The aim of this study was to examine antibiotic resistance (AR) dissemination in coastal water, considering the contribution of different sources of faecal contamination. Samples were collected in Berlenga, an uninhabited island classified as Natural Reserve and visited by tourists for aquatic recreational activities. To achieve our aim, AR in Escherichia coli isolates from coastal water was compared to AR in isolates from two sources of faecal contamination: human-derived sewage and seagull faeces. Isolation of E. coli was done on Chromocult agar. Based on genetic typing 414 strains were established. Distribution of E. coli phylogenetic groups was similar among isolates of all sources. Resistances to streptomycin, tetracycline, cephalothin and amoxicillin were the most frequent. Higher rates of AR were found among seawater and faeces isolates, except for last-line antibiotics used in human medicine. Multi-resistance rates in isolates from sewage and seagull faeces (29% and 32%) were lower than in isolates from seawater (39%). Seawater AR profiles were similar to those from seagull faeces and differed significantly from sewage AR profiles. Nucleotide sequences matching resistance genes blaTEM, sul1, sul2, tet(A) and tet(B), were present in isolates of all sources. Genes conferring resistance to 3rd generation cephalosporins were detected in seawater (blaCTX-M-1 and blaSHV-12) and seagull faeces (blaCMY-2). Plasmid-mediated determinants of resistance to quinolones were found: qnrS1 in all sources and qnrB19 in seawater and seagull faeces. Our results show that seawater is a relevant reservoir of AR and that seagulls are an efficient vehicle to spread human-associated bacteria and resistance genes. The E. coli resistome recaptured from Berlenga coastal water was mainly modulated by seagulls-derived faecal pollution. The repertoire of resistance genes covers antibiotics critically important for humans, a potential risk for human health.

Pine Wilt Disease (PWD) has a significant impact on Eurasia pine forests. The microbiome of the nematode (the primary cause of the disease), its insect vector, and the host tree may be relevant for the disease mechanism. The aim of this study was to characterize these microbiomes, from three PWD-affected areas in Portugal, using Denaturing Gradient Gel Electrophoresis, 16S rRNA gene pyrosequencing, and a functional inference-based approach (PICRUSt). The bacterial community structure of the nematode was significantly different from the infected trees but closely related to the insect vector, supporting the hypothesis that the nematode microbiome might be in part inherited from the insect. Sampling location influenced mostly the tree microbiome (P < 0.05). Genes related both with plant growth promotion and phytopathogenicity were predicted for the tree microbiome. Xenobiotic degradation functions were predicted in the nematode and insect microbiomes. Phytotoxin biosynthesis was also predicted for the nematode microbiome, supporting the theory of a direct contribution of the microbiome to tree-wilting. This is the first study that simultaneously characterized the nematode, tree and insect-vector microbiomes from the same affected areas, and overall the results support the hypothesis that the PWD microbiome plays an important role in the disease's development.

Monochamus beetles are the dispersing vectors of the nematode Bursaphelenchus xylophilus, the causative agent of pine wilt disease (PWD). PWD inflicts significant damages in Eurasian pine forests. Symbiotic microorganisms have a large influence in insect survival. The aim of this study was to characterize the bacterial community associated to PWD vectors in Europe and East Asia using a culture-independent approach. Twenty-three Monochamus galloprovincialis were collected in Portugal (two different locations); twelve Monochamus alternatus were collected in Japan. DNA was extracted from the insects’ tracheas for 16S rDNA analysis through denaturing gradient gel electrophoresis and barcoded pyrosequencing. Enterobacteriales, Pseudomonadales, Vibrionales and Oceanospirilales were present in all samples. Enterobacteriaceae was represented by 52.2% of the total number of reads. Twenty-three OTUs were present in all locations. Significant differences existed between the microbiomes of the two insect species while for M. galloprovincialis there were no significant differences between samples from different Portuguese locations. This study presents a detailed description of the bacterial community colonizing the Monochamus insects’ tracheas. Several of the identified bacterial groups were described previously in association with pine trees and B. xylophilus, and their previously described functions suggest that they may play a relevant role in PWD.

To understand the contribution of animal- and human-derived fecal pollution sources in shaping integron prevalence and diversity in beach waters, 414 Escherichia coli strains were collected from beach waters (BW, n=166), seagull feces (SF, n=179) and wastewaters (WW, n=69), on the World Biosphere Reserve of the Berlenga Island, Portugal. Statistical differences were found between the prevalence of integrons in BW (21%) and WW (10%), but not between BW and SF (19%). The majority of integrase-positive (intI+)-strains affiliated to commensal phylogroups B1 (37%), A0 (24%) and A1 (20%). Eighteen different gene cassette arrays were detected, most of them coding for resistances to aminoglycosides, trimethoprim, chloramphenicol and quaternary ammonia compounds. Common arrays were found among strains from different sources. Multi-resistance to three or more different classes of antibiotics was observed in 89%, 82% and 57% of intI+-strains from BW, SF and WW, respectively. Plasmids were detected in 79% of strains (60/76) revealing a high diversity of replicons in all sources, mostly belonging to IncF (Frep, FIA and FIB subgroups), IncI1, IncN, IncY and IncK incompatibility groups. In 20% (15/76) of strains, integrons were successfully mobilized through conjugation to E. coli CV601. Results obtained support the existence of a diverse integron pool in the E. coli strains from this coastal environment, associated with different resistance traits and plasmid incompatibility groups, mainly shaped by animal fecal pollution inputs. These findings underscore the role of wild life in dissemination of integrons and antibiotic resistance traits in natural environments.