Gian Marco Luna

Deep-Sea Biodiversity in the Mediterranean Sea: The Known, the Unknown, and the Unknowable

Deep-sea ecosystems represent the largest biome of the global biosphere, but knowledge of their biodiversity is still scant. The Mediterranean basin has been proposed as a hot spot of terrestrial and coastal marine biodiversity but has been supposed to be impoverished of deep-sea species richness. We summarized all available information on benthic biodiversity (Prokaryotes, Foraminifera, Meiofauna, Macrofauna, and Megafauna) in different deep-sea ecosystems of the Mediterranean Sea (200 to more than 4,000 m depth), including open slopes, deep basins, canyons, cold seeps, seamounts, deep-water corals and deep-hypersaline anoxic basins and analyzed overall longitudinal and bathymetric patterns. We show that in contrast to what was expected from the sharp decrease in organic carbon fluxes and reduced faunal abundance, the deep-sea biodiversity of both the eastern and the western basins of the Mediterranean Sea is similarly high. All of the biodiversity components, except Bacteria and Archaea, displayed a decreasing pattern with increasing water depth, but to a different extent for each component. Unlike patterns observed for faunal abundance, highest negative values of the slopes of the biodiversity patterns were observed for Meiofauna, followed by Macrofauna and Megafauna. Comparison of the biodiversity associated with open slopes, deep basins, canyons, and deep-water corals showed that the deep basins were the least diverse. Rarefaction curves allowed us to estimate the expected number of species for each benthic component in different bathymetric ranges. A large fraction of exclusive species was associated with each specific habitat or ecosystem. Thus, each deep-sea ecosystem contributes significantly to overall biodiversity. From theoretical extrapolations we estimate that the overall deep-sea Mediterranean biodiversity (excluding prokaryotes) reaches approximately 2805 species of which about 66% is still undiscovered. Among the biotic components investigated (Prokaryotes excluded), most of the unknown species are within the phylum Nematoda, followed by Foraminifera, but an important fraction of macrofaunal and megafaunal species also remains unknown. Data reported here provide new insights into the patterns of biodiversity in the deep-sea Mediterranean and new clues for future investigations aimed at identifying the factors controlling and threatening deep-sea biodiversity.

Large Fraction of Dead and Inactive Bacteria in Coastal Marine Sediments: Comparison of Protocols for Determination and Ecological Significance

ABSTRACT It is now universally recognized that only a portion of aquatic bacteria is actively growing, but quantitative information on the fraction of living versus dormant or dead bacteria in marine sediments is completely lacking. We compared different protocols for the determination of the dead, dormant, and active bacterial fractions in two different marine sediments and at different depths into the sediment core. Bacterial counts ranged between (1.5 ± 0.2) × 108 cells g−1 and (53.1 ± 16.0) × 108 cells g−1 in sandy and muddy sediments, respectively. Bacteria displaying intact membrane (live bacterial cells) accounted for 26 to 30% of total bacterial counts, while dead cells represented the most abundant fraction (70 to 74%). Among living bacterial cells, nucleoid-containing cells represented only 4% of total bacterial counts, indicating that only a very limited fraction of bacterial assemblage was actively growing. Nucleoid-containing cells increased with increasing sediment organic content. The number of bacteria responsive to antibiotic treatment (direct viable count; range, 0.3 to 4.8% of the total bacterial number) was significantly lower than nucleoid-containing cell counts. An experiment of nutrient enrichment to stimulate a response of the dormant bacterial fraction determined a significant increase of nucleoid-containing cells. After nutrient enrichment, a large fraction of dormant bacteria (6 to 11% of the total bacterial number) was “reactivated.” Bacterial turnover rates estimated ranged from 0.01 to 0.1 day−1 but were 50 to 80 times higher when only the fraction of active bacteria was considered (on average 3.2 day−1). Our results suggest that the fraction of active bacteria in marine sediments is controlled by nutrient supply and availability and that their turnover rates are at least 1 order of magnitude higher than previously reported.

Comparison of Two Fingerprinting Techniques, Terminal Restriction Fragment Length Polymorphism and Automated Ribosomal Intergenic Spacer Analysis, for Determination of Bacterial Diversity in Aquatic Environments

ABSTRACT We investigated bacterial diversity in different aquatic environments (including marine and lagoon sediments, coastal seawater, and groundwater), and we compared two fingerprinting techniques (terminal restriction fragment length polymorphism [T-RFLP] and automated ribosomal intergenic spacer analysis [ARISA]) which are currently utilized for estimating richness and community composition. Bacterial diversity ranged from 27 to 99 phylotypes (on average, 56) using the T-RFLP approach and from 62 to 101 genotypes (on average, 81) when the same samples were analyzed using ARISA. The total diversity encountered in all matrices analyzed was 144 phylotypes for T-RFLP and 200 genotypes for ARISA. Although the two techniques provided similar results in the analysis of community structure, bacterial richness and diversity estimates were significantly higher using ARISA. These findings suggest that ARISA is more effective than T-RFLP in detecting the presence of bacterial taxa accounting for <5% of total amplified product. ARISA enabled also distinction among aquatic bacterial isolates of Pseudomonas spp. which were indistinguishable using T-RFLP analysis. Overall, the results of this study show that ARISA is more accurate than T-RFLP analysis on the 16S rRNA gene for estimating the biodiversity of aquatic bacterial assemblages.

Preservation, origin and genetic imprint of extracellular DNA in permanently anoxic deep‐sea sediments

Molecular approaches that target the total DNA pool recovered from permanently anoxic marine ecosystems have revealed an extraordinary diversity of prokaryotes and unicellular eukaryotes. However, the presence of gene sequences contained within the extracellular DNA pool is still largely neglected. We have investigated the preservation, origin and genetic imprint of extracellular DNA recovered from permanently anoxic deep‐sea sediments of the Black Sea. Despite high DNase activities, huge amounts of total extracellular DNA were found in both the surface and subsurface sediment layers, suggesting reduced availability of the extracellular DNA pool to nuclease degradation. The reduced degradation of the total extracellular DNA was confirmed by its low decay rate and the high accumulation in the deeper sediment layers. The copy numbers of 16S and 18S rDNA contained within the extracellular DNA pool in both the surface and subsurface sediment layers was very high, indicating that permanently anoxic sediments of the deep Black Sea are hot spots of preserved extracellular gene sequences. The extracellular DNA recovered from these sediment layers also contained highly diversified 18S rDNA sequences. These were not only representative of the major protistan lineages, but also of new very divergent lineages, branching as independent clades at the base of the tree. Our findings indicate that the extracellular DNA pool is a major archive of present/past eukaryotic gene sequences, and they highlight the importance of integrating molecular cell‐oriented approaches with molecular analyses of the extracellular DNA pool, for a better assessment of microbial diversity and temporal changes in marine benthic ecosystems.

Aquaculture can promote the presence and spread of antibiotic-resistant Enterococci in marine sediments.

Aquaculture is an expanding activity worldwide. However its rapid growth can affect the aquatic environment through release of large amounts of chemicals, including antibiotics. Moreover, the presence of organic matter and bacteria of different origin can favor gene transfer and recombination. Whereas the consequences of such activities on environmental microbiota are well explored, little is known of their effects on allochthonous and potentially pathogenic bacteria, such as enterococci. Sediments from three sampling stations (two inside and one outside) collected in a fish farm in the Adriatic Sea were examined for enterococcal abundance and antibiotic resistance traits using the membrane filter technique and an improved quantitative PCR. Strains were tested for susceptibility to tetracycline, erythromycin, ampicillin and gentamicin; samples were directly screened for selected tetracycline [tet(M), tet(L), tet(O)] and macrolide [erm(A), erm(B) and mef] resistance genes by newly-developed multiplex PCRs. The abundance of benthic enterococci was higher inside than outside the farm. All isolates were susceptible to the four antimicrobials tested, although direct PCR evidenced tet(M) and tet(L) in sediment samples from all stations. Direct multiplex PCR of sediment samples cultured in rich broth supplemented with antibiotic (tetracycline, erythromycin, ampicillin or gentamicin) highlighted changes in resistance gene profiles, with amplification of previously undetected tet(O), erm(B) and mef genes and an increase in benthic enterococcal abundance after incubation in the presence of ampicillin and gentamicin. Despite being limited to a single farm, these data indicate that aquaculture may influence the abundance and spread of benthic enterococci and that farm sediments can be reservoirs of dormant antibiotic-resistant bacteria, including enterococci, which can rapidly revive in presence of new inputs of organic matter. This reservoir may constitute an underestimated health risk and deserves further investigation.

Extraintestinal Escherichia coli Carrying Virulence Genes in Coastal Marine Sediments

ABSTRACT Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx1, stx2, aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.

Antibiotic-Resistant Enterococci in Seawater and Sediments from a Coastal Fish Farm

The aim of this study was to detect and characterize antibiotic-resistant enterococci in seawater and sediment from a Mediterranean aquaculture site where no antibiotics are used. Colonies (650) grown on Slanetz-Bartley (SB) agar were amplified on antibiotic-supplemented SB, and erythromycin (ERY), tetracycline (TET), and ampicillin (AMP) MICs were determined. Of 75 resistant isolates (17 to TET, 5 to ERY, and 45 to AMP), 5 Enterococcus faecalis, 25 E. faecium, 5 E. casseliflavus, 1 E. gallinarum, 1 E. durans, and 23 Enterococcus spp. were identified by genus- and species-specific polymerase chain reaction (PCR). tet(M), tet(O), tet(L), tet(K), erm(B), erm(A), erm(C), mef, msr, blaZ, and int(Tn916) were sought by PCR, including an improved multiplex PCR assay targeting tet(M), tet(L), and erm(B). Tet(M) was the most frequent TET resistance gene; msr(C) was the sole ERY resistance gene detected. blaZ was found in 29/45 AMP-resistant isolates; however, no β-lactamase production was detected. Antibiotic-resistant enterococci were recovered 2 km off the coast despite the absence of selective pressure exerted by antibiotic use. The occurrence of resistant strains in the absence of the tested genes may indicate the presence of less common resistance determinants. This first evidence of resistant enterococci at a Mediterranean aquaculture site suggests the existence of a marine reservoir of antibiotic resistances potentially transmissible to virulent strains that could be affected by mariculture in an antibiotic-independent manner.

Patterns of benthic bacterial diversity in coastal areas contaminated by heavy metals, polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs)

Prokaryotes in coastal sediments are fundamental players in the ecosystem functioning and regulate processes relevant in the global biogeochemical cycles. Nevertheless, knowledge on benthic microbial diversity patterns across spatial scales, or as function to anthropogenic influence, is still limited. We investigated the microbial diversity in two of the most chemically polluted sites along the coast of Italy. One site is the Po River Prodelta (Northern Adriatic Sea), which receives contaminant discharge from one of the largest rivers in Europe. The other site, the Mar Piccolo of Taranto (Ionian Sea), is a chronically polluted area due to steel production plants, oil refineries, and intense maritime traffic. We collected sediments from 30 stations along gradients of contamination, and studied prokaryotic diversity using Illumina sequencing of amplicons of a 16S rDNA gene fragment. The main sediment variables and the concentration of eleven metals, polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs) were measured. Chemical analyses confirmed the high contamination in both sites, with concentrations of PCBs particularly high and often exceeding the sediment guidelines. The analysis of more than 3 millions 16S rDNA sequences showed that richness decreased with higher contamination levels. Multivariate analyses showed that contaminants significantly shaped community composition. Assemblages differed significantly between the two sites, but showed wide within-site variations related with spatial gradients in the chemical contamination, and the presence of a core set of OTUs shared by the two geographically distant sites. A larger importance of PCB-degrading taxa was observed in the Mar Piccolo, suggesting their potential selection in this historically polluted site. Our results indicate that sediment contamination by multiple contaminants significantly alter benthic prokaryotic diversity in coastal areas, and suggests considering the potential contribution of the resident microbes to contaminant bioremediation actions.

New Sequence Types and Multidrug Resistance among Pathogenic Escherichia coli Isolates from Coastal Marine Sediments

ABSTRACT The spread of antibiotic-resistant microorganisms is widely recognized, but data about their sources, presence, and significance in marine environments are still limited. We examined 109 Escherichia coli strains from coastal marine sediments carrying virulence genes for antibiotic susceptibility, specific resistance genes, prevalence of class 1 and 2 integrons, and sequence type. Antibiotic resistance was found in 35% of strains, and multiple resistances were found in 14%; the resistances detected most frequently were against tetracycline (28%), ampicillin (16.5%), trimethoprim-sulfamethoxazole (13%), and streptomycin (7%). The highest prevalence of resistant strains was in phylogenetic group A, whereas phylogroup B2 exhibited a significantly lower frequency than all the other groups. Sixty percent of multiresistant strains harbored class 1 or 2 integrase genes, and about 50% carried resistance genes (particularly dfrA and aadA) linked to a class 1 integron. Multilocus sequence typing of 14 selected strains identified eight different types characteristic of extraintestinal pathogens and three new allelic combinations. Our data suggest that coastal marine sediment may be a suitable environment for the survival of pathogenic and antimicrobial-resistant E. coli strains capable of contributing to resistance spread via integrons among benthic bacteria, and they highlight a role for these strains in the emergence of new virulent genotypes.

Epidemic Escherichia coli ST131 and Enterococcus faecium ST17 in Coastal Marine Sediments from an Italian Beach

Fecal indicator bacteria (FIB) are used worldwide to assess water quality in coastal environments, but little is known about their genetic diversity and pathogenicity. This study examines the prevalence, antimicrobial resistance, virulence, and genetic diversity of FIB isolated from marine sediments from a central Adriatic seaside resort. FIB, recovered from 6 out of 7 sites, were significantly more abundant at sampling stations 300 m offshore than close to the shore. Escherichia coli accounted for 34.5% of fecal coliforms, and Enterococcus faecalis accounted for 32% of enterococci. Most isolates (27% of E. coli and 22% of enterococci) were recovered from the sediments that had the highest organic content. Multidrug-resistant E. coli (31%) and enterococci (22%) were found at nearly all sites, whereas 34.5% of E. coli and 28% of enterococci harboring multiple virulence factors were recovered from just two sites. Pulsed-field gel electrophoresis typing showed wide genetic diversity among isolates. Human epidemic clones ( E. coli ST131 and Enterococcus faecium ST17) were identified for the first time by multilocus sequence typing in an area where bathing had not been prohibited. These clones were from sites far removed from riverine inputs, suggesting a wide diffusion of pathogenic FIB in the coastal environment and a high public health risk.