamination of seafood by Chakraborty et al. (2008) in India and by Costa Sobrinho et al. (2011) in Brazil were reported as 64% and 100%, respectively.However, the incidence (28%) of V. parahaemolyticus in retail seafood reported here was relatively higher than that reported by Baffone et al. (2000) (14.8%) and by Ottaviani et al. (2005) (24.3%).
The single isolate of V. vulnificus in our study originated from fish.Canigral et al. ( 2010) also reported that this bacterium was recovered from two oyster samples in Spain.Studies conducted by J i et al. ( 2011) suggested that 140 out of 239 shrimp samples from Chinese seafood markets were contaminated with V. vulnificus.Vibrio cholerae, the causative agent of cholera in humans, was detected in only one shrimp sample (1%).Higher incidence rate (3.7%) of V. cholerae in seafood products was suggested in a study conducted by Baffone et al. (2000).On the other hand, V. cholerae was not isolated from any of the shrimp samples investigated by Hosseini et al. (2004).
Although V. parahaemolyticus, V. cholerae and V. vulnificus are the main species associated with seafood-borne infections, others including V. alginolyticus, V. mimicus and Grimontia hollisae (formerly V. hollisae) have been sporadically found in outbreaks (Cariani et al. 2012).In this study, V. alginolyticus was detected in 27 fish, 6 mussel and 4 shrimp samples, with a total incidence rate of 37%.In a study on fish, mussels and clams in Italy (Baffone et al. 2000), 81.5% of the samples were positive for V. alginolyticus.In India, an investigation showed that V. alginolyticus was present in 19% of 30 west coast shrimp samples analysed (Gopal et al. 2005).
Our study revealed that of 100 seafood products, six (6%) samples harboured S. aureus.Three isolates of S. aureus were obtained from fish samples, one isolate from mussels and two isolates from shrimp (Table 2).Five of the isolates were found to be coagulase positive except for one isolate from mussels.Much higher incidence rates of S. aureus were reported for fresh (43%) and frozen (30%) fishery products in Spain (Vazquez-Sanchez et al. 2012), and for low salt sardine (86.7%), feseikh (93.3%) and molouha (90%) fish samples in Egypt (Ezzeldeen et al. 2011).
Salmonella was not detected in the samples analysed in this study, which was in agreement with previous studies (Popovic et al. 2010;Rodriguez et al. 2011) in seafood products.Conversely, different workers (Shabarinath et al. 2007;Kumar et al. 2009;Bakr et al. 2011) reported varying incidence rates of Salmonella in seafood.
In summary, this work revealed the presence of potentially pathogenic bacteria in seafood including fish, mussel and shrimp, and a probable health risk for consumers of raw seafood.The data presented here are useful for risk assessment and management of pathogenic Vibrio spp.and S. aureus in seafood.Improvement of the effective sanitary conditions in handling and processing operations from fishing to marketing is needed to minimize the risk of infections associated with consumption of these products.

Table 1 .
Incidence and species distribution of Vibrio spp.from seafood.

Table 2 .
Incidence of S. aureus and Salmonella in seafood.