A concept for international societally relevant microbiology education and microbiology knowledge promulgation in society.

EXECUTIVE SUMMARY: Microbes are all pervasive in their distribution and influence on the functioning and well-being of humans, life in general and the planet. Microbially-based technologies contribute hugely to the supply of important goods and services we depend upon, such as the provision of food, medicines and clean water. They also offer mechanisms and strategies to mitigate and solve a wide range of problems and crises facing humanity at all levels, including those encapsulated in the sustainable development goals (SDGs) formulated by the United Nations. For example, microbial technologies can contribute in multiple ways to decarbonisation and hence confronting global warming, provide sanitation and clean water to the billions of people lacking them, improve soil fertility and hence food production and develop vaccines and other medicines to reduce and in some cases eliminate deadly infections. They are the foundation of biotechnology, an increasingly important and growing business sector and source of employment, and the centre of the bioeconomy, Green Deal, etc. But, because microbes are largely invisible, they are not familiar to most people, so opportunities they offer to effectively prevent and solve problems are often missed by decision-makers, with the negative consequences this entrains. To correct this lack of vital knowledge, the International Microbiology Literacy Initiative-the IMiLI-is recruiting from the global microbiology community and making freely available, teaching resources for a curriculum in societally relevant microbiology that can be used at all levels of learning. Its goal is the development of a society that is literate in relevant microbiology and, as a consequence, able to take full advantage of the potential of microbes and minimise the consequences of their negative activities. In addition to teaching about microbes, almost every lesson discusses the influence they have on sustainability and the SDGs and their ability to solve pressing problems of societal inequalities. The curriculum thus teaches about sustainability, societal needs and global citizenship. The lessons also reveal the impacts microbes and their activities have on our daily lives at the personal, family, community, national and global levels and their relevance for decisions at all levels. And, because effective, evidence-based decisions require not only relevant information but also critical and systems thinking, the resources also teach about these key generic aspects of deliberation. The IMiLI teaching resources are learner-centric, not academic microbiology-centric and deal with the microbiology of everyday issues. These span topics as diverse as owning and caring for a companion animal, the vast range of everyday foods that are produced via microbial processes, impressive geological formations created by microbes, childhood illnesses and how they are managed and how to reduce waste and pollution. They also leverage the exceptional excitement of exploration and discovery that typifies much progress in microbiology to capture the interest, inspire and motivate educators and learners alike. The IMiLI is establishing Regional Centres to translate the teaching resources into regional languages and adapt them to regional cultures, and to promote their use and assist educators employing them. Two of these are now operational. The Regional Centres constitute the interface between resource creators and educators-learners. As such, they will collect and analyse feedback from the end-users and transmit this to the resource creators so that teaching materials can be improved and refined, and new resources added in response to demand: educators and learners will thereby be directly involved in evolution of the teaching resources. The interactions between educators-learners and resource creators mediated by the Regional Centres will establish dynamic and synergistic relationships-a global societally relevant microbiology education ecosystem-in which creators also become learners, teaching resources are optimised and all players/stakeholders are empowered and their motivation increased. The IMiLI concept thus embraces the principle of teaching societally relevant microbiology embedded in the wider context of societal, biosphere and planetary needs, inequalities, the range of crises that confront us and the need for improved decisioning, which should ultimately lead to better citizenship and a humanity that is more sustainable and resilient. ABSTRACT: The biosphere of planet Earth is a microbial world: a vast reactor of countless microbially driven chemical transformations and energy transfers that push and pull many planetary geochemical processes, including the cycling of the elements of life, mitigate or amplify climate change (e.g., Nature Reviews Microbiology, 2019, 17, 569) and impact the well-being and activities of all organisms, including humans. Microbes are both our ancestors and creators of the planetary chemistry that allowed us to evolve (e.g., Life's engines: How microbes made earth habitable, 2023). To understand how the biosphere functions, how humans can influence its development and live more sustainably with the other organisms sharing it, we need to understand the microbes. In a recent editorial (Environmental Microbiology, 2019, 21, 1513), we advocated for improved microbiology literacy in society. Our concept of microbiology literacy is not based on knowledge of the academic subject of microbiology, with its multitude of component topics, plus the growing number of additional topics from other disciplines that become vitally important elements of current microbiology. Rather it is focused on microbial activities that impact us-individuals/communities/nations/the human world-and the biosphere and that are key to reaching informed decisions on a multitude of issues that regularly confront us, ranging from personal issues to crises of global importance. In other words, it is knowledge and understanding essential for adulthood and the transition to it, knowledge and understanding that must be acquired early in life in school. The 2019 Editorial marked the launch of the International Microbiology Literacy Initiative, the IMiLI. HERE, WE PRESENT: our concept of how microbiology literacy may be achieved and the rationale underpinning it; the type of teaching resources being created to realise the concept and the framing of microbial activities treated in these resources in the context of sustainability, societal needs and responsibilities and decision-making; and the key role of Regional Centres that will translate the teaching resources into local languages, adapt them according to local cultural needs, interface with regional educators and develop and serve as hubs of microbiology literacy education networks. The topics featuring in teaching resources are learner-centric and have been selected for their inherent relevance, interest and ability to excite and engage. Importantly, the resources coherently integrate and emphasise the overarching issues of sustainability, stewardship and critical thinking and the pervasive interdependencies of processes. More broadly, the concept emphasises how the multifarious applications of microbial activities can be leveraged to promote human/animal, plant, environmental and planetary health, improve social equity, alleviate humanitarian deficits and causes of conflicts among peoples and increase understanding between peoples (Microbial Biotechnology, 2023, 16(6), 1091-1111). Importantly, although the primary target of the freely available (CC BY-NC 4.0) IMiLI teaching resources is schoolchildren and their educators, they and the teaching philosophy are intended for all ages, abilities and cultural spectra of learners worldwide: in university education, lifelong learning, curiosity-driven, web-based knowledge acquisition and public outreach. The IMiLI teaching resources aim to promote development of a global microbiology education ecosystem that democratises microbiology knowledge.

Plankton and marine aggregates as transmission vectors for V. aestuarianus 02/041 infecting the pacific oyster Crassostrea gigas.

Vibrio aestuarianus is a bacterium related to mass mortality outbreaks of the Pacific oyster, Crassostrea gigas in Europe. In this study, the role of different planktonic substrates (phytoplankton cells, marine aggregates and chitin fragments) in mediating V. aestuarianus 02/041 infection of oysters was evaluated by controlled infection experiments. It was shown that phytoplankton cells and, to a greater extent, marine aggregates, significantly promote V. aestuarianus 02/041 intake by C. gigas maintained under stressful conditions in the laboratory. Such intake is associated with higher concentration of the pathogen in the bivalve hemolymph and compromised health status of infected oysters. In contrast, chitin particles do not play a significant role as transmission vector for V. aestuarianus 02/041 infecting its bivalve host. Interestingly, incorporation into marine aggregates foster extracellular proteases (ECPs) activity and a higher expression of bacterial virulence genes, that are potentially involved in bivalve infection. Results from this study contribute to elucidate transmission patterns of V. aestuarianus 02/041 to C. gigas that may be useful for the development of efficient measures to prevent and control oyster disease outbreaks.

A deep-sea bacterium related to coastal marine pathogens.

Evolution of virulence traits from adaptation to environmental niches other than the host is probably a common feature of marine microbial pathogens, whose knowledge might be crucial to understand their emergence and pathogenetic potential. Here, we report genome sequence analysis of a novel marine bacterial species, Vibrio bathopelagicus sp. nov., isolated from warm bathypelagic waters (3309 m depth) of the Mediterranean Sea. Interestingly, V. bathopelagicus sp. nov. is closely related to coastal Vibrio strains pathogenic to marine bivalves. V. bathopelagicus sp. nov. genome encodes genes involved in environmental adaptation to the deep-sea but also in virulence, such as the R5.7 element, MARTX toxin cluster, Type VI secretion system and zinc-metalloprotease, previously associated with Vibrio infections in farmed oysters. The results of functional in vitro assays on immunocytes (haemocytes) of the Mediterranean mussel Mytilus galloprovincialis and the Pacific oyster Crassostrea gigas, and of the early larval development assay in Mytilus support strong toxicity of V. bathopelagicus sp. nov. towards bivalves. V. bathopelagicus sp. nov., isolated from a remote Mediterranean bathypelagic site, is an example of a planktonic marine bacterium with genotypic and phenotypic traits associated with animal pathogenicity, which might have played an evolutionary role in the origin of coastal marine pathogens.

Aquatic reservoir of Vibrio cholerae in an African Great Lake assessed by large scale plankton sampling and ultrasensitive molecular methods.

The significance of large tropical lakes as environmental reservoirs of Vibrio cholerae in cholera endemic countries has yet to be established. By combining large scale plankton sampling, microbial culture and ultrasensitive molecular methods, namely Droplet Digital PCR (ddPCR) and targeted genomics, the presence of Vibrio cholerae was investigated in a 96,600 L volume of surface water collected on a 322 nautical mile (596 km) transect in Lake Tanganyika. V. cholerae was detected and identified in a large area of the lake. In contrast, toxigenic strains of V. cholerae O1 or O139 were not detected in plankton samples possibly in relation to environmental conditions of the lake ecosystem, namely very low salinity compared to marine brackish and coastal environments. This represents to our knowledge, the largest environmental study to determine the role of tropical lakes as a reservoir of V. cholerae.

Vibrio-bivalve interactions in health and disease.

In the marine environment, bivalve mollusks constitute habitats for bacteria of the Vibrionaceae family. Vibrios belong to the microbiota of healthy oysters and mussels, which have the ability to concentrate bacteria in their tissues and body fluids, including the hemolymph. Remarkably, these important aquaculture species respond differently to infectious diseases. While oysters are the subject of recurrent mass mortalities at different life stages, mussels appear rather resistant to infections. Thus, Vibrio species are associated with the main diseases affecting the worldwide oyster production. Here, we review the current knowledge on Vibrio-bivalve interaction in oysters (Crassostrea sp.) and mussels (Mytilus sp.). We discuss the transient versus stable associations of vibrios with their bivalve hosts as well as technical issues limiting the monitoring of these bacteria in bivalve health and disease. Based on the current knowledge of oyster/mussel immunity and their interactions with Vibrio species pathogenic for oyster, we discuss how differences in immune effectors could contribute to the higher resistance of mussels to infections. Finally, we review the multiple strategies evolved by pathogenic vibrios to circumvent the potent immune defences of bivalves and how key virulence mechanisms could have been positively or negatively selected in the marine environment through interactions with predators.

Global emergence of environmental non-O1/O139 Vibrio cholerae infections linked with climate change: a neglected research field?

The bacterium Vibrio cholerae is a natural inhabitant of aquatic ecosystems across the planet. V. cholerae serogroups O1 and O139 are responsible for cholera outbreaks in developing countries accounting for 3-5 million infections worldwide and 28.800-130.000 deaths per year according to the World Health Organization. In contrast, V. cholerae serogroups other than O1 and O139, also designated as V. cholerae non-O1/O139 (NOVC), are not associated with epidemic cholera but can cause other illnesses that may range in severity from mild (e.g. gastroenteritis, otitis, etc.) to life-threatening (e.g. necrotizing fasciitis). Although generally neglected, NOVC-related infections are on the rise and represent one of the most striking examples of emerging human diseases linked to climate change. NOVC strains are also believed to potentially contribute to the emergence of new pathogenic strains including strains with epidemic potential as a direct consequence of genetic exchange mechanisms such as horizontal gene transfer and genetic recombination. Besides general features concerning the biology and ecology of NOVC strains and their associated diseases, this review aims to highlight the most relevant aspects related to the emergence and potential threat posed by NOVC strains under a rapidly changing environmental and climatic scenario.

Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next-generation sequencing strategy.

Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real-time PCR and 16SrRNA gene-based microbial profiling. A new target enrichment next-generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas.

Responses of Mytilus galloprovincialis to challenge with the emerging marine pathogen Vibrio coralliilyticus.

Vibrio coralliilyticus has emerged as a coral pathogen of concern throughout the Indo-Pacific reef. The interest towards understanding its ecology and pathogenic potential has increased since V. coralliilyticus was shown to be strongly virulent also for other species; in particular, it represents a serious threat for bivalve aquaculture, being one of the most important emerging pathogen responsible for oyster larval mortalities worldwide. V. coralliilyticus has a tightly regulated temperature-dependent virulence and it has been related to mass mortalities events of benthic invertebrates also in the temperate northwestern Mediterranean Sea. However, no data are available on the effects of V. coralliilyticus in the mussel Mytilus galloprovincialis, the most abundant aquacultured species in this area. In this work, responses of M. galloprovincialis to challenge with V. coralliilyticus (ATCC BAA-450) were investigated. In vitro, short term responses of mussel hemocytes were evaluated in terms of lysosomal membrane stability, bactericidal activity, lysozyme release, ROS and NO production, and ultrastructural changes, evaluated by TEM. In vivo, hemolymph parameters were measured in mussels challenged with V. coralliilyticus at 24h p.i. Moreover, the effects of V. coralliilyticus on mussel early embryo development (at 48 hpf) were evaluated. The results show that both in vitro and in vivo, mussels were unable to activate immune response towards V. coralliilyticus, and that challenge mainly induced lysosomal stress in the hemocytes. Moreover, V. coralliilyticus showed a strong and concentration-dependent embryotoxicity. Overall, the results indicate that, although M. galloprovincialis is considered a resistant species to vibrio infections, the emerging pathogen V. coralliilyticus can represent a potential threat to mussel aquaculture.

ddPCR applied on archived Continuous Plankton Recorder samples reveals long-term occurrence of class 1 integrons and a sulphonamide resistance gene in marine plankton communities.

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.

Components in Lentinus edodes mushroom with anti-biofilm activity directed against bacteria involved in caries and gingivitis.

The present study investigated the compounds present in the low molecular mass fraction of Lentinus edodes mushroom (shiitake) extract and their anti-virulence activity against oral pathogens (reference and clinical Streptococcus mutans, Actinomyces naeslundii, and Prevotella intermedia strains). Oxalic, succinic, and quinic acids, and adenine, inosine, and uridine were identified by HPLC-DAD-ESI-MS/MS. Their anti-biofilm production and preformed biofilm disaggregation activities were studied using commercial standard compounds at different concentrations. As regards S. mutans, the highest activity was shown by adenine at 5 mg mL-1 both in the biofilm inhibition (BI 50%) and biofilm disaggregation tests (BD 20%). Considering A. naeslundii, BI values close to 80% were registered for oxalic acid at 1 mg mL-1 and 2 mg mL-1 and BD 50% for quinic acid at 3 mg mL-1. A weaker activity was found against P. intermedia. Furthermore, different mixtures of the commercial standards were tested showing that the activity of a compound can be strongly and sometimes negatively affected by the presence of the other compounds.

Autophagic processes in Mytilus galloprovincialis hemocytes: Effects of Vibrio tapetis.

Autophagy is a highly conserved and regulated catabolic process involved in maintaining cell homeostasis in response to different stressors. The autophagic machinery is also used as an innate immune mechanism against microbial infection. In invertebrates, that lack acquired immunity, autophagy may thus play a key role in the protection against potential pathogens. In aquatic molluscs, evidence has been provided for induction of autophagy by starvation and different environmental stressors; however, no information is available on autophagic pathways in the immune cells, the hemocytes. In this work, the autophagic processes were investigated in the hemocytes of the marine bivalve, the mussel Mytilus galloprovincialis. The effects of classical inducers/inhibitors of mammalian autophagy were first tested. Rapamycin induced a decrease in lysosomal membrane stability-LMS that was prevented by the autophagy inhibitor Wortmannin. Increased MDC fluorescence and expression of LC3-II were also observed. Moreover, responses to in vitro challenge with the bivalve pathogen Vibrio tapetis were evaluated. Mussel hemocytes were unable to activate the immune response towards V. tapetis; however, bacterial challenge induced a moderate decrease in LMS, corresponding to lysosomal activation but no cytotoxicity; the effect was prevented by Wortmannin. TEM observations showed that V. tapetis resulted in rapid formation of autophagosomes and autolysosomes. Accordingly, increased LC3-II expression, decreased levels of phosphorylated mTor and of p62 were observed. The results represent the first evidence for autophagic processes in bivalve hemocytes in response to bacterial challenge, and underline the protective role of autophagy towards potential pathogenic vibrios.

Climate influence on Vibrio and associated human diseases during the past half-century in the coastal North Atlantic.

Climate change is having a dramatic impact on marine animal and plant communities but little is known of its influence on marine prokaryotes, which represent the largest living biomass in the world oceans and play a fundamental role in maintaining life on our planet. In this study, for the first time to our knowledge, experimental evidence is provided on the link between multidecadal climatic variability in the temperate North Atlantic and the presence and spread of an important group of marine prokaryotes, the vibrios, which are responsible for several infections in both humans and animals. Using archived formalin-preserved plankton samples collected by the Continuous Plankton Recorder survey over the past half-century (1958-2011), we assessed retrospectively the relative abundance of vibrios, including human pathogens, in nine areas of the North Atlantic and North Sea and showed correlation with climate and plankton changes. Generalized additive models revealed that long-term increase in Vibrio abundance is promoted by increasing sea surface temperatures (up to ∼1.5 °C over the past 54 y) and is positively correlated with the Northern Hemisphere Temperature (NHT) and Atlantic Multidecadal Oscillation (AMO) climatic indices (P < 0.001). Such increases are associated with an unprecedented occurrence of environmentally acquired Vibrio infections in the human population of Northern Europe and the Atlantic coast of the United States in recent years.

Killing of Vibrio cholerae and Escherichia coli Strains Carrying D-mannose-sensitive Ligands by Mytilus Hemocytes is Promoted by a Multifunctional Hemolymph Serum Protein.

In aquatic environments, bivalve mollusks represent an important ecological niche for microorganisms. Persistence of bacteria in bivalve tissues partly depends on their capacity to survive the bactericidal activity of the hemolymph due to both cellular (hemocyes) and soluble serum factors (e.g., enzymes, lectins, opsonins). The extrapallial protein (EP) present in serum of Mytilus galloprovincialis (MgEP) has been recently shown to work as an opsonin promoting D-mannose sensitive (MS) interactions of the bivalve pathogen Vibrio aestuarianus 01/032 strain with the hemocytes. In this study, the role of MgEP in adhesion and killing of other bacteria carrying MS sensitive ligands was investigated. MgEP enhanced adhesion to and killing by hemocytes of Vibrio cholerae ElTor N16961, expressing the MS hemagglutin (MSHA), as well as of Escherichia coli MG1655, carrying type 1 fimbriae. These results further support the recent finding that the multifunctional MgEP also acts as an opsonin involved in mussel defense towards bacteria carrying MS ligands. In addition, these results contribute to elucidate the ecology of bacterial pathogens that can be transmitted to humans via shellfish consumption.

Effects of Global Warming on Vibrio Ecology.

Vibrio-related infections are increasing worldwide both in humans and aquatic animals. Rise in global sea surface temperature (SST), which is approximately 1 °C higher now than 140 years ago and is one of the primary physical impacts of global warming, has been linked to such increases. In this chapter, major known effects of increasing SST on the biology and ecology of vibrios are described. They include the effects on bacterial growth rate, both in the field and in laboratory, culturability, expression of pathogenicity traits, and interactions with aquatic organisms and abiotic surfaces. Special emphasis is given to the effect of ocean warming on Vibrio interactions with zooplankters, which represent one of the most important aquatic reservoirs for these bacteria. The reported findings highlight the biocomplexity of the interactions between vibrios and their natural environment in a climate change scenario, posing the need for interdisciplinary studies to properly understand the connection between ocean warming and persistence and spread of vibrios in sea waters and the epidemiology of the diseases they cause.

gbpA as a Novel qPCR Target for the Species-Specific Detection of Vibrio cholerae O1, O139, Non-O1/Non-O139 in Environmental, Stool, and Historical Continuous Plankton Recorder Samples.

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.

Susceptibility of Vibrio aestuarianus 01/032 to the antibacterial activity of Mytilus haemolymph: identification of a serum opsonin involved in mannose-sensitive interactions.

The interactions of Vibrio aestuarianus 01/032 with haemolymph of the bivalves Mytilus galloprovincialis and Crassostrea gigas were investigated to understand if haemolymph components (haemocytes and soluble factors) could be involved in the higher resistance to microbial infection shown by mussels in comparison with oysters. Although 01/032 bacteria adhered to haemocytes of both bivalves, they were sensitive to the bactericidal activity of whole haemolymph from mussel, but not from oyster; in addition, adhesion to mussel (but not oyster) haemocytes was affected by D-mannose. Mussel serum opsonins directed towards D-mannose-binding bacterial ligands were purified by affinity chromatography and were shown to mediate 01/032 interactions with M. galloprovincialis haemocytes. Nano-High Performance Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry (HPLC-ESI-MS/MS) analysis showed that the purified opsonin matched the protein precursor of Mytilus edulis extrapallial protein (EP). In the presence of M. galloprovincialis EP protein (MgEP), C. gigas haemocytes killed V. aestuarianus 01/032 almost as efficiently as mussel phagocytes. These findings suggest that the different sensitivity of 01/032 strain to the antibacterial activity of oyster and mussel haemolymph might partly depend on the fact that C. gigas serum lacks MgEP-like opsonins. These results represent the basis for understanding the different sensitivity to microbial infections shown by the two bivalve species.

Aquatic ecology of the oyster pathogens Vibrio splendidus and Vibrio aestuarianus.

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 anti-adhesive mode of action of a purified mushroom (Lentinus edodes) extract with anticaries and antigingivitis properties in two oral bacterial phatogens.

BACKGROUND: In previous works we have shown that a low-molecular-mass (LMM) fraction from mushroom (Lentinus edodes) homogenate interferes with binding of Streptococcus mutans to hydroxyapatite and Prevotella intermedia to gingival cells. Additionally, inhibition of biofilm formation of both odonto- and periodonto-pathogenic bacteria and detachment from preformed biofilms have been described for this compound. Further purification of mushroom extract has been recently achieved and a sub-fraction (i.e. # 5) has been identified as containing the majority of the mentioned biological activities. The aim of this study was to characterise the bacterial receptors for the purified mushroom sub-fraction #5 in order to better elucidate the mode of action of this compound when interfering with bacterial adhesion to host surfaces or with bacteria-bacteria interactions in the biofilm state. METHODS: Candidate bacterial molecules to act as target of this compound were bacterial surface molecules involved in cell adhesion and biofilm formation, and, thus, we have considered cell wall associated proteins (CWPs), teichoic acid (TA) and lipoteichoic acid (LTA) of S. mutans, and outer membrane proteins (OMPs) and lipopolysaccharide (LPS) of P. intermedia. RESULTS: Fifteen S. mutans CWPs and TA were capable of binding sub-fraction #5, while LTA did not. As far as P. intermedia is concerned, we show that five OMPs interact with sub-fraction # 5. Capacity of binding to P. intermedia LPS was also studied but in this case negative results were obtained. CONCLUSIONS: Binding sub-fraction # 5 to surface molecules of S. mutans or P. intermedia may result in inactivation of their physiological functions. As a whole, these results indicate, at molecular level, the bacterial surface alterations affecting adhesion and biofim formation. For these antimicrobial properties, the compound may find use in daily oral hygiene.