Biomolecular identification of fish species by PCR and analysis of microbiological risk linked to the consumption of ready to eat fishery products

The present study regarded the isolation and the characterisation of Staphylococcus aureus and Listeria monocytogenes from ready to eat (RTE) fishery products and the development and the improvement of novel PCR protocols for the identification of fish species. For the detection of S. aureus and L. monocytogenes, 99 and 135 RTE samples, respectively, were collected at local retail outlets and analysed according to ISO procedures in the laboratories of Food Microbiology of Experimental Zooprophylactic Institute of Apulia and Basilicata located in Foggia. RTE fishery products for the isolation of S. aureus and L. monocytogenes consisted of 33 and 45 samples of marinated anchovies fillets (Engraulis encrasicolus), 33 and 45 samples of smoked salmon (Salmo salar), 33 and 45 samples of seafood salad, respectively. As regards the identification of fish species, novel species-specific primers were developed by the program "Primer Express 3.0" and by the software “Primer-BLAST” to amplify fragments of 200 bp, 250 bp, 300 and 562 bp, 350 bp, 400 bp and 522 bp within COI gene for Merluccius merluccius, Lates niloticus, Gadus morhua, Ruvettus pretiosus, Pangasius hypophthalmus, Epinephelus spp., respectively. Ten samples of each fish species of interest were obtained from wholesale fishery plants. DNA was extracted from individual sample and quantified. DNA isolates were subjected to end-point PCR analysis and PCR products were sequenced. Out of 33 samples of smoked salmon, S. aureus was isolated from one sample (3.03%). The S. aureus strains carried the icaA, seb and sec genes and were resistant to ampicillin and tetracycline. L. monocytogenes was isolated from 2 of 45 samples of smoked salmon (4.44%). The strains of L. monocytogenes, isolated from both samples, resulted to belong to the serovar 1/2a and to be susceptible to all antibiotics tested. Single PCRs were performed using DNA isolates and the developed primers for each fish species of interest. After sequencing, the isolates were compared with the selected sequences of COI gene and showed a similarity ranging from 99 to 100%. Duplex and Triplex PCR protocols were developed for the simultaneous analysis of more fish species using the designed primers with several combinations. In addition, a survey on fish products was carried out to evaluate the application of labelling laws and to detect fraudulent actions using the developed PCR protocols. Forty-three fishery products were collected, in particular 18 and 25 samples at hypermarket stores, and at local fisheries and fish marketplaces, respectively. Fishery products purchased at local fisheries and fish marketplaces consisted of 20 fish fillets and 5 fish slices. After PCR analysis and sequencing, 19 (44.2%) resulted mislabelled, with 18 (41.9%) mislabelled samples from local fisheries and fish marketplaces and 1 (2.32%) from hypermarket stores. As regards fish samples purchased at local fisheries and fish marketplaces, fraudulent actions regarded more fish slices (100%) than fish fillets (65%). Regarding fish fillets, 3 out of four samples labelled as grouper, three (75%) resulted to be Lates niloticus and one (25%) Pangasianodon hypophthalmus. Two fillets marketed as cod (100%) were substituted with Lates niloticus (100%). Five samples labelled as “fillet” and two samples labelled as “perch” were identified as P. hypophthalmus. As regards fish slices, all samples marketed as grouper slices (E. marginatus) were slices of Ruvettus pretiosus (100%). The single case of mislabelling detected from fishery products purchased at hypermarket stores regarded a sample of “Spinycheek grouper” (Epinephelus diacanthus) that was indicated on label as “Grouper” (Epinephelus marginatus). In conclusion, our work highlights the need of a continuous surveillance on the commercialisation of fishery products, in order to reduce the food-borne risk linked to the presence of S. aureus and L. monocytogenes in RTE fishery products. Furthermore, our protocols based on PCR techniques could be useful for quality controls of fresh finfish and to strengthen controls on the most frequent fraudulent actions of marketed fishery products.