Anti-Biofilm Extracts and Molecules from the Marine Environment.

Pathogenic bacteria and their biofilms are involved in many diseases and represent a major public health problem, including the development of antibiotic resistance. These biofilms are known to cause chronic infections for which conventional antibiotic treatments are often ineffective. The search for new molecules and innovative solutions to combat these pathogens and their biofilms has therefore become an urgent need. The use of molecules with anti-biofilm activity would be a potential solution to these problems. The marine world is rich in micro- and macro-organisms capable of producing secondary metabolites with original skeletons. An interest in the chemical strategies used by some of these organisms to regulate and/or protect themselves against pathogenic bacteria and their biofilms could lead to the development of bioinspired, eco-responsible solutions. Through this original review, we listed and sorted the various molecules and extracts from marine organisms that have been described in the literature as having strictly anti-biofilm activity, without bactericidal activity.

Antibiofilm Activity of the Marine Probiotic Bacillus subtilis C3 Against the Aquaculture-Relevant Pathogen Vibrio harveyi.

V. harveyi is a well-known pathogen-inducing vibriosis, especially for shrimp, fish, and invertebrates. Its virulence is related to biofilm formation and this negatively impacts the aquaculture industry. Therapeutic strategies such as the utilization of probiotic bacteria may slow down Vibrio infections. In this study, we investigated the potential antibiofilm activity of the probiotic Bacillus subtilis C3 for aquaculture. First, B. subtilis C3 biofilm was characterized by confocal laser scanning microscopy (CLSM) before testing its bioactivities. We demonstrated antibiofilm activity of B. subtilis C3 culture supernatant, which is mainly composed-among other molecules-of lipopeptidic surfactants belonging to the surfactin family as identified by ultra-high-performance liquid chromatography (UHPLC)-MS/MS. Their antibiofilm activity was confirmed on V. harveyi ORM4 (pFD086) biofilm by CLSM. These findings suggest that the marine probiotic B. subtilis C3 might inhibit or reduce Vibrio colonization and thus decrease the associated animal mortalities.

Genetic Insights into Biofilm Formation by a Pathogenic Strain of Vibrio harveyi.

The Vibrio genus includes bacteria widely distributed in aquatic habitats and the infections caused by these bacteria can affect a wide range of hosts. They are able to adhere to numerous surfaces, which can result in biofilm formation that helps maintain them in the environment. The involvement of the biofilm lifestyle in the virulence of Vibrio pathogens of aquatic organisms remains to be investigated. Vibrio harveyi ORM4 is a pathogen responsible for an outbreak in European abalone Haliotis tuberculata populations. In the present study, we used a dynamic biofilm culture technique coupled with laser scanning microscopy to characterize the biofilm formed by V. harveyi ORM4. We furthermore used RNA-seq analysis to examine the global changes in gene expression in biofilm cells compared to planktonic bacteria, and to identify biofilm- and virulence-related genes showing altered expression. A total of 1565 genes were differentially expressed, including genes associated with motility, polysaccharide synthesis, and quorum sensing. The up-regulation of 18 genes associated with the synthesis of the type III secretion system suggests that this virulence factor is induced in V. harveyi ORM4 biofilms, providing indirect evidence of a relationship between biofilm and virulence.

Sensitivity of Legionella pneumophila to phthalates and their substitutes.

Phthalates constitute a family of anthropogenic chemicals developed to be used in the manufacture of plastics, solvents, and personal care products. Their dispersion and accumulation in many environments can occur at all stages of their use (from synthesis to recycling). However, many phthalates together with other accumulated engineered chemicals have been shown to interfere with hormone activities. These compounds are also in close contact with microorganisms that are free-living, in biofilms or in microbiota, within multicellular organisms. Herein, the activity of several phthalates and their substitutes were investigated on the opportunistic pathogen Legionella pneumophila, an aquatic microbe that can infect humans. Beside showing the toxicity of some phthalates, data suggested that Acetyl tributyl citrate (ATBC) and DBP (Di-n-butyl phthalate) at environmental doses (i.e. 10-6 M and 10-8 M) can modulate Legionella behavior in terms of motility, biofilm formation and response to antibiotics. A dose of 10-6 M mostly induced adverse effects for the bacteria, in contrast to a dose of 10-8 M. No perturbation of virulence towards Acanthamoeba castellanii was recorded. These behavioral alterations suggest that L. pneumophila is able to sense ATBC and DBP, in a cross-talk that either mimics the response to a native ligand, or dysregulates its physiology.

Extracts from Wallis Sponges Inhibit Vibrio harveyi Biofilm Formation.

Pathogenic bacteria and their biofilms are involved in many human and animal diseases and are a major public health problem with, among other things, the development of antibiotic resistance. These biofilms are known to induce chronic infections for which classical treatments using antibiotic therapy are often ineffective. Sponges are sessile filter-feeding marine organisms known for their dynamic symbiotic partnerships with diverse microorganisms and their production of numerous metabolites of interest. In this study, we investigated the antibiofilm efficacy of different extracts from sponges, isolated in Wallis, without biocidal activity. Out of the 47 tested extracts, from 28 different genera, 11 showed a strong activity against Vibrio harveyi biofilm formation. Moreover, one of these extracts also inhibited two quorum-sensing pathways of V. harveyi.

Effect of Phthalates and Their Substitutes on the Physiology of Pseudomonas aeruginosa.

Phthalates are used in a variety of applications-for example, as plasticizers in polyvinylchloride products to improve their flexibility-and can be easily released into the environment. In addition to being major persistent organic environmental pollutants, some phthalates are responsible for the carcinogenicity, teratogenicity, and endocrine disruption that are notably affecting steroidogenesis in mammals. Numerous studies have thus focused on deciphering their effects on mammals and eukaryotic cells. While multicellular organisms such as humans are known to display various microbiota, including all of the microorganisms that may be commensal, symbiotic, or pathogenic, few studies have aimed at investigating the relationships between phthalates and bacteria, notably regarding their effects on opportunistic pathogens and the severity of the associated pathologies. Herein, the effects of phthalates and their substitutes were investigated on the human pathogen, Pseudomonas aeruginosa, in terms of physiology, virulence, susceptibility to antibiotics, and ability to form biofilms. We show in particular that most of these compounds increased biofilm formation, while some of them enhanced the bacterial membrane fluidity and altered the bacterial morphology.

Alterins, a new family of marine antibacterial cyclolipopeptides.

Five strains of Pseudoalteromonas, isolated from oyster haemolymph, have exhibited antibacterial activity against several Gram-negative bacteria. Bioactive compounds have been identified in their cell-free supernatant and characterised as alterins, which are cyclolipopeptides comprising a heptapeptidic ring connected to a fatty acid chain. Using ultra-performance liquid chromatography-high-resolution mass spectrometry, this paper describes 37 structural analogues differing from each other by one or more amino acid residue, the length of the fatty acid chain, its hydroxylation and the presence of unsaturation.

Pseudoalteromonas ostreae sp. nov., a new bacterial species harboured by the flat oyster Ostrea edulis.

Three bacterial strains, named hOe-66T, hOe-124 and hOe-125, were isolated from the haemolymph of different specimens of the flat oyster Ostrea edulis collected in Concarneau bay (Finistère, France). These strains were characterized by a polyphasic approach, including (i) whole genome analyses with 16S rRNA gene sequence alignment and pangenome analysis, determination of the G+C content, average nucleotide identity (ANI), and in silico DNA-DNA hybridization (isDDH), and (ii) fatty acid methyl ester and other phenotypic analyses. Strains hOe-66T, hOe-124 and hOe-125 were closely related to both type strains Pseudoalteromonas rhizosphaerae RA15T and Pseudoalteromonas neustonica PAMC 28425T with less than 93.3% ANI and 52.3% isDDH values. Regarding their phenotypic traits, the three strains were Gram-negative, 1-2 µm rod-shaped, aerobic, motile and non-spore-forming bacteria. Cells grew optimally at 25 °C in 2.5% NaCl and at 7-8 pH. The most abundant fatty acids were summed feature 3 (C16:1 ω7c/C16:1 ω6c), C16:0 and C17:1 ω8c. The strains carried a genome average size of 4.64 Mb and a G+C content of 40.28 mol%. The genetic and phenotypic results suggested that strains hOe-66T, hOe-124 and hOe-125 belong to a new species of the genus Pseudoalteromonas. In this context, we propose the name Pseudoalteromonas ostreae sp. nov. The type strain is hOe-66T (=CECT 30303T=CIP 111911T).

Virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata involves both quorum sensing and a type III secretion system.

Environmental Vibrio strains represent a major threat in aquaculture, but the understanding of their virulence mechanisms heavily relies on the transposition of knowledge from human-pathogen vibrios. Here, the genetic bases of the virulence of Vibrio harveyi ORM4 towards the European abalone Haliotis tuberculata were characterized. We demonstrated that luxO, encoding a major regulator of the quorum sensing system, is crucial for the virulence of this strain, and that its deletion leads to a decrease in swimming motility, biofilm formation, and exopolysaccharide production. Furthermore, the biofilm formation by V. harveyi ORM4 was increased by abalone serum, which required LuxO. The absence of LuxO in V. harveyi ORM4 yielded opposite phenotypes compared with other Vibrio species including V. campbellii (still frequently named V. harveyi). In addition, we report a full type III secretion system (T3SS) gene cluster in the V. harveyi ORM4 genome. LuxO was shown to negatively regulate the promoter activity of exsA, encoding the major regulator of the T3SS genes, and the deletion of exsA abolished the virulence of V. harveyi ORM4. These results unveil virulence mechanisms set up by this environmentally important bacterial pathogen and pave the way for a better molecular understanding of the regulation of its pathogenicity.

The Temperature-Regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX.

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis.

Anti-Biofilm Activity of a Low Weight Proteinaceous Molecule from the Marine Bacterium Pseudoalteromonas sp. IIIA004 against Marine Bacteria and Human Pathogen Biofilms.

Pseudoalteromonas bacteria are known as potential bioactive metabolite producers. Because of the need to obtain natural molecules inhibiting the bacterial biofilms, we investigated the biofilm inhibitory activity of the marine bacterium Pseudoalteromonas sp. IIIA004 against the pioneer surface colonizer Roseovarius sp. VA014. The anti-biofilm activity from the culture supernatant of Pseudoalteromonas sp. IIIA004 (SNIIIA004) was characterized in microtiter plates (static conditions/polystyrene surface) and in flow cell chambers (dynamic conditions/glass surface). The Pseudoalteromonas exoproducts exhibited an inhibition of Roseovarius sp. VA014 biofilm formation as well as a strong biofilm dispersion, without affecting the bacterial growth. Microbial adhesion to solvent assays showed that SNIIIA004 did not change the broad hydrophilic and acid character of the Roseovarius strain surface. Bioassay-guided purification using solid-phase extraction and C18 reverse-phase-high-performance liquid chromatography (RP-HPLC) was performed from SNIIIA004 to isolate the proteinaceous active compound against the biofilm formation. This new anti-biofilm low weight molecule (< 3kDa), named P004, presented a wide spectrum of action on various bacterial biofilms, with 71% of sensitive strains including marine bacteria and human pathogens. Pseudoalteromonas sp. IIIA004 is a promising source of natural anti-biofilm compounds that combine several activities.

Alterocin, an Antibiofilm Protein Secreted by Pseudoalteromonas sp. Strain 3J6.

We sought to identify and study the antibiofilm protein secreted by the marine bacterium Pseudoalteromonas sp. strain 3J6. The latter is active against marine and terrestrial bacteria, including Pseudomonas aeruginosa clinical strains forming different biofilm types. Several amino acid sequences were obtained from the partially purified antibiofilm protein, named alterocin. The Pseudoalteromonas sp. 3J6 genome was sequenced, and a candidate alt gene was identified by comparing the genome-encoded proteins to the sequences from purified alterocin. Expressing the alt gene in another nonactive Pseudoalteromonas sp. strain, 3J3, demonstrated that it is responsible for the antibiofilm activity. Alterocin is a 139-residue protein that includes a predicted 20-residue signal sequence, which would be cleaved off upon export by the general secretion system. No sequence homology was found between alterocin and proteins of known functions. The alt gene is not part of an operon and adjacent genes do not seem related to alterocin production, immunity, or regulation, suggesting that these functions are not fulfilled by devoted proteins. During growth in liquid medium, the alt mRNA level peaked during the stationary phase. A single promoter was experimentally identified, and several inverted repeats could be binding sites for regulators. alt genes were found in about 30% of the Pseudoalteromonas genomes and in only a few instances of other marine bacteria of the Hahella and Paraglaciecola genera. Comparative genomics yielded the hypothesis that alt gene losses occurred within the Pseudoalteromonas genus. Overall, alterocin is a novel kind of antibiofilm protein of ecological and biotechnological interest.IMPORTANCE Biofilms are microbial communities that develop on solid surfaces or interfaces and are detrimental in a number of fields, including for example food industry, aquaculture, and medicine. In the latter, antibiotics are insufficient to clear biofilm infections, leading to chronic infections such as in the case of infection by Pseudomonas aeruginosa of the lungs of cystic fibrosis patients. Antibiofilm molecules are thus urgently needed to be used in conjunction with conventional antibiotics, as well as in other fields of application, especially if they are environmentally friendly molecules. Here, we describe alterocin, a novel antibiofilm protein secreted by a marine bacterium belonging to the Pseudoalteromonas genus, and its gene. Alterocin homologs were found in about 30% of Pseudoalteromonas strains, indicating that this new family of antibiofilm proteins likely plays an important albeit nonessential function in the biology of these bacteria. This study opens up the possibility of a variety of applications.

PHA Production and PHA Synthases of the Halophilic Bacterium Halomonas sp. SF2003.

Among the different tools which can be studied and managed to tailor-make polyhydroxyalkanoates (PHAs) and enhance their production, bacterial strain and carbon substrates are essential. The assimilation of carbon sources is dependent on bacterial strain's metabolism and consequently cannot be dissociated. Both must wisely be studied and well selected to ensure the highest production yield of PHAs. Halomonas sp. SF2003 is a marine bacterium already identified as a PHA-producing strain and especially of poly-3-hydroxybutyrate (P-3HB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P-3HB-co-3HV). Previous studies have identified different genes potentially involved in PHA production by Halomonas sp. SF2003, including two phaC genes with atypical characteristics, phaC1 and phaC2. At the same time, an interesting adaptability of the strain in front of various growth conditions was highlighted, making it a good candidate for biotechnological applications. To continue the characterization of Halomonas sp. SF2003, the screening of carbon substrates exploitable for PHA production was performed as well as production tests. Additionally, the functionality of both PHA synthases PhaC1 and PhaC2 was investigated, with an in silico study and the production of transformant strains, in order to confirm and to understand the role of each one on PHA production. The results of this study confirm the adaptability of the strain and its ability to exploit various carbon substrates, in pure or mixed form, for PHA production. Individual expression of PhaC1 and PhaC2 synthases in a non-PHA-producing strain, Cupriavidus necator H16 PHB¯4 (DSM 541), allows obtaining PHA production, demonstrating at the same time, functionality and differences between both PHA synthases. All the results of this study confirm the biotechnological interest in Halomonas sp. SF2003.

Antibiofilm activity in the culture supernatant of a marine Pseudomonas sp. bacterium.

In the marine environment, most solid surfaces are covered by microbial biofilms, mainly composed of bacteria and diatoms. The negative effects of biofilms on materials and equipment are numerous and pose a major problem for industry and human activities. Since marine micro-organisms are an important source of bioactive metabolites, it is possible that they synthesize natural ecofriendly molecules that inhibit the adhesion of organisms. In this work, the antibiofilm potential of marine bacteria was investigated using Flavobacterium sp. II2003 as a target. This strain is potentially a pioneer strain of bacteria that was previously selected from marine biofilms for its strong biofilm-forming ability. The culture supernatants of 86 marine heterotrophic bacteria were tested for their ability to inhibit Flavobacterium sp. II2003 biofilm formation and the Pseudomonas sp. IV2006 strain was identified as producing a strong antibiofilm activity. The Pseudomonas sp. IV2006 culture supernatant (SNIV2006) inhibited Flavobacterium sp. II2003 adhesion without killing the bacteria or inhibiting its growth. Moreover, SNIV2006 had no effect on the Flavobacterium sp. II2003 cell surface hydrophilic/hydrophobic and general Lewis acid-base characteristics, but modified the surface properties of glass, making it on the whole more hydrophilic and more alkaline and significantly reducing bacterial cell adhesion. The glass-coating molecules produced by Pseudomonas sp. IV2006 were found to probably be polysaccharides, whereas the antibiofilm molecules contained in SNIV2006 and acting during the 2 h adhesion step on glass and polystyrene surfaces would be proteinaceous. Finally, SNIV2006 exhibited a broad spectrum of antibiofilm activity on other marine bacteria such as Flavobacterium species that are pathogenic for fish, and human pathogens in both the medical environment, such as Staphylococcus aureus and Pseudomonas aeruginosa, and in the food industry, such as Yersinia enterocolitica. Thus, a wide range of applications could be envisaged for the SNIV2006 compounds, both in aquaculture and human health.

Draft Genome Sequences of Four Pseudomonas aeruginosa Clinical Strains with Various Biofilm Phenotypes.

Biofilms produced by Pseudomonas aeruginosa present a serious threat to cystic fibrosis patients. Here, we report the draft genome sequences of four cystic fibrosis isolates displaying various mucoid and biofilm phenotypes. The estimated average genome size was about 6,255,986 ± 50,202 bp with a mean G+C content of 66.52 ± 0.06%.

Evaluation of immunomodulatory activities of essential oils by high content analysis.

Nowadays, research concerning immunomodulatory products are of great interest, particularly in the treatment of inflammatory diseases or the prevention of infectious diseases. These activities are usually evaluated on cell cultures, by tracking different factors requiring dedicated manipulation. Evaluation of the immunomodulatory activities of essential oils and pure compounds using several technics adapted to high content analysis is described in this study. This approach allows a multiparametric evaluation on a single cell batch, in order to obtain an overall response. The developed method is based on the simultaneous evaluation of phagocytosis, production of iNOS and secretion of IL-6, induced by contact of RAW 264.7 cells with LPS. The results highlight the immunomodulatory activities of cinnamon and clove essential oils. They also provide information, particularly concerning the inhibitory activity of mint essential oil, which inhibits the LPS-induced phagocytosis of RAW 264.7 cells by 42%, at 100 µg/ml. This work presents for the first time the adaptation of high content analyses for the monitoring of immunomodulatory activities of essential oils. This protocol could be adjustable to other cell types and supplemented by the evaluation of additional parameters.

Complete genome sequence of the halophilic PHA-producing bacterium Halomonas sp. SF2003: insights into its biotechnological potential.

A halophilic Gram-negative eubacterium was isolated from the Iroise Sea and identified as an efficient producer of polyhydroxyalkanoates (PHA). The strain, designated SF2003, was found to belong to the Halomonas genus on the basis of 16S rRNA gene sequence similarity. Previous biochemical tests indicated that the Halomonas sp. strain SF2003 is capable of supporting various culture conditions which sometimes can be constraining for marine strains. This versatility could be of great interest for biotechnological applications. Therefore, a complete bacterial genome sequencing and de novo assembly were performed using a PacBio RSII sequencer and Hierarchical Genome Assembly Process software in order to predict Halomonas sp. SF2003 metabolisms, and to identify genes involved in PHA production and stress tolerance. This study demonstrates the complete genome sequence of Halomonas sp. SF2003 which contains a circular 4,36 Mbp chromosome, and replaces the strain in a phylogenetic tree. Genes related to PHA metabolism, carbohydrate metabolism, fatty acid metabolism and stress tolerance were identified and a comparison was made with metabolisms of relative species. Genes annotation highlighted the presence of typical genes involved in PHA biosynthesis such as phaA, phaB and phaC and enabled a preliminary analysis of their organization and characteristics. Several genes of carbohydrates and fatty acid metabolisms were also identified which provided helpful insights into both a better knowledge of the intricacies of PHA biosynthetic pathways and of production purposes. Results show the strong versatility of Halomonas sp. SF2003 to adapt to various temperatures and salinity which can subsequently be exploited for industrial applications such as PHA production.

Extracytoplasmic function sigma factors in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa, like all members of the genus Pseudomonas, has the capacity to thrive in very different environments, ranging from water, plant roots, to animals, including humans to whom it can cause severe infections. This remarkable adaptability is reflected in the number of transcriptional regulators, including sigma factors in this bacterium. Among those, the 19 to 21 extracytoplasmic sigma factors (ECFσ) are endowed with different regulons and functions, including the iron starvation σ (PvdS, FpvI, HasI, FecI, FecI2 and others), the cell wall stress ECFσ AlgU, SigX and SbrI, and the unorthodox σVreI involved in the expression of virulence. Recently published data show that these ECFσ have separate regulons although presenting some cross-talk. We will present evidence that these different ECFσ are involved in the expression of different phenotypes, ranging from cell-wall stress response, production of extracellular polysaccharides, formation of biofilms, to iron acquisition.

Host Peptidic Hormones Affecting Bacterial Biofilm Formation and Virulence.

Bacterial biofilms constitute a critical problem in hospitals, especially in resuscitation units or for immunocompromised patients, since bacteria embedded in their own matrix are not only protected against antibiotics but also develop resistant variant strains. In the last decade, an original approach to prevent biofilm formation has consisted of studying the antibacterial potential of host communication molecules. Thus, some of these compounds have been identified for their ability to modify the biofilm formation of both Gram-negative and Gram-positive bacteria. In addition to their effect on biofilm production, a detailed study of the mechanism of action of these human hormones on bacterial physiology has allowed the identification of new bacterial pathways involved in biofilm formation. In this review, we focus on the impact of neuropeptidic hormones on bacteria, address some future therapeutic issues, and provide a new view of inter-kingdom communication.