Luciano Beneduce, Department of Food Science, University of Foggia, via Napoli 25, 71122 Foggia, Italy. E-mail: email@example.com
Aims: Monitoring the occurrence of the human pathogen Vibrio vulnificus in a mussel farm located in the lagoon of Varano (Italy).
Methods and Results: A total of 72 samples of mussel, water and sediment, collected from two locations of Varano lagoon in the Gargano peninsula, during a 7- month survey, were analysed. Isolation and PCR characterization of six V. vulnificus environmental genotype strains revealed that this pathogen was isolated when with T was above 22°C and salinity ranged between 22·7 and 26·4‰. No significant correlation of the occurrence of V. vulnificus with water pH or salinity was observed. Moreover, 8% of mussel samples were found to be contaminated by V. vulnificus. All of that positive mussel samples originated from the same sampling station.
Conclusion: It is suggested that warmer season are risky to eat raw or undercooked bivalve molluscs in the local area.
Significance and Impact of the Study: To increase knowledge about environmental conditions that may affect the occurrence of waterborne pathogen Vibrio vulnificus in seafood.
The presence of V. vulnificus in water and shellfish is generally not correlated with levels of conventional indicator organisms, which are intended to predict the presence of faecal pollution, whereas Vibrio spp. are normal flora in these waters (Marino et al. 2003). Therefore, sensitive and specific methods for detection of this pathogen are extremely important for the protection of human health (Mattéet al. 1994; Sunén et al. 1995; Harwood et al. 2004; Oliver et al., 2006b). The observation that V. vulnificus could enter into a viable but nonculturable (VBNC) state when exposed to cold temperatures (Whitesides and Oliver 1997; Kong et al. 2004; Oliver 2009) further complicates the epidemiological and ecological considerations for this organism, including its isolation from environmental samples. Molecular techniques, such as the polymerase chain reaction (PCR), may provide the sensitivity and specificity necessary to accurately detect low numbers of V. vulnificus. Probes based on the cytolysin–haemolysin gene (vvhA) of V. vulnificus are able to identify presumptive V. vulnificus recovered from environmental samples (Morris et al. 1987). In this study, the occurrence of V. vulnificus in a mussel farming area in a coastal lagoon in Italy was studied by conventional cultural methods and by combining a culture-based approach with a DNA-based technique (PCR).
Materials and methods
Samples were taken from two stations located in the Varano lagoon, located in the North of the Gargano peninsula in the Province of Foggia (Fig. 1a). The lagoon has a total surface area of c. 65 km2 and is separated from the Adriatic Sea by a narrow sand dune. The average depth is 5·5 m, and two channels allow the lagoon communication with the sea. Several freshwater inputs are provided by submarine springs; these characteristically make the lagoon an interesting environment for micro- and macro-biota, because of its high variability in salinity and nutrient input. The lagoon is intensively used for farming of mussels and other aquaculture products. In the present work, two sampling points were selected, the first close to the sand dune that splits the lagoon from the sea and the second c. 6 km from the coast (Fig. 1b).
Sample collection and preparation
A total of 72 samples (24 each of mussels, sediment and water) were analysed. Samples were taken from the sampling sites monthly between May and November. Each mussel sample was comprised of eight individuals, taken from three different depths. The mussels were placed in sterile stomacher plastic bags for transportation. Water samples were aseptically collected in presterilized 1·0-l flasks at a 0·5 m depth in proximity to the mussel implants. Sediment samples were collected from the top 50 cm. All samples were transported in refrigerated containers and analysed within 2 h of sampling.
Chemical and physical assays
At each sampling time, temperature, pH and salinity (S‰) were measured in situ using a portable YSI probe model 6920 equipped with a model 650 multi-probe system.
Each mussel was rinsed in sterile distilled water to remove any debris from the shell then aseptically opened. The contents of each mussel sample (flesh and intervalve liquid) were mashed in a stomacher (Stomacher Lab-Blender 400, PBI, Italy) in the presence of the diluent for 5 min. Twenty-five grams of mashed mussel sample were suspended in 225 ml of alkaline peptone water (1% NaCl + 1% peptone; Oxoid, Basingstoke, UK), and serial dilutions prepared in the same diluent. Sediment samples were processed in the same manner.
Seawater samples (1·0, 10·0 and 100·0 ml) were filtered through 0·2- μm pore size, sterile membrane filters that were directly placed on the surface of culture media plates.
The diluted samples (mussels and sediment) were analysed by the spread-plate technique for total Vibrio spp. counts on thiosulfate-citrate-bile salts-sucrose agar (TCBS agar; Oxoid) and incubated at 37°C for 24 h. Total marine bacteria were enumerated by spreading on nonselective marine agar (meat extract 1%, peptone 1%, agar 1%, sterilized sea water 750 ml l−1) and then incubating at 25°C for 24–48 h. Membrane filters from water samples were incubated on the same media and at the same condition as described earlier for mussels and sediment.
The isolation of V. vulnificus strains was conducted following a pre-enrichment step of mashed mussels and sediment (and filter membranes from 100 ml filtered seawater samples) for 12 h in alkaline peptone water and then spreading 100, 10 and 1 μl of enrichment broth onto cellobiose-polymyxin B-colistin (CPC) agar plates (Fluka Biochemika, Seinheim, Germany), following the instructions reported by Massad and Oliver (1987). Plates were then incubated at 40°C for 24 h. Characteristic yellow and green colonies on TCBS agar were considered to be Vibrio spp., and yellow-orange colonies on CPC agar, with 1–5 mm diameter and a darker centre, were considered presumptive V. vulnificus. Five colonies from each CPC agar plate were picked and further analysed by PCR. A total of 84 bacterial colonies were tested in this manner.
Molecular identification of Vibrio vulnificus strains
Genomic DNA of presumptive V. vulnificus colonies was extracted using the Utlraclean Microbial DNA extraction kit (Mo-bio, Carlsbrand, CA, USA), according to manufacturer’s instructions.
The vvhAF and vvhAR primers described by Brauns et al. (1991; Table 1), which produce a 340- bp fragment, were used to confirm the identification of V. vulnificus isolates. For these studies, about 100 ng of genomic DNA was added to a 50 μl PCR mixture containing 1·25 U Taq polymerase (Qiagen, Milan, Italy), 0·2 mmol l−1 of each dATP, dTTP, dGTP, dCTP, 10 mmol l−1 Tris–HCl pH 8·3, 50 mmol l−1 KCl, 1·5 mmol l−1 MgCl2 and 0·4 μmol l−1 of primers targeting the vvhA gene. The reaction mix was cycled through the following temperature profile: denaturation at 95°C for 3 min, 30 cycles of 95°C for 1 min, 55°C for 40 s and 72°C for 40 s. The PCR was terminated at 72°C for 5 min and thereafter cooled at 4°C.
Strains confirmed by PCR as V. vulnificus were further characterized by a second PCR test that allows differentiation between strains having the ‘environmental’ (E) or ‘clinical’ genotype (Rosche et al. 2005, 2010). Primers P1–P3 and P1–P2 (Table 1) were used to identify clinical and environmental strains, respectively. These primers result in a 277- bp amplicon with only a single primer pair, providing confirmation that the strain was C- or E-genotype. The amplification protocol used was that reported by Rosche et al. (2005).
The bacterial concentrations were log transformed and statistical analysis was performed using StatsofT Statistica version 6.0 (Statsoft, Tulsa, OK, USA).
Seawater temperature patterns during sampling (Fig. 2) were almost identical for the two sampling stations, varying from 10·5 (November) to 30·0°C (July and August). Salinity ranged from 21·7 to 28·5‰ (Fig. 2) and pH from 8·0 to 8·2 (data not shown).
Figure 3(a,b) reports viable cell counts of total marine micro-organisms and Vibrio spp., respectively. In general, total marine counts were higher in sediment and mussels taken from Station 2 than from Station 1, while water from both stations had almost the same counts with the exception of August and September, when significantly higher values were seen at Station 2. As expected, total marine counts were significantly higher for mussels than for water and sediments for both sampling stations. In contrast, Vibrio spp. counts showed similar values in water and sediment samples from the two stations, while Vibrio spp. counts from mussel samples were higher at Station 2 than at Station 1.
No correlation was observed between the total marine counts and total Vibrio counts. Moreover, the highest Vibrio spp. counts were found in July, with subsequent decreases until November, whereas total marine counts increased from August to October.
A total of 84 presumptive V. vulnificus, isolated on CPC agar after enrichment in alkaline peptone water, were obtained during our 7-month study. Of these, 46 strains were isolated from water, 24 from mussels and 14 from sediments samples.
Molecular identification by PCR analysis
Of the 84 presumptive V. vulnificus colonies, six were confirmed to be V. vulnificus by PCR analysis (Table 2). All of these were found to be of the environmental genotype. The six V. vulnificus strains were isolated from mussels (four isolates) and water (two isolates), with none found in sediment samples. Interestingly, all six confirmed strains were found in samples from Station 1. The six isolates were detected in June and July when temperatures ranged from 22 to 29°C and salinities between 22·7 and 26·4‰. In one case (June), V. vulnificus was detected both in water and mussel samples. In total, 8% of the mussels examined were positive for V. vulnificus.
Table 2. PCR identification and characterization of putative Vibrio vulnificus strain isolated in the survey
V. vulnificus isolated strains
E/C type PCR†
*Species specific for identification of V. vulnificus.
†E, environmental type strain; C, clinical type strain.
‡All PCR positive strains were isolated from Station 1 survey.
Numbers in bold represent samples identified as V. vulnificus by species specific PCR.
Before this study, few reports have been published on the presence of V. vulnificus associated with seawater, mussels or sediment samples in Italy and none on the molecular characterization of V. vulnificus in the Puglia region.
In our study, V. vulnificus was only detected when water temperatures were high, consistent with numerous studies on this aspect (Kaspar and Tamplin 1993; Pfeffer et al. 2003; Maugeri et al. 2006). Because the abundance of V. vulnificus in coastal environments is linked to water temperature, the reported risk of food poisoning associated with the consumption of vibrio-contaminated seafood is higher during this period (Baffone et al., 2000; Wong et al. 2004). That V. vulnificus is rarely isolated from cold waters may be because of the fact that V. vulnificus is known to enter into a viable but nonculturable (VBNC) state at low temperatures (Oliver 1995).
While the isolation of V. vulnificus from the mussel samples was low, the presence of this pathogenic species represents a potential health hazard for individuals who consume raw or undercooked mussels. The risk of V. vulnificus infection is especially high during the spring and summer months, considering that a large number of mussels are harvested and eaten from the Varano lagoon during that period.
In agreement with other researchers (Urdaci et al. 1988; Schintu et al. 1994; Baffone et al., 2000; Huss et al. 2000), we believe that to reduce the incidence of foodborne diseases because of the consumption of bivalve molluscs, it is advisable to inform consumers about the health hazards associated with the consumption of these products, either raw or undercooked.
The authors are grateful to Dr Sergio Pelosi and Dr Matteo Francavilla at ISMAR CNR, Lesina (Italy) for their logistic support and scientific advice. This work was partially supported by the ex 60% PAR programme at Foggia University.