Horizontal gene transfer in the marine environment: non pathogenic vibrio strains carrying virulence and antibiotic resistant genes and constituting a potential risk for human health

Environmental bacterial strains belonging to non pathogenic Vibrio species have been considered as deprived of pathogenic potential and not able of causing infectious diseases. In recent years, however, a series of infection cases due to non pathogenic vibrios have been reported in persons swimming in European areas. Moreover, indications that horizontal gene transfer may occur in the aquatic environment and few reports on the presence of a number of virulence genes in non pathogenic bacteria, is changing the vision of these microorganisms as potential risk for public health. A number of studies indicate that genes involved in pathogenicity, ecological fitness and antibiotic resistance can be mobilized in extra-chromosomal elements or in pathogenicity islands thus having the possibility of mix or recombine in the aquatic environment and to originate new strains with increased fitness and virulence potential. Horizontal gene transfer (HGT) and the acquisition of foreign DNA is a fundamental process in the evolution of most bacterial species. In this study, a collection of marine Vibrio strains isolated in the area of the North Adriatic Sea was screened for the presence of virulence, fitness genes and antibiotic (AR) resistance traits usually present in V. cholerae and V. parahaemolyticus pathogenic strains. To this end, a collection of environmental strains belonging to non pathogenic Vibrio species such as V. alginolyticus, V. harveyi, V. mimicus and many others, was obtained from marine samples of water, plankton and sediments withdrawn in a series of coastal sites, mainly in correspondence to the riverine mouths. To evaluate the incidence of antibiotic resistant strains, samples were also obtained in different fish farms in the described area, as they represent restricted ecological niches under the selective pressure caused by the use of antibiotics to treat fish outbreaks. Among strains derived from marine (water, plankton and sediment) samples, 20% carried fitness genes and 10% virulence determinants. A number of strains carried more than one virulence or fitness genes and some carried contemporary genes involved in pathogenicity and in ecological fitness. Globally, the percentage of strains carrying virulence and/or fitness genes in the marine samples was 28%. The percentages of strains showing the specific genes varied significantly when fish farm samples are considered: 64% of the strains carried fitness genes and 29% of the isolates carried virulence genes Taken all together, strains carrying virulence and/or fitness genes in samples from aquaculture were 74%. Some of the genes detected in the marine non pathogenic Vibrio strains here examined are classical virulence factors of human pathogenic Vibrio species such as V. cholera (neuraminidase-encoding gene) or V. parahaemolyticus (tdh, trh, secretion system genes). Also fitness genes have an important role in virulence in that they are involved in the persistence of bacteria in a specific environment thus facilitating a possible contact with an appropriate host. In this study, it has been demonstrated a high incidence of genes involved in important mechanisms of survival in the natural environment; among these, the formation of a biofilm, the synthesis of flagella mediating motility and the possibility of surviving via the symbiotic association with other marine organisms. The emergence of antibiotic resistant bacteria in the environment is a public health concern due to a largely use of antimicrobial agents in aquaculture to prevent and treat diseases in fish and shellfish. Antibiotics and antibiotic-resistant bacteria can persist in the aquatic environment also for several months after their administration. The risk for human health is then due to a direct action of the antibiotic but also to the infection of the human host with antibiotic resistant bacteria present in water and fish products. To the best of our knowledge, this is the first study which evaluates the incidence of quinolone resistance genes in vibrios directly isolated from the marine environment. Of the 156 strains examined, 13 strains (8%) resulted to carry at least one gene involved in quinolone resistance. Genes qnr were the most frequently present in these strains with six strains carrying qnrA, 3 strains with qnrS, and one presenting qnrD. Strains NPV42 and NPV44 carried contemporary genes qnrA and qnrS. Other four environmental strains showed the presence of the gene qepA while only one carried the aac(6’)-Ib-cr gene. The gene qnrC was not detected at all. Interestingly, many of the genes detected in the marine Vibrio strains were located in mobile genetic elements such as integrons and pathogenicity islands, known elements capturing different types of genes. Of the 28 strains showing to carry the integrase associated to the class 1 integron, some demonstrated to bear integrons of different sizes. Two of these isolates carried in the integron two cassettes involved in chloramphenicol and in amikacin resistance although only one of the strains expressed antibiotic resistance. None of the environmental strains resulted positive to the search for integrative conjugative elements. Particularly interesting the presence, in a number of strains, of multiple virulence genes located in pathogenicity islands originally described in V. cholerae and V. parahaemolyticus. As an example, the strains carrying the VPI-2 with the virulence gene nanH and fragments of the V. parahaemolyticus Vp-PAI including genes belonging to secretion systems and the tdh. Data collected in this study clearly support the vision of non pathogenic Vibrio strains as a significant reservoir of virulence and antibiotic resistance genes and confirm that exchange of DNA material occurs among bacteria in the aquatic environment. The exchange of this genetic material can occurs among marine autochthonous bacteria but also among members of the marine microflora and human pathogens reversed, via anthropogenic activities, in the aquatic environment. The emergence of human bacterial pathogens with new virulence and /or antibiotic resistance traits is a direct concern for public health and a risk for human health.