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Summary 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.

The low molecular mass (LMM) extract of Cichorium intybus var. silvestre (red chicory) has been shown to inhibit virulence-linked properties of oral pathogens including Streptococcus mutans, Actinomyces naeslundii and Prevotella intermedia. In the present study HPLC-DAD-ESI/MS(2) was used to investigate the compounds contained in this extract for their anti-virulence activity. The extract contained a number of components, including oxalic, succinic, shikimic and quinic acids, which interfere with the growth and virulence traits (i.e., biofilm formation, adherence to epithelial cells and hydroxyapatite) of oral pathogens involved in gingivitis and tooth decay. Succinic and quinic acid seem to be the most potent, mainly by interfering with the ability of oral pathogens to form biofilms (either through inhibition of their development or promotion of their disruption). Our findings suggest that one or more of these compounds may modulate plaque formation in vivo, which is a prerequisite for the development of both caries and gingivitis.

Summary Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin binding protein and a virulence factor involved in the colonization of human intestine. We investigated the distribution and genetic variations of gbpA in 488 V. cholerae strains of environmental and clinical origin, belonging to different serogroups and biotypes. We found that the gene is consistently present and highly conserved including an environmental V. cholerae-related strain of ancestral origin. The gene was also consistently expressed in a number of representative V. cholerae strains cultured in laboratory aquatic microcosms under conditions simulating those found in temperate marine environments. Functional analysis carried out on V. cholerae O1 El Tor N16961 showed that GbpA is not involved in adhesion to inorganic surfaces but promotes interaction with environmental biotic substrates (plankton and bivalve hepatopancreas cells) representing known marine reservoir or host for the bacterium. It is suggested that the ability of GbpA to colonize human intestinal cells most probably originated from its primary function in the aquatic environment.

Antibacterial strategies targeting bacterial adhesion to substrates are considered a valuable alternative to traditional antibiotic therapy, in view of the great advantage they bring in combating the infectious process at the very early stage without selecting for drug resistant cells. Amongst bioactive compounds with activity against bacterial adhesion, several are found in natural food and beverages, such as cranberry, tea, coffee, wine and milk. For the analysis of their anti-infective potential, successful experimental models can be conducted using different substrates from the oral cavity. Studies conducted so far in this field allowed the discovery of a variety of anti-adhesive fractions and compounds proven to be effective against bacterial traits involved in the development of oral pathologies such as caries and gingivitis/periodontitis. Discovering new anti-adhesive compounds from natural products, unravelling and testing their prophylactic and therapeutic values, and improving their use in the general population are promising new frontiers in the global fight against human infectious diseases.

Vibrio cholerae O1 El Tor attachment to chitin and biofilm formation on polyvinylchloride surfaces via the N-acetylglucosamine-binding protein A (GbpA) and the mannose-sensitive haemagglutinin (MSHA) were investigated under different temperature and salinity conditions simulating those found in the aquatic environment. In vitro tests showed that mshA and gbpA defective V. cholerae N16961 strains displayed a significant reduction (P

Polymerase chain reaction (PCR) efficiency is the rate at which a PCR amplicon is generated. More efficient amplification of the DNA fragment (amplicon) will generate more products in fewer cycles, thus improving accuracy and sensitivity of quantitative PCR. The efficiency of Real-Time PCR to detect and quantify microbial cells was compared in marine sediment samples of different origin and chemical composition, and in standard samples. Real-Time PCR efficiencies of marine sediment samples ranged from 1.48±0.1 to 1.83±0.1 and were significantly different from those of standard samples, most probably due to different concentrations of PCR inhibitors. The Real-Time PCR efficiency was higher using species-specific primers (>1.7), and lower using universal primers (

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 10 2 genomic units/l for drinking and seawater samples, 10 1 genomic units/g for sediment and 10 2 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 complex interactions occurring between farmed bivalves and their potential pathogens in the circumstances of global climate changes are current matter of study, owing to the recurrent production breakdowns reported in Europe and other regions of the world. In the frame of Project FP7-KBBE-2010-4 BIVALIFE, we investigated the occurrence of mortality and potential pathogens during the Spring–Summer transition in Crassostrea gigas and Mytilus galloprovincialis cohabiting in the shallow waters of one northern Italian lagoon (Sacca di Goro, Adriatic Sea) and regarded as susceptible and resistant species, respectively. In 2011, limited bivalve mortality was detected in the open-field trial performed with 6–12 month old spat whereas subsequent trials with 2–3 month old spat produced almost complete (2012) and considerable (2013) oyster mortality. Macroscopical examination and histology excluded the presence of notifiable pathogens but, in the sampling preceding the massive oyster spat mortality of 2012, a μdeleted variant of OsHV-1 DNA was found in wide-ranging amounts in all analyzed oysters in conjunction with substantial levels of Vibrio splendidus and Vibrio aestuarianus. The large oyster spat mortality with borderline OsHV-1 positivity recorded in 2013 supports the multi-factorial etiology of the syndrome. This is the first report of a OsHV-1 (under a form interpreted as the variant μVar) in the Goro lagoon. Transcriptional host footprints are under investigation to better understand the bivalve response to environmental factors, included viral and bacterial pathogens, in relation to the observed mortalities.

Vibrio cholerae O1 El Tor attachment to chitin and biofilm formation on polyvinylchloride surfaces via the N-acetylglucosamine-binding protein A (GbpA) and the mannose-sensitive haemagglutinin (MSHA) were investigated under different temperature and salinity conditions simulating those found in the aquatic environment. In vitro tests showed that mshA and gbpA defective V. cholerae N16961 strains displayed a significant reduction (P 0.05) in attachment to chitin in comparison with the parent in all the environmental conditions tested. The lack of mshA, but not gbpA, resulted in a significant decrease (P 0.05) of V. cholerae N16961 strain ability to form biofilm. Wild-type attachment to chitin and biofilm formation increased from 15°C to 25°C as did gbpA and mshA expression. In situ data obtained analysing zooplankton and water samples collected in coastal waters of NW Mediterranean Sea over an annual cycle showed that the percentage of plankton-associated V. cholerae was positive correlated with sea surface temperature, and increased dramatically at temperature values above 22°C. It is suggested that temperature plays a major role in affecting persistence of V. cholerae in the aquatic environment by promoting colonization of environmental surfaces, via an enhanced expression of both mshA and gbpA.

Vibrio cholerae N-acetyl glucosamine-binding protein A (GbpA) is a chitin binding protein and a virulence factor involved in the colonization of human intestine. We investigated the distribution and genetic variations of gbpA in 488 V. cholerae strains of environmental and clinical origin, belonging to different serogroups and biotypes. We found that the gene is consistently present and highly conserved including an environmental V. cholerae-related strain of ancestral origin. The gene was also consistently expressed in a number of representative V. cholerae strains cultured in laboratory aquatic microcosms under conditions simulating those found in temperate marine environments. Functional analysis carried out on V. cholerae O1 El Tor N16961 showed that GbpA is not involved in adhesion to inorganic surfaces but promotes interaction with environmental biotic substrates (plankton and bivalve hepatopancreas cells) representing known marine reservoir or host for the bacterium. It is suggested that the ability of GbpA to colonize human intestinal cells most probably originated from its primary function in the aquatic environment.

Edible bivalves (e.g., mussels, oysters) can accumulate large amount of bacteria in their tissues and act as passive carriers of pathogens to humans. Bacterial persistence inside bivalves depends, at least in part, on hemolymph anti-bacterial activity that is exerted by both serum soluble factors and phagocytic cells (i.e., the hemocytes). It was previously shown that Mytilus galloprovincialis hemolymph serum contains opsonins that mediate D-mannose-sensitive interactions between hemocytes and Vibrio cholerae O1 El Tor bacteria that carry the mannose-sensitive hemagglutinin (MSHA). These opsonins enhance phagocytosis and killing of vibrios by facilitating their binding to hemocytes. Since V. cholerae strains not carrying the MSHA ligand (O1 classical, non-O1/O139) are present in coastal water and can be entrapped by mussels, we studied whether in mussel serum, in addition to opsonins directed toward MSHA, other components can mediate opsonization of these bacteria. By comparing interactions of O1 classical and non-O1/O139 strains with hemocytes in artificial sea water and serum, it was found that M. galloprovincialis serum contains components that increase by at approximately twofold their adhesion to, association with, and killing by hemocytes. Experiments conducted with high and low molecular mass fractions obtained by serum ultrafiltration indicated that these compounds have molecular mass higher than 5000 Da. Serum exposure to high temperature (80°C) abolished its opsonizing capability suggesting that the involved serum active components are of protein nature. Further studies are needed to define the chemical properties and specificity of both the involved bacterial ligands and hemolymph opsonins. This information will be central not only to better understand V. cholerae ecology, but also to improve current bivalve depuration practices and properly protect human health.

Recent results for Vibrio cholerae interactions with bivalves and chitin-containing substrates are reviewed. Chitin, composed of b-1,4-linked N -acetylglucosamine residues, is one of the most abundant biopolymers in nature and the most abundant in the marine environment. V. cholerae connection to chitin is a well known phenomenon and one of the best documented examples of a successful bacteriasubstrate interaction, affecting both the lifestyle of the microorganisms and natural system functioning. In sea water, vibrios can be entrapped by filter-feeding invertebrates that sieve suspended food particles from the aquatic environment. Persistence of bacteria in bivalves depends on different factors including their sensitivity to the bactericidal activity of hemolymph. Both V. cholerae survival inside bivalves and chitin colonization depend on cell surface properties, with contribution of different ligands (e.g. chitin-binding proteins, MSHA). The role of these ligands in interactions with human surfaces is discussed and evidence is presented supporting (i) the link between vibrio persistence in the environment (both inside bivalves and adhering to chitin-containing surfaces) and infection of the human host, and (ii) the hypothesis that virulence mechanisms of bacteria that have environmental reservoirs represent a consequence of adaptive mechanisms to the environment. Key words: Vibrio cholerae, chitin, virulence, adaptive mechanisms, environmental reservoir

In this study, demethylfruticuline A (dfA) and fruticuline A (fA), two quinones representing the major diterpenoid components of the exudate produced by the aerial parts of Salvia corrugata, were assessed for their ability to modify surface characteristics, such as hydrophobicity, and to inhibit synthesis of biofilm in vitro by multiresistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis. Five strains of S. aureus (three meticillin-resistant and two meticillin-susceptible), five strains of S. epidermidis (four meticillin-resistant and one meticillin-susceptible) and eight vancomycin-resistant E. faecalis, all recently isolated from clinical specimens and capable of slime production, were studied. fA decrease by at least two-fold the hydrophobic properties of the S. aureus cell membrane but did not affect S. epidermidis or E. faecalis. Biofilm formation on polystyrene plates was quantified spectrophotometrically by established methodologies. Inhibition of biofilm formation was also confirmed by the Congo red agar plate assay. dfA and fA were more effective against S. aureus strains (>70% effect at subinhibitory concentrations) than against S. epidermidis in inhibiting slime synthesis. Against E. faecalis, dfA at subinhibitory concentration induced an inhibition of biofilm production of ca. 60%; fA was less active and more strain-dependent. Moreover, the two compounds were shown to possess chelating activity on divalent and trivalent metal cations. Interactions of fA and dfA with bacteria could be very complex, possibly being species-specific, and could depend not only on inhibition of exopolysaccharide synthesis but also on their chelating activity and on changes in the microorganism's surface, including cell hydrophobicity.

Low molecular mass (LMM) fractions obtained from extracts of raspberry, red chicory, and Shiitake mushrooms have been shown to be an useful source of specific antibacterial, antiadhesion/coaggregation, and antibiofilm agent(s) that might be used for protection towards caries and gingivitis. In this paper, the effects of such LMM fractions on human gingival KB cells exposed to the periodontal pathogensPrevotella intermediaandActinomyces naeslundiiwere evaluated. Expression of cytokeratin 18 (CK18) andβ4 integrin (β4INT) genes, that are involved in cell proliferation/differentiation and adhesion, and of the antimicrobial peptideβ2 defensin (HβD2) in KB cells was increased upon exposure to either live or heat-killed bacteria. All LMM fractions tested prevented or reduced the induction of gene expression byP. intermediaandA. naeslundiidepending on the experimental conditions. Overall, the results suggested that LMM fractions could modulate the effects of bacteria associated with periodontal disease in gingival cells.

Gingivitis is a preventable disease characterised by inflammation of the gums due to the buildup of a microbial biofilm at the gingival margin. It is implicated as a precursor to periodontitis, a much more serious problem which includes associated bone loss. Unfortunately, due to poor oral hygiene among the general population, gingivitis is prevalent and results in high treatment costs. Consequently, the option of treating gingivitis using functional foods, which promote oral health, is an attractive one. Medicinal mushrooms, including shiitake, have long been known for their immune system boosting as well as antimicrobial effects; however, they have not been employed in the treatment of oral disease. In the current study, the effectiveness of shiitake mushroom extract was compared to that of the active component in the leading gingivitis mouthwash, containing chlorhexidine, in an artificial mouth model (constant depth film fermenter). The total bacterial numbers as well as numbers of eight key taxa in the oral community were investigated over time using multiplex qPCR. The results indicated that shiitake mushroom extract lowered the numbers of some pathogenic taxa without affecting the taxa associated with health, unlike chlorhexidine which has a limited effect on all taxa.

The aim of the current study was to investigate the anticariogenic potential of the (sub)fractions obtained from the edible mushroom shiitake (Lentinula edodes) inin vitrocaries model. We used a modified constant depth film fermentor (CDFF) with pooled saliva as the inoculum and bovine dentin as a substratum. The test compounds were low molecular weight fraction (MLMW) of the shiitake extract and subfractions 4 and 5 (SF4 and SF5) of this fraction. Chlorhexidine (CHX) and water served as a positive and a negative control, respectively. Dentin mineral loss was quantified (TMR), microbial shifts within the microcosms were determined (qPCR), and the acidogenicity of the microcosms was assessed (CIA). From the compounds tested, the SF4 of shiitake showed strong inhibiting effect on dentin demineralization and induced microbial shifts that could be associated with oral health. The acid producing potential was increased, suggesting uncoupling of the glycolysis of the microbiota by the exposure to SF4. In conclusion, the results suggest that SF4 of shiitake has an anticariogenic potential.

In previous studies we demonstrated that green and roasted coffee contains low molecular weight (LMW) compounds capable of inhibiting the ability of Streptococcus mutans, the major causative agent of human dental caries, to adhere to hydroxyapatite (HA) beads. This study addressed the ability of the whole high molecular weight coffee fraction (cHMW) and of its melanoidin and non-melanoidin components (GFC1-5), applied at concentrations that occur in coffee beverages, to (i) inhibit S. mutans growth; (ii) affect S. mutans sucrose-dependent adhesion to and detachment from saliva-coated HA beads (sHA); and (iii) inhibit biofilm development on microtiter plates. The results indicated that only cHMW is endowed with antimicrobial activity. The cHMW fraction and each of the five GFC components inhibited S. mutans adhesion, the strongest effect being exerted by cHMW (91%) and GFC1 (88%). S. mutans detachment from sHA was four times greater (∼20%) with cHMW and the GFC1 and GFC4 melanoidins than with controls. Finally, biofilm production by S. mutans was completely abolished by cHMW and was reduced by 20% by the melanoidin components GFC2 and GFC4 and by the non-melanoidin component GFC5 compared with controls. Altogether these findings show that coffee beverage contains both LMW compounds and HMW melanoidin and non-melanoidin components with a strong ability to interfere in vitro with the S. mutans traits relevant for cariogenesis.

It was shown that barley coffee (BC) interferes with Streptococcus mutans adsorption to hydroxyapatite. After BC component fractionation by dialysis and gel filtration chromatography (GFC), it was found that the low molecular mass (< 1,000 Da) fraction (LMM fraction) containing polyphenols, zinc and fluoride ions and, above all, a high molecular mass (HMM > 1,000 kDa) melanoidin fraction display strong anti-adhesive properties towards S. mutans. In this study, we have further examined the potential of BC, BC LMM fraction and BC HMM melanoidin fraction as caries controlling agents by evaluating their anti-biofilm activity.The effects of BC and BC fractions on biofilm formation by S. mutans ATCC 25175 and its detachment from pre-developed biofilms were evaluated by microtiter plate assay. It was found that BC and its fractions, at concentrations ranging from 60 to 15 mg ml(-1) that are devoid of antimicrobial activity, inhibited S. mutans biofilm formation. An increase of S. mutans ATCC 25175 detachment from 24 h developed biofilm was observed at the highest tested concentrations. Interestingly, BC and BC fractions also showed anti-biofilm activity towards a variety of S. mutans clinical strains isolated from saliva, plaque and caries lesions of adult donors. In general, the HMM melanoidin fraction was more active than the LMM fraction. These findings, classifying BC LMM fraction and BC HMM melanoidin fractions as natural anti-biofilm agents, represent the basis for studying their possible use as anti-caries agents.

Red wine is a widely consumed beverage with multiple beneficial effects on human health. In the present paper, the anticaries properties of red wine were studied in vitro and ex vivo. Our in vitro findings shows that dealcoholised red wine, besides exerting antibacterial activity, strongly interferes with Streptococcus mutans adhesion to saliva-coated hydroxyapatite (sHA) beads, promotes its detachment from sHA, and powerfully inhibits in vitro biofilm formation. The main components responsible for such activities were found to be proanthocyanidins. The ability of red wine to inhibit ex vivo S. mutans biofilm formation on the occlusal surface of natural human teeth also was demonstrated. Our data indicates that protection of the oral cavity from the cariogenic action of S. mutans may be another beneficial effect of the moderate consumption of red wine.