Distribution of Escherichia coli in a coastal lagoon (Venice, Italy): Temporal patterns, genetic diversity and the role of tidal forcing.

Despite its worldwide importance as fecal indicator in aquatic systems, little is known about the diversity of Escherichia coli in the environment and the factors driving its spatial distribution. The city of Venice (Italy), lying at the forefront of a large European lagoon, is an ideal site to study the mechanisms driving the fate of fecal bacteria, due to the huge fluxes of tourists, the city's unique architecture (causing poor efficiency of sewages treatment), and the long branching network of canals crossing the city. We summarize the results of a multi-year investigation to study the temporal dynamics of E. coli around the city, describe the population structure (by assigning isolates to their phylogenetic group) and the genotypic diversity, and explore the role of environmental factors in determining its variability. E. coli abundance in water was highly variable, ranging from being undetectable up to 10(4) Colony Forming Units (CFU) per 100 ml. Abundance did not display significant relationships with the water physico-chemical variables. The analysis of the population structure showed the presence of all known phylogroups, including extra-intestinal and potentially pathogenic ones. The genotypic diversity was very high, as likely consequence of the heterogeneous input of fecal bacteria from the city, and showed site-specific patterns. Intensive sampling during the tidal fluctuations highlighted the prominent role of tides, rather than environmental variables, as source of spatial variation, with a more evident influence in water than sediments. These results, the first providing information on the genetic properties, spatial heterogeneity and influence of tides on E. coli populations around Venice, have implications to manage the fecal pollution, and the associated waterborne disease risks, in coastal cities lying in front of lagoons and semi-enclosed basins.

Extracellular DNA can preserve the genetic signatures of present and past viral infection events in deep hypersaline anoxic basins.

Deep hypersaline anoxic basins (DHABs) of the Mediterranean Sea are among the most extreme ecosystems on Earth and host abundant, active and diversified prokaryotic assemblages. However, factors influencing biodiversity and ecosystem functioning are still largely unknown. We investigated, for the first time, the impact of viruses on the prokaryotic assemblages and dynamics of extracellular DNA pool in the sediments of La Medee, the largest DHAB found on Earth. We also compared, in La Medee and L'Atalante sediments, the diversity of prokaryotic 16S rDNA sequences contained in the extracellular DNA released by virus-induced prokaryotic mortality. We found that DHAB sediments are hot-spots of viral infections, which largely contribute to the release of high amounts of extracellular DNA. DNase activities in DHAB sediments were much higher than other extracellular enzymatic activities, suggesting that extracellular DNA released from killed prokaryotes can be the most suitable trophic resource for benthic prokaryotes. Preserved extracellular DNA pools, which contained novel and diversified gene sequences, were very similar between the DHABs but dissimilar from the respective microbial DNA pools. We conclude that the strong viral impact in DHAB sediments influences the genetic composition of extracellular DNA, which can preserve the signatures of present and past infections.

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.

Impact of aquaculture on benthic virus-prokaryote interactions in the Mediterranean Sea.

We investigated the effects of organic enrichment due to the biodeposition from fish farms on benthic prokaryotic and viral abundance and production, viral-induced prokaryotic mortality, enzymatic activities and bacterial diversity. We compared four areas across the Mediterranean Sea, from Cyprus to Spain, and two different habitats: sediments covered by the seagrass Posidonia oceanica and soft-bottom unvegetated sediments. In several cases, the sediments beneath the cages showed higher prokaryotic and viral abundance and production, and higher rates of organic matter decomposition. However, the differences between impact and control sediments were not consistent at all regions and habitats. Benthic bacterial diversity was always lower below the cages, where high viral-induced bacterial mortality rates were also observed. The δ- and γ-Proteobacteria dominated in both impacted and control sediments, but the relative importance of sulphate-reducing δ-Proteobacteria increased beneath the cages. We conclude that aquaculture can have a significant impact on benthic prokaryotes and viruses, by stimulating prokaryotic metabolism and viral infections, reducing bacterial diversity and altering assemblage composition. However, these impacts vary depending upon the sediment type and the habitat characteristics.

New sequence types and multidrug resistance among pathogenic Escherichia coli isolates from coastal marine sediments.

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.

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.

Extraintestinal Escherichia coli carrying virulence genes in coastal marine sediments.

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, stx(1), stx(2), 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.

Prokaryote diversity and virus abundance in shallow hydrothermal vents of the Mediterranean Sea (Panarea Island) and the Pacific Ocean (north Sulawesi-Indonesia).

Despite their ubiquitous distribution in tectonically active coastal zones, shallow water hydrothermal vents have been less investigated than deep-sea vents. In the present study, we investigated the role of viral control and fluid emissions on prokaryote abundance, diversity, and community structure (total Archaea, total Bacteria, and sulphate-reducing bacteria) in waters and sediments surrounding the caldera of four different shallow-water hydrothermal vents (three located in the Mediterranean Sea and one in the Pacific Ocean). All vents, independent of their location, generally displayed a significant decrease of benthic prokaryote abundance, as well as its viable fraction, with increasing distance from the vent. Prokaryote assemblages were always dominated by Bacteria. Benthic Archaea accounted for 23-33% of total prokaryote abundance in the Mediterranean Sea and from 13 to 29% in the Pacific Ocean, whereas in the water column they accounted for 25-38%. The highest benthic bacterial ribotype richness was observed in close proximity of the vents (i.e., at 10-cm distance from the emissions), indicating that vent fluids might influence bacterial diversity in surrounding sediments. Virioplankton and viriobenthos abundances were low compared to other marine systems, suggesting that temperature and physical-chemical conditions might influence viral survival in these vent systems. We thus hypothesize that the high bacterial diversity observed in close proximity of the vents is related with the highly variable vent emissions, which could favor the coexistence of several prokaryotic species.

Bacteria associated with the rapid tissue necrosis of stony corals.

The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio-specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for > 15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology.

Comparison of two fingerprinting techniques, terminal restriction fragment length polymorphism and automated ribosomal intergenic spacer analysis, for determination of bacterial diversity in aquatic environments.

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.

Exo-enzymatic activities and dissolved organic pools in relation with mucilage development in the Northern Adriatic Sea.

We tested the hypothesis that the appearance of mucilage in the Northern Adriatic Sea was related with the accumulation of dissolved organic compounds released by intensive enzymatic activities and not utilized as direct substrate for microbial growth. To do this enzymatic activities and dissolved organic and inorganic pools in periods characterized by the presence of mucilage and in the same seasons but in absence of mucilage were compared. Extracellular enzymatic activities (aminopeptidase, beta-glucosidase and alkaline phosphatase), nutrient pool concentrations (total dissolved nitrogen, dissolved organic nitrogen, total dissolved phosphorus, dissolved organic phosphorus) and the biochemical composition of particulate and dissolved organic matter (in terms of proteins and carbohydrates) were determined on a monthly basis over a period of 3 years. Aminopeptidase and alkaline phosphatase activities displayed higher values in springs preceding the appearance of mucilage than in spring when no mucilage was observed. Beta-Glucosidase activity showed significantly higher values in summer periods characterized by the massive production of mucilage than in summers without mucilage events. The months preceding mucilage events were also characterized by an increase of the alkaline phosphatase to aminopeptidase activity ratio and by a significant accumulation of dissolved proteins. These findings, together with the significant increase of the DON/DOP ratio, suggest that mucilage formation is favoured by the deficiency of organic P. The present study provides compelling evidences that mucilage formation is favoured by the unbalance between organic matter mobilization by enzymatic activities and the accumulation of labile dissolved organic-N compounds.

Sustainable impact of mussel farming in the Adriatic Sea (Mediterranean Sea): evidence from biochemical, microbial and meiofaunal indicators.

We have investigated the impact of a large mussel farm on the benthic environment using a battery of benthic indicators of environmental quality (including biochemical, microbial and meiofaunal parameters). These were analysed through a multi-control sampling strategy over one year. The differences across the seasons are typically higher than those between the impacted and the control stations. No effects are seen in terms of the sediment oxygen penetration and the downward fluxes (as the total mass, organic and phytopigment fluxes). The indicators based on the biochemical compositions of the sediment organic matter and the microbial parameters also show no evidence of the eutrophication process, except as a slight increase in the bacterial density in the sediments beneath the long-lines of the farm during the period of highest mussel stocks. Finally, no effects are observed in terms of the benthic faunal indicators, as the meiofaunal abundance, the community structure and the taxa richness are all indistinguishable between the farm sediments and the controls. These results show that mussel farming in the investigated system is eco-sustainable and does not significantly alter the coastal marine ecosystem, both in terms of the functioning and the trophic state. The battery of indicators selected in this study represents a useful tool for the monitoring of the potential ecological impact of mussel farms, towards guaranteeing the sustainable development of aquacultures in shallow coastal environments.

Bacterial diversity in deep Mediterranean sediments: relationship with the active bacterial fraction and substrate availability.

We investigated vertical distribution and depth-related patterns (from 670 to 2,570 metres) of bacterial diversity in sediment samples collected along a transect in the warm deep Mediterranean sea. Analyses of bacterial diversity were compared with the abundance of benthic bacteria, their metabolically active fraction and the substrates potentially available for their growth. The number of active bacteria was dependent upon the availability of organic substrate in the sediment deriving from phytopigment inputs from the photic layer. The T-RFLP analysis revealed that the surface layers of all sediments analysed were dominated by the same ribotypes, but clear shifts in bacterial community structure were observed in deeper sediment layers. High values of bacterial diversity (expressed as D, H') and evenness (as J) were observed at all stations (a total of 61 ribotypes was identified), and as a result of the large fraction of rare ribotypes (c. 35%), the overall bacterial diversity in the deep sea region investigated was among the highest reported so far in literature. Biodiversity parameters did not display any relationship with water depth, but ribotype richness was related with the number and percentage of active bacteria, suggesting a coupling between organic inputs stimulating bacterial growth and deep-sea bacterial diversity.

Large fraction of dead and inactive bacteria in coastal marine sediments: comparison of protocols for determination and ecological significance.

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) x 10(8) cells g(-1) and (53.1 +/- 16.0) x 10(8) 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.