Marine microorganisms producing carbapenemases and β-lactamases as environmental reservoirs of antibiotic resistance genes constituting a risk for human health

Nowadays, infections due to multi drug resistant bacteria represent a real global concern, not only in the nosocomial environment but also and above all in the veterinary, agri-food and environmental sectors. It is in this context that my doctoral project was placed. The use of Next Generation Sequencing technology allowed to sequence the whole genome (Whole Genome Sequencing) of 12 strains belonging to the Shewanellaceae (n=10; Shewanella algae) and Vibrionaceae (n=2; Vibrio anguillarum, Vibrio parahaemolyticus) families isolated in Italian aquaculture centers located at different sites along the coast of the Adriatic Sea. The aim of the thesis was to evaluate the role of these microorganisms as possible reservoirs of antibiotic resistance genes in the aquatic environment. Moreover, the ability to transfer these genes to other potentially pathogenic bacterial species which can temporarily and/or accidentally share the same environment was investigated as well. The resistome showed the presence of genes conferring resistance to different classes of antibiotics including β-lactams, quinolones, tetracyclines, macrolides, polymyxins, chloramphenicol and heavy metals. The mechanisms of resistance involved were not only enzymatic but a complex system of efflux pumps was also found. In particular, β-lactam resistance was mediated by the presence of genes coding for blaOXA-55-like, blaAmpC and mexB-OprM in S. algae strains, while for vibrios the genes coding for blaAmpC in V. anguillarum, mexA-OprM and blaCARB-19 in V. parahaemolyticus were identified. All the analyzed strains showed to possess the eptA, the phosphoethanolamine transferase that contributed to the resistance to polymyxins, while the resistance to tetracyclines was mainly due to the presence of efflux pumps such as TetR, Tet34 and Tet35 in vibrios. The mobilome analysis showed the presence of mobile genetic elements that could be associated to the class I integron in all the analyzed S. algae and in the V. anguillarum strains. Regarding the insertion sequences (ISs), different families were detected in shewanellae and vibrios. Of particular interest was the 353M S. algae strain isolated in the open sea and containing the conjugative and integrative elements involved in the partition (ParAB), replication (repAB, traI), translocation (traD), the type IV secretion system assembly (traC, traN) and integration (XerD). Moreover, in some strains of S. algae (144bCP, 146bCP, 178CP, 353M, 38LV, 57CP, 83CP) some elements of about 200 kbp containing well-structured genetic modules were detected. In addition, some complete and incomplete phage elements were identified. The virulome analysis showed the presence of bacteriocins and hemolysins (hlyD, hemolysin III) in all the S. algae strains. Regarding the vibrio strains, other hemolysins (tlh, hemolysin III), rtxA and hemagglutinin were detected in V. anguillarum, while hemolysin D and thermostable hemolysin delta-VPH were found in V. parahaemolyticus. Finally, type I, II, III, IV and VI secretion systems were identified. In the second part of the project, I proceeded with the phylogenetic analysis of the predicted OXA-55-like, AmpC and CARB-19 amino acid sequences in order to evaluate their similarity with the sequences stored in Genbank belonging to other species of Shewanella spp., Vibrio spp. and of clinical interest (Enterobacteriaceae and Acinetobacter spp.). The results showed that OXA-55-like represented a cluster phylogenetically related to the other OXA variants present in the genus Shewanella, but of interest was its close correlation with OXA-20 and OXA-21 identified in clinical strains if compared to other variants isolated in Acinetobacter spp. Furthermore, the presence of specific mutations in certain positions within the AmpC sequences in the S. algae strains allowed to group the strains according to the sampling sites and dates, indicating the presence along the Italian Adriatic coast of clones containing several mutations of the same beta-lactamase. Conversely, CARB-19 sequence analysis reported some point mutations that did not change its amino acid sequence. Finally, blaAmpC and blaCARB-19 cloning in Escherichia coli TOP10 was performed. This has allowed to mobilize these two beta-lactamases to study how their expression level vary on Enterobacteriaceae family members.