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Antibiotic resistance is a rising threat for human health. Although in clinical settings and terrestrial environments the rise of antibiotic resistant bacteria is well documented, their dissemination and spread in the marine environment, covering almost two-thirds of the Earth's surface, is still poorly understood. In this study, the presence and abundance of sulphonamide resistance gene (sul2) and class 1 integron-integrase gene (intI1), used as markers for the occurrence and spread of antibiotic resistance genes since the beginning of the antibiotic era, were investigated. Twenty-nine archived formalin-fixed samples, collected by the Continuous Plankton Recorder (CPR) survey in the Atlantic Ocean and North Sea from 1970 to 2011, were analysed using Droplet Digital PCR (ddPCR) applied for the first time on CPR samples. The two marker genes were present in a large fraction of the samples (48% for sul2 and 76% for intI1). In contrast, results from Real-Time PCR performed on the same samples greatly underestimate their occurrence (21% for sul2 and 52% for intI1). Overall, besides providing successful use of ddPCR for the molecular analysis of CPR samples, this study reveals long-term occurrence and spread of sul2 gene and class 1 integrons in the plankton-associated bacterial communities in the ocean., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin-binding protein involved in V. cholerae attachment to environmental chitin surfaces and human intestinal cells. We previously investigated the distribution and genetic variations of gbpA in a large collection of V. cholerae strains and found that the gene is consistently present and highly conserved in this species. Primers and probe were designed from the gbpA sequence of V. cholerae and a new Taq-based qPCR protocol was developed for diagnostic detection and quantification of the bacterium in environmental and stool samples. In addition, the positions of primers targeting the gbpA gene region were selected to obtain a short amplified fragment of 206 bp and the protocol was optimized for the analysis of formalin-fixed samples, such as historical Continuous Plankton Recorder (CPR) samples. Overall, the method is sensitive (50 gene copies), highly specific for V. cholerae and failed to amplify strains of the closely-related species Vibrio mimicus. The sensitivity of the assay applied to environmental and stool samples spiked with V. cholerae ATCC 39315 was comparable to that of pure cultures and was of 102 genomic units/l for drinking and seawater samples, 101 genomic units/g for sediment and 102 genomic units/g for bivalve and stool samples. The method also performs well when tested on artificially formalin-fixed and degraded genomic samples and was able to amplify V. cholerae DNA in historical CPR samples, the earliest of which date back to August 1966. The detection of V. cholerae in CPR samples collected in cholera endemic areas such as the Benguela Current Large Marine Ecosystem (BCLME) is of particular significance and represents a proof of concept for the possible use of the CPR technology and the developed qPCR assay in cholera studies.

The ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus in the brackish aquatic environment was extensively investigated in this study. By conducting laboratory experiments under natural setting conditions, it was shown that V. splendidus LGP32 strain generally exhibits longer persistence in both seawater and sediment than V. aestuarianus 01/32 strain. Both strains maintained viability and culturability for longer times in the sediment, suggesting that this compartment may represent a suitable niche for their persistence in the environment. In addition, both strains attached to chitin particles and copepods, the efficiency of attachment being higher in V. splendidus than in V. aestuarianus. Similarly, LGP32 strain showed a greater capability to form biofilm on poly-vinyl chloride (PVC) surfaces than 01/32 strain. LGP32 and 01/32 strains were also capable of entering a viable but non-culturable state after extended incubation at 5°C, a condition commonly found during cold season in the aquatic brackish environment. These results are consistent with field data collected during a 2-year sampling campaign in the northern Adriatic Sea and provide background information on the mechanisms promoting V. splendidus and V. aestuarianus persistence in coastal water, thus contributing to a better understanding of the epidemiology of the associated diseases., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Vibrios are among the most common bacteria that inhabit surface waters throughout the world and are responsible for a number of severe infections both in humans and animals. Several reports recently showed that human Vibrio illnesses are increasing worldwide including fatal acute diarrheal diseases, such as cholera, gastroenteritis, wound infections, and septicemia. Many scientists believe this increase may be associated with global warming and rise in sea surface temperature (SST), although not enough evidence is available to support a causal link between emergence of Vibrio infections and climate warming. The effect of increased SST in promoting spread of vibrios in coastal and brackish waters is considered a causal factor explaining this trend. Field and laboratory studies carried out over the past 40 years supported this hypothesis, clearly showing temperature promotes Vibrio growth and persistence in the aquatic environment. Most recently, a long-term retrospective microbiological study carried out in the coastal waters of the southern North Sea provided the first experimental evidence for a positive and significant relationship between SST and Vibrio occurrence over a multidecadal time scale. As a future challenge, macroecological studies of the effects of ocean warming on Vibrio persistence and spread in the aquatic environment over large spatial and temporal scales would conclusively support evidence acquired to date combined with studies of the impact of global warming on epidemiologically relevant variables, such as host susceptibility and exposure. Assessing a causal link between ongoing climate change and enhanced growth and spread of vibrios and related illness is expected to improve forecast and mitigate future outbreaks associated with these pathogens.

The long-term effects of ocean warming on prokaryotic communities are unknown because of lack of historical data. We overcame this gap by applying a retrospective molecular analysis to the bacterial community on formalin-fixed samples from the historical Continuous Plankton Recorder archive, which is one of the longest and most geographically extensive collections of marine biological samples in the world. We showed that during the last half century, ubiquitous marine bacteria of the Vibrio genus, including Vibrio cholerae, increased in dominance within the plankton-associated bacterial community of the North Sea, where an unprecedented increase in bathing infections related to these bacteria was recently reported. Among environmental variables, increased sea surface temperature explained 45% of the variance in Vibrio data, supporting the view that ocean warming is favouring the spread of vibrios and may be the cause of the globally increasing trend in their associated diseases.

Mass mortality events of benthic invertebrates in the temperate north-western (NW) Mediterranean Sea have been observed in recent seasons. A 16 month in situ study in the Ligurian Sea (NW Mediterranean Sea) demonstrated that the occurrence of Paramuricea clavata mortality episodes were concomitant to a condition of prolonged high sea surface temperatures, low chlorophyll concentrations and the presence of culturable Vibrio spp. in seawater. The occurrence of Vibrio spp. at the seasonal scale was correlated with temperature; with few vibrios retrieved on specific media when the temperature dropped below 18 degrees C and a sharp increase of vibrios abundance (up to 3.4 x 10(4) MPN l(-1)) when the temperature was greater than or equal to 22 degrees C. Phylogenetic and phenotypic analysis of Vibrio isolates associated with healthy and diseased P. clavata colonies collected during a mortality episode showed that these bacteria were significantly more abundant in diseased than in healthy corals and were related to the V. harveyi, V. splendidus and V. coralliilyticus groups, the latter only identified in diseased organisms. Inoculation of bacterial isolates from these groups onto healthy P. clavata in aquaria caused disease signs and death in a range of Vibrio concentrations, temperature values and trophic conditions consistent with those recorded in the field. It is concluded that Vibrio infections may act as an additional triggering mechanism of mass mortality events in the coastal Mediterranean Sea and that their occurrence is climate-linked. Predicted global warming leading to long-lasting hot summer periods together with stratification resulting in energetic constraints represent a major threat to the survival of benthic invertebrates in the temperate NW Mediterranean Sea due to potential disease outbreak associated with Vibrio pathogens.

We carried out a 16-month in situ study to investigate the ecology of Vibrio spp. and pathogenic Vibrio species in coastal sediments of the Mediterranean Sea, employing multiple-regression analysis to reveal the major environmental factors controlling their occurrence in the benthic environment. In addition, association between vibrios and sediment-inhabiting meiofauna, which is a major component of benthic ecosystems, was investigated. Culturable and total Vibrio spp. estimates by most-probable-number technique coupled with standard polymerase chain reaction (PCR) and real-time PCR methods, respectively, were at least one order of magnitude higher in sediment than in seawater. In addition, potential human pathogenic species Vibrio cholerae, Vibrio vulnificus and Vibrio parahaemolyticus occurred in the sediment with V. parahaemolyticus being the most frequently found. In the pelagic environment, 60% of total variance in culturable Vibrio data was explained by sea surface temperature (40%), salinity (13%) and organic matter concentration (7%). In the benthic environment, sea surface temperature was the only factor that significantly affected culturable Vibrio occurrence although it explained only 25% of total variance, suggesting that additional unexplored factors may play a role as well. No correlation was found between culturable Vibrio spp. concentrations and the abundance of harpacticoid copepods in the sediment whilst a negative correlation was found between Vibrio spp. and nematode abundance which accounted for almost 90% of the total meiofaunal density. Taxonomic analysis revealed that selective bacterial feeders accounted for nearly 50% of the total nematode community and included genera such as Terschellingia, Molgolaimus and Halalaimus, suggesting that top-down control by nematode grazing may be an important factor affecting Vibrio occurrence in these sediments. It is concluded that the benthic marine environment may function as a reservoir of Vibrio spp. and potential pathogenic vibrios whose ecological features appeared substantially different from the ones recognised in the pelagic environment.

In the marine environment, vibrios adhere to a number of substrates including chitin-rich organisms such as crustaceans. Their wide diffusion in coastal waters and pathogenic potential require knowledge of the lifestyle and environmental reservoirs of these bacteria. To test the presence of culturable vibrios in coastal areas and their association with benthic crustaceans, vibrios were isolated from water, sediments and crustaceans (copepods and anphipods) at three stations placed in front of heavily used tourist beaches of the Adriatic Sea. We observed significant correlations between vibrios and temperature. Benthic and planktonic copepods harboured vibrios in summer, while benthic amphipods harboured these bacteria in spring and autumn. Vibrio alginolyticus and Vibrio parahaemolyticus strains gave positive results using primers for Vibrio cholerae toxR and toxS. Sedimentary crustaceans may extend Vibrio persistence in seawater and may represent an additional aquatic reservoir of these bacteria.

Culturable vibrios were isolated from water and plankton fractions collected during an 18-month sampling study performed along the north-central coast of the Adriatic Sea (Italy). Unculturable Vibrio vulnificus and V. parahaemolyticus were detected in plankton fractions by polymerase chain reaction amplification of DNA sequences for cytotoxin-haemolysin and thermolabile haemolysin respectively. The presence of V. parahaemolyticus, V. vulnificus and V. cholerae virulence genes and the expression of pathogenicity-associated traits were analysed in all isolates. The results showed the spreading of these properties among the environmental isolates and confirm the need of both monitoring the presence of vibrios in coastal areas and studying their pathogenicity potential in order to properly protect human health.

The presence of enterococci in lake and seawater in an 18-month survey comparing molecular (PCR and quantitative PCR) and culture methods was evaluated, as well as the possibility that zooplankton could act as reservoirs for enterococci. Samples of both water and zooplankton were collected monthly from a Lake Garda site and an Adriatic Sea site. In lake water, the positive samples numbered 13 of 54 (24%) by culture and 32 of 54 (59%) when PCR was applied. In seawater, they numbered 0 of 51 by culture and 18 of 51 (35%) by PCR. Enterococci were found either totally bound to plankton or totally in water, depending on the presence or absence of plankton, respectively. These results clearly indicate that the PCR assay is a powerful tool for detecting fecal indicators and pathogens in the environment, thus providing a much more sensitive method than culture.

The effect of exposure to artificial sea water (ASW) on the ability of classical Vibrio cholerae O1 cells to interact with chitin-containing substrates and human intestinal cells was studied. Incubation of vibrios in ASW at 5 degrees C and 18 degrees C resulted in two kinds of cell responses: the viable but non-culturable (VBNC) state (i.e. <0.1 colony forming unit ml-1) at 5 degrees C, and starvation (i.e. maintenance of culturability of the population) at 18 degrees C. The latter remained rod shaped and, after 40 days' incubation, presented a 47-58% reduction in the number of cells attached to chitin, a 48-53% reduction in the number of bacteria adhering to copepods, and a 48-54% reduction in the number of bacteria adhering to human cultured intestinal cells, compared to control cells not suspended in ASW. Bacteria suspended in ASW at 5 degrees C became coccoid and, after 40 days, showed 34-42% fewer cells attached to chitin, 52-55% fewer adhering to copep-ods, and 45-48% fewer cells adhering to intestinal cell monolayers, compared to controls. Sarkosyl-insoluble membrane proteins that bind chitin particles were isolated and analysed by SDS-PAGE. After 40 days incubation in ASW at both 5 degrees C and 18 degrees C vibrios expressed chitin-binding ligands similar to bacteria harvested in the stationary growth phase. It is concluded that as vibrios do not lose adhesive properties after long-term exposure to ASW, it is important to include methods for VBNC bacteria when testing environmental and clinical samples for purposes of public health safety.