Molecular characterization, antibiotic resistance pattern and biofilm formation of vibrio parahaemolyticus isolated from mussels and clams in the Marmara, aegean and black sea

In recent years, with the change in consumer habits in Turkey, a great increase is observed in the consumption amounts of other seafood other than fish, especially bivalve seafood. In addition to its nutritional value, bivalves are also very important for public health by being included in the foods associated with epidemics. Mussels are considered as potential sources of contamination and pose a risk to consumers as a reservoir for Vibrio parahaemolyticus, which is considered to be the leading cause of seafood-borne gastroenteritis in the world. In this study, 135 mussels (Mytilus galloprovincialis) and 120 clams from different species (Chamelea gallina, Ruditapes philippinarum and Donax trunculus) were collected from Aegean, Marmara and Black Sea between May 2021 and May 2022. The culture method according to ISO 21872-1:2017 standard was used for isolation and identification of V. parahaemolyticus, confirmation of V. parahaemolyticus in suspected isolates was conducted by PCR based on the toxR gene. Molecular characterization of confirmed V. parahaemolyticus isolates was performed by determining the presence of the main virulence genes (tdh and trh). Antimicrobial resistance profiles of the isolates were determined by the Kirby-Bauer disc diffusion method, and then the isolates selected from pathogenic and non-pathogenic strains were incubated at 3 different temperatures (10°C, 25°C and 37°C) and 2 different incubation time (24 and 48 hours) to investigate the biofilm formation capacity. According to the results, 32.2% of the all samples were confirmed as V. parahaemolyticus, and 30.8% of them were from clams; 33.3% from mussels. The incidence of the isolates was the Black Sea (52.5%), the Marmara Sea (34.8%) and the Aegean Sea (3.6%), respectively, and the difference between the seas was statistically significant (p<0.05). It was determined that the highest positivity rate in the results according to the seasons was in the samples collected in the summer season (60%), while the spring samples had the lowest rate (15.9%). 5.9% of the 255 samples collected were identified as pathogens. 73.3% of the pathogen isolates were from clams; 26.7% were from mussel, and the difference between mussels in terms of pathogen isolate was statistically significant (p<0.05). It is noteworthy that 60% of the pathogen isolates were from the Black Sea and 40% from the Marmara Sea, and no pathogen isolates were found in the Aegean Sea. In addition, it was determined that most of the pathogen isolates (46.7%) were isolated in winter. The antimicrobial resistance profiles of a total of 82 V. parahaemolyticus strains isolated from mussel samples were investigated, and the mean MAR index value was determined as 0.67. A high risk of contamination in Turkish waters was shown by detecting multi-antibiotic resistance. Two of the isolates were resistant to all nine antibiotics tested and all isolates were resistant to at least three antibiotic groups. In addition, all isolates (100%) were resistant to piperacillin and tetracycline. Any difference could not be determined between pathogenic and non-pathogenic strains in their biofilm forming capacities. It was determined that the isolates formed better biofilms at a 48-hour incubation period than at a 24-hour incubation period, and at 25°C compared to 10°C and 37°C temperatures. While strong biofilm formation was not observed in any of the strains, moderate biofilm formation was observed only in strains isolated from clams (7.1%). 103 Determining the prevalence of Vibrio species, which is one of the most important causes of seafood-borne gastroenteritis cases, and the presence of pathogenic strains is the first step to develop treatment, protection and control strategies for these cases. This study is important as it is the first comprehensive study in Turkey to determine the presence of Vibrio parahaemolyticus and pathogen strains in mussels and clams obtained from three different seas of Turkey (Aegean, Marmara and Black Sea), to reveal antibiotic resistance profiles in mussels and clams samples, and to investigate the biofilm forming capacities of strains.