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.

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.

Biocontrol of Biofilm Formation: Jamming of Sessile-Associated Rhizobial Communication by Rhodococcal Quorum-Quenching.

Biofilms are complex structures formed by a community of microbes adhering to a surface and/or to each other through the secretion of an adhesive and protective matrix. The establishment of these structures requires a coordination of action between microorganisms through powerful communication systems such as quorum-sensing. Therefore, auxiliary bacteria capable of interfering with these means of communication could be used to prevent biofilm formation and development. The phytopathogen Rhizobium rhizogenes, which causes hairy root disease and forms large biofilms in hydroponic crops, and the biocontrol agent Rhodococcus erythropolis R138 were used for this study. Changes in biofilm biovolume and structure, as well as interactions between rhizobia and rhodococci, were monitored by confocal laser scanning microscopy with appropriate fluorescent biosensors. We obtained direct visual evidence of an exchange of signals between rhizobia and the jamming of this communication by Rhodococcus within the biofilm. Signaling molecules were characterized as long chain (C14) N-acyl-homoserine lactones. The role of the Qsd quorum-quenching pathway in biofilm alteration was confirmed with an R. erythropolis mutant unable to produce the QsdA lactonase, and by expression of the qsdA gene in a heterologous host, Escherichia coli. Finally, Rhizobium biofilm formation was similarly inhibited by a purified extract of QsdA enzyme.

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.

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.

Bioaccumulation of metallic trace elements and organic pollutants in marine sponges from the South Brittany Coast, France.

The purpose of this study was to compare the accumulation of metallic and organic pollutants in marine sponges with the oyster Crassostrea gigas used as sentinel species. The concentrations of 12 Metallic Trace Elements (MTEs), 16 Polycyclic Aromatic Hydrocarbons (PAHs), 7 PolyChlorinated Biphenyls (PCBs), and 3 organotin derivatives were measured in 7 marine sponges collected in the Etel River (South Brittany, France). Results indicated Al, Co, Cr, Fe, Pb, and Ti particularly accumulated in marine sponges such as Hymeniacidon perlevis and Raspailia ramosa at higher levels compared to oysters. At the opposite, Cu and Zn accumulated significantly at higher concentrations in oysters. Among PAHs analyzed, benzo(a)pyrene bioaccumulated in H. perlevis at levels up to 17-fold higher than in oysters. In contrast, PCBs bioaccumulated preferentially in oysters. Significant differences exist in the abilities of marine phyla and sponge species to accumulate organic and metallic pollutants however, among the few sponge species studied, H. perlevis showed impressive bioaccumulation properties. The use of this species as bioindicator and/or bioremediator near shellfish farming areas is also discussed.

Marine bacteria from the French Atlantic coast displaying high forming-biofilm abilities and different biofilm 3D architectures.

BACKGROUND: Few studies have reported the species composition of bacterial communities in marine biofilms formed on natural or on man-made existing structures. In particular, the roles and surface specificities of primary colonizers are largely unknown for most surface types. The aim of this study was to obtain potentially pioneering bacterial strains with high forming-biofilm abilities from two kinds of marine biofilms, collected from two different surfaces of the French Atlantic coast: an intertidal mudflat which plays a central role in aquaculture and a carbon steel structure of a harbour, where biofilms may cause important damages. RESULTS: A collection of 156 marine heterotrophic aerobic bacteria isolated from both biofilms was screened for their ability to form biofilms on polystyrene 96-well microtiter plates. Out of 25 strains able to build a biofilm in these conditions, only four bacteria also formed a thick and stable biofilm on glass surfaces under dynamic conditions. These strains developed biofilms with four different three-dimensional architectures when observed by confocal laser scanning microscopy: Flavobacterium sp. II2003 biofilms harboured mushroom-like structures, Roseobacter sp. IV3009 biofilms were quite homogeneous, Shewanella sp. IV3014 displayed hairy biofilms with horizontal fibres, whereas Roseovarius sp. VA014 developed heterogeneous and tousled biofilms. CONCLUSIONS: This work led for the first time to the obtaining of four marine bacterial strains, potentially pioneering bacteria in marine biofilms, able to adhere to at least two different surfaces (polystyrene and glass) and to build specific 3D biofilms. The four selected strains are appropriate models for a better understanding of the colonization of a surface as well as the interactions that can occur between bacteria in a marine biofilm, which are crucial events for the initiation of biofouling.

The Pseudomonas aeruginosa rhlG and rhlAB genes are inversely regulated and RhlG is not required for rhamnolipid synthesis.

BACKGROUND: Pseudomonas aeruginosa produces rhamnolipid biosurfactants involved in numerous phenomena including virulence. The transcriptional study of the rhlAB operon encoding two key enzymes for rhamnolipid synthesis led to the discovery of the quorum sensing system RhlRI. The latter positively controls the transcription of rhlAB, as well as of rhlC, which is required for di-rhamnolipid synthesis. The rhlG gene encodes an NADPH-dependent ß-ketoacyl reductase. Although it was reported to be required for the biosynthesis of the fatty acid part of rhamnolipids, its function in rhamnolipid synthesis was later questioned. The rhlG transcription and its role in rhamnolipid production were investigated here. RESULTS: Using 5'-RACE PCR, a luxCDABE-based transcriptional fusion, and quantitative reverse transcription-PCR, we confirmed two previously identified σ70- and σ54-dependent promoters and we identified a third promoter recognized by the extra-cytoplasmic function sigma factor AlgU. rhlG was inversely regulated compared to rhlAB and rhlC: the rhlG transcription was down-regulated in response to N-butyryl-l-homoserine lactone, the communication molecule of the RhlRI system, and was induced by hyperosmotic stress in an AlgU-dependent manner. Consistently with this transcriptional pattern, the single or double deletions of rhlG and PA3388, which forms an operon with rhlG, did not dramatically impair rhamnolipid synthesis. CONCLUSION: This first detailed study of rhlG transcription reveals a complex regulation involving three sigma factors and N-butyryl-l-homoserine lactone. We furthermore present evidences that RhlG does not play a key role in rhamnolipid synthesis.

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.

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.

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.

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.

Transcription of the oprF gene of Pseudomonas aeruginosa is dependent mainly on the SigX sigma factor and is sucrose induced.

The OprF porin is the major outer membrane protein of Pseudomonas aeruginosa. OprF is involved in several crucial functions, including cell structure, outer membrane permeability, environmental sensing, and virulence. The oprF gene is preceded by the sigX gene, which encodes the poorly studied extracytoplasmic function (ECF) sigma factor SigX. Three oprF promoters were previously identified. Two intertwined promoters dependent on σ(70) and SigX are located in the sigX-oprF intergenic region, whereas a promoter dependent on the ECF AlgU lies within the sigX gene. An additional promoter was found in the cmpX-sigX intergenic region. In this study, we dissected the contribution of each promoter region and of each sigma factor to oprF transcription using transcriptional fusions. In Luria-Bertani (LB) medium, the oprF-proximal region (sigX-oprF intergenic region) accounted for about 80% of the oprF transcription, whereas the AlgU-dependent promoter had marginal activity. Using the sigX mutant PAOSX, we observed that the SigX-dependent promoter was largely predominant over the σ(70)-dependent promoter. oprF transcription was increased in response to low NaCl or high sucrose concentrations, and this induced transcription was strongly impaired in the absence of SigX. The lack of OprF itself increased oprF transcription. Since these conditions led to cell wall alterations, oprF transcription could be activated by signals triggered by perturbation of the cell envelope.

Proteomic studies highlight outer-membrane proteins related to biofilm development in the marine bacterium Pseudoalteromonas sp. D41.

Bacterial biofilm development is conditioned by complex processes involving bacterial attachment to surfaces, growth, mobility, and exoproduct production. The marine bacterium Pseudoalteromonas sp. strain D41 is able to attach strongly onto a wide variety of substrates, which promotes subsequent biofilm development. Study of the outer-membrane and total soluble proteomes showed ten spots with significant intensity variations when this bacterium was grown in biofilm compared to planktonic cultures. MS/MS de novo sequencing analysis allowed the identification of four outer-membrane proteins of particular interest since they were strongly induced in biofilms. These proteins are homologous to a TonB-dependent receptor (TBDR), to the OmpW and OmpA porins, and to a type IV pilus biogenesis protein (PilF). Gene expression assays by quantitative RT-PCR showed that the four corresponding genes were upregulated during biofilm development on hydrophobic and hydrophilic surfaces. The Pseudomonas aeruginosa mutants unable to produce any of the OmpW, OmpA, and PilF homologues yielded biofilms with lower biovolumes and altered architectures, confirming the involvement of these proteins in the biofilm formation process. Our results indicate that Pseudoalteromonas sp. D41 shares biofilm formation mechanisms with human pathogenic bacteria, but also relies on TBDR, which might be more specific to the marine environment.

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.

Full virulence of Pseudomonas aeruginosa requires OprF.

OprF is a general outer membrane porin of Pseudomonas aeruginosa, a well-known human opportunistic pathogen associated with severe hospital-acquired sepsis and chronic lung infections of cystic fibrosis patients. A multiphenotypic approach, based on the comparative study of a wild-type strain of P. aeruginosa, its isogenic oprF mutant, and an oprF-complemented strain, showed that OprF is required for P. aeruginosa virulence. The absence of OprF results in impaired adhesion to animal cells, secretion of ExoT and ExoS toxins through the type III secretion system (T3SS), and production of the quorum-sensing-dependent virulence factors pyocyanin, elastase, lectin PA-1L, and exotoxin A. Accordingly, in the oprF mutant, production of the signal molecules N-(3-oxododecanoyl)-l-homoserine lactone and N-butanoyl-l-homoserine lactone was found to be reduced and delayed, respectively. Pseudomonas quinolone signal (PQS) production was decreased, while its precursor, 4-hydroxy-2-heptylquinoline (HHQ), accumulated in the cells. Taken together, these results show the involvement of OprF in P. aeruginosa virulence, at least partly through modulation of the quorum-sensing network. This is the first study showing a link between OprF, PQS synthesis, T3SS, and virulence factor production, providing novel insights into virulence expression.

C-type natriuretic peptide modulates quorum sensing molecule and toxin production in Pseudomonas aeruginosa.

Pseudomonas aeruginosa coordinates its virulence expression and establishment in the host in response to modification of its environment. During the infectious process, bacteria are exposed to and can detect eukaryotic products including hormones. It has been shown that P. aeruginosa is sensitive to natriuretic peptides, a family of eukaryotic hormones, through a cyclic nucleotide-dependent sensor system that modulates its cytotoxicity. We observed that pre-treatment of P. aeruginosa PAO1 with C-type natriuretic peptide (CNP) increases the capacity of the bacteria to kill Caenorhabditis elegans through diffusive toxin production. In contrast, brain natriuretic peptide (BNP) did not affect the capacity of the bacteria to kill C. elegans. The bacterial production of hydrogen cyanide (HCN) was enhanced by both BNP and CNP whereas the production of phenazine pyocyanin was strongly inhibited by CNP. The amount of 2-heptyl-4-quinolone (HHQ), a precursor to 2-heptyl-3-hydroxyl-4-quinolone (Pseudomonas quinolone signal; PQS), decreased after CNP treatment. The quantity of 2-nonyl-4-quinolone (HNQ), another quinolone which is synthesized from HHQ, was also reduced after CNP treatment. Conversely, both BNP and CNP significantly enhanced bacterial production of acylhomoserine lactone (AHL) [e.g. 3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and butanoylhomoserine lactone (C4-HSL)]. These results correlate with an induction of lasI transcription 1 h after bacterial exposure to BNP or CNP. Concurrently, pre-treatment of P. aeruginosa PAO1 with either BNP or CNP enhanced PAO1 exotoxin A production, via a higher toxA mRNA level. At the same time, CNP led to elevated amounts of algC mRNA, indicating that algC is involved in C. elegans killing. Finally, we observed that in PAO1, Vfr protein is essential to the pro-virulent effect of CNP whereas the regulator PtxR supports only a part of the CNP pro-virulent activity. Taken together, these data reinforce the hypothesis that during infection natriuretic peptides, particularly CNP, could enhance the virulence of PAO1. This activity is relayed by Vfr and PtxR activation, and a general diagram of the virulence activation cascade involving AHL, HCN and exotoxin A is proposed.

The anti-biofilm activity secreted by a marine Pseudoalteromonas strain.

Bacterial biofilms occur on all submerged structures in marine environments. The authors previously reported that the marine bacterium Pseudoalteromonas sp. 3J6 secretes antibiofilm activity. Here, it was discovered that another Pseudoalteromonas sp. strain, D41, inhibited the development of strain 3J6 in mixed biofilms. Confocal laser scanning microscope observations revealed that the culture supernatant of strain D41 impaired biofilm formation of strain 3J6 and another marine bacterium. A microtiter plate assay of the antibiofilm activity was set up and validated with culture supernatants of Pseudoalteromonas sp. 3J6. This assay was used to determine the spectra of action of strains D41 and 3J6. Each culture supernatant impaired the biofilm development of 13 marine bacteria out of 18. However, differences in the spectra of action and the physical behaviours of the antibiofilm molecules suggest that the latter are not identical. They nevertheless share the originality of being devoid of antibacterial activity against planktonic bacteria.