Bacteroides thetaiotaomicron uses a widespread extracellular DNase to promote bile-dependent biofilm formation.

Bacteroides thetaiotaomicron is a gut symbiont that inhabits the mucus layer and adheres to and metabolizes food particles, contributing to gut physiology and maturation. Although adhesion and biofilm formation could be key features for B. thetaiotaomicron stress resistance and gut colonization, little is known about the determinants of B. thetaiotaomicron biofilm formation. We previously showed that the B. thetaiotaomicron reference strain VPI-5482 is a poor in vitro biofilm former. Here, we demonstrated that bile, a gut-relevant environmental cue, triggers the formation of biofilm in many B. thetaiotaomicron isolates and common gut Bacteroidales species. We determined that bile-dependent biofilm formation involves the production of the DNase BT3563 or its homologs, degrading extracellular DNA (eDNA) in several B. thetaiotaomicron strains. Our study therefore shows that, although biofilm matrix eDNA provides a biofilm-promoting scaffold in many studied Firmicutes and Proteobacteria, BT3563-mediated eDNA degradation is required to form B. thetaiotaomicron biofilm in the presence of bile.

Gliding motility proteins GldJ and SprB contribute to Flavobacterium columnare virulence.

Flavobacterium columnare causes columnaris disease in fish. Columnaris disease is incompletely understood, and adequate control measures are lacking. The type IX secretion system (T9SS) is required for F. columnare gliding motility and virulence. The T9SS and gliding motility machineries share some, but not all, components. GldN (required for gliding and for secretion) and PorV (involved in secretion but not required for gliding) are both needed for virulence, implicating T9SS-mediated secretion in virulence. The role of motility in virulence is uncertain. We constructed and analyzed sprB, sprF, and gldJ mutants that were defective for motility but that maintained T9SS function to understand the role of motility in virulence. Wild-type cells moved rapidly and formed spreading colonies. In contrast, sprB and sprF deletion mutants were partially defective in gliding and formed nonspreading colonies. Both mutants exhibited reduced virulence in rainbow trout fry. A gldJ deletion mutant was nonmotile, secretion deficient, and avirulent in rainbow trout fry. To separate the roles of GldJ in secretion and in motility, we generated gldJ truncation mutants that produce nearly full-length GldJ. Mutant gldJ563, which produces GldJ truncated at amino acid 563, was defective for gliding but was competent for secretion as measured by extracellular proteolytic activity. This mutant displayed reduced virulence in rainbow trout fry, suggesting that motility contributes to virulence. Fish that survived exposure to the sprB deletion mutant or the gldJ563 mutant exhibited partial resistance to later challenge with wild-type cells. The results aid our understanding of columnaris disease and may suggest control strategies.IMPORTANCEFlavobacterium columnare causes columnaris disease in many species of freshwater fish in the wild and in aquaculture systems. Fish mortalities resulting from columnaris disease are a major problem for aquaculture. F. columnare virulence is incompletely understood, and control measures are inadequate. Gliding motility and protein secretion have been suggested to contribute to columnaris disease, but evidence directly linking motility to disease was lacking. We isolated and analyzed mutants that were competent for secretion but defective for motility. Some of these mutants exhibited decreased virulence. Fish that had been exposed to these mutants were partially protected from later exposure to the wild type. The results contribute to our understanding of columnaris disease and may aid development of control strategies.

Analysis of in-patient evolution of Escherichia coli reveals potential links to relapse of bone and joint infections.

Bone and joint infections (BJIs) are difficult to treat and affect a growing number of patients, in which relapses are observed in 10-20% of the case. These relapses, which call for prolonged antibiotic treatment and increase resistance emergence risk, may originate from ill understood adaptation of the pathogen to the host. Here, we investigated three pairs of Escherichia coli strains from BJI cases and their relapses to unravel in-patient adaptation. Whole genome comparison presented evidence for positive selection and phenotypic characterization showed that biofilm formation remained unchanged, contrary to what is usually described in such cases. Although virulence was not modified, we identified the loss of two virulence factors contributing to immune system evasion in one of the studied strains. Other strategies, including global growth optimization and colicin production, likely allowed the strains to outcompete competitors. This work highlights the variety of strategies allowing in-patient adaptation in BJIs.

Gnotobiotic rainbow trout (Oncorhynchus mykiss) model reveals endogenous bacteria that protect against Flavobacterium columnare infection.

The health and environmental risks associated with antibiotic use in aquaculture have promoted bacterial probiotics as an alternative approach to control fish infections in vulnerable larval and juvenile stages. However, evidence-based identification of probiotics is often hindered by the complexity of bacteria-host interactions and host variability in microbiologically uncontrolled conditions. While these difficulties can be partially resolved using gnotobiotic models harboring no or reduced microbiota, most host-microbe interaction studies are carried out in animal models with little relevance for fish farming. Here we studied host-microbiota-pathogen interactions in a germ-free and gnotobiotic model of rainbow trout (Oncorhynchus mykiss), one of the most widely cultured salmonids. We demonstrated that germ-free larvae raised in sterile conditions displayed no significant difference in growth after 35 days compared to conventionally-raised larvae, but were extremely sensitive to infection by Flavobacterium columnare, a common freshwater fish pathogen causing major economic losses worldwide. Furthermore, re-conventionalization with 11 culturable species from the conventional trout microbiota conferred resistance to F. columnare infection. Using mono-re-conventionalized germ-free trout, we identified that this protection is determined by a commensal Flavobacterium strain displaying antibacterial activity against F. columnare. Finally, we demonstrated that use of gnotobiotic trout is a suitable approach for the identification of both endogenous and exogenous probiotic bacterial strains protecting teleostean hosts against F. columnare. This study therefore establishes an ecologically-relevant gnotobiotic model for the study of host-pathogen interactions and colonization resistance in farmed fish.

Type IX Secretion System Effectors and Virulence of the Model Flavobacterium columnare Strain MS-FC-4.

Flavobacterium columnare causes columnaris disease in wild and cultured freshwater fish and is a major problem for sustainable aquaculture worldwide. The F. columnare type IX secretion system (T9SS) secretes many proteins and is required for virulence. The T9SS component GldN is required for secretion and gliding motility over surfaces. Genetic manipulation of F. columnare is inefficient, which has impeded identification of secreted proteins that are critical for virulence. Here, we identified a virulent wild-type F. columnare strain (MS-FC-4) that is highly amenable to genetic manipulation. This facilitated isolation and characterization of two deletion mutants lacking core components of the T9SS. Deletion of gldN disrupted protein secretion and gliding motility and eliminated virulence in zebrafish and rainbow trout. Deletion of porV disrupted secretion and virulence but not motility. Both mutants exhibited decreased extracellular proteolytic, hemolytic, and chondroitin sulfate lyase activities. They also exhibited decreased biofilm formation and decreased attachment to fish fins and other surfaces. Using genomic and proteomic approaches, we identified proteins secreted by the T9SS. We deleted 10 genes encoding secreted proteins and characterized the virulence of mutants lacking individual or multiple secreted proteins. A mutant lacking two genes encoding predicted peptidases exhibited reduced virulence in rainbow trout, and mutants lacking a predicted cytolysin showed reduced virulence in zebrafish and rainbow trout. The results establish F. columnare strain MS-FC-4 as a genetically amenable model to identify virulence factors. This may aid development of measures to control columnaris disease and impact fish health and sustainable aquaculture. IMPORTANCE Flavobacterium columnare causes columnaris disease in wild and aquaculture-reared freshwater fish and is a major problem for aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. The type IX secretion system (T9SS) secretes many proteins and is required for virulence, but the secreted virulence factors are not known. We identified a strain of F. columnare (MS-FC-4) that is well suited for genetic manipulation. The components of the T9SS and the proteins secreted by this system were identified. Deletion of core T9SS genes eliminated virulence. Genes encoding 10 secreted proteins were deleted. Deletion of two peptidase-encoding genes resulted in decreased virulence in rainbow trout, and deletion of a cytolysin-encoding gene resulted in decreased virulence in rainbow trout and zebrafish. Secreted peptidases and cytolysins are likely virulence factors and are targets for the development of control measures.

Autotransporters Drive Biofilm Formation and Autoaggregation in the Diderm Firmicute Veillonella parvula.

The Negativicutes are a clade of the Firmicutes that have retained the ancestral diderm character and possess an outer membrane. One of the best studied Negativicutes, Veillonella parvula, is an anaerobic commensal and opportunistic pathogen inhabiting complex human microbial communities, including the gut and the dental plaque microbiota. Whereas the adhesion and biofilm capacities of V. parvula are expected to be crucial for its maintenance and development in these environments, studies of V. parvula adhesion have been hindered by the lack of efficient genetic tools to perform functional analyses in this bacterium. Here, we took advantage of a recently described naturally transformable V. parvula isolate, SKV38, and adapted tools developed for the closely related Clostridia spp. to perform random transposon and targeted mutagenesis to identify V. parvula genes involved in biofilm formation. We show that type V secreted autotransporters, typically found in diderm bacteria, are the main determinants of V. parvula autoaggregation and biofilm formation and compete with each other for binding either to cells or to surfaces, with strong consequences for V. parvula biofilm formation capacity. The identified trimeric autotransporters have an original structure compared to classical autotransporters identified in Proteobacteria, with an additional C-terminal domain. We also show that inactivation of the gene coding for a poorly characterized metal-dependent phosphohydrolase HD domain protein conserved in the Firmicutes and their closely related diderm phyla inhibits autotransporter-mediated biofilm formation. This study paves the way for further molecular characterization of V. parvula interactions with other bacteria and the host within complex microbiota environments.IMPORTANCEVeillonella parvula is an anaerobic commensal and opportunistic pathogen whose ability to adhere to surfaces or other bacteria and form biofilms is critical for it to inhabit complex human microbial communities such as the gut and oral microbiota. Although the adhesive capacity of V. parvula has been previously described, very little is known about the underlying molecular mechanisms due to a lack of genetically amenable Veillonella strains. In this study, we took advantage of a naturally transformable V. parvula isolate and newly adapted genetic tools to identify surface-exposed adhesins called autotransporters as the main molecular determinants of adhesion in this bacterium. This work therefore provides new insights on an important aspect of the V. parvula lifestyle, opening new possibilities for mechanistic studies of the contribution of biofilm formation to the biology of this major commensal of the oral-digestive tract.

Biofilm microenvironment induces a widespread adaptive amino-acid fermentation pathway conferring strong fitness advantage in Escherichia coli.

Bacterial metabolism has been studied primarily in liquid cultures, and exploration of other natural growth conditions may reveal new aspects of bacterial biology. Here, we investigate metabolic changes occurring when Escherichia coli grows as surface-attached biofilms, a common but still poorly characterized bacterial lifestyle. We show that E. coli adapts to hypoxic conditions prevailing within biofilms by reducing the amino acid threonine into 1-propanol, an important industrial commodity not known to be naturally produced by Enterobacteriaceae. We demonstrate that threonine degradation corresponds to a fermentation process maintaining cellular redox balance, which confers a strong fitness advantage during anaerobic and biofilm growth but not in aerobic conditions. Whereas our study identifies a fermentation pathway known in Clostridia but previously undocumented in Enterobacteriaceae, it also provides novel insight into how growth in anaerobic biofilm microenvironments can trigger adaptive metabolic pathways edging out competition with in mixed bacterial communities.

Biofilms 2018: A diversity of microbes and mechanisms.

The 8th ASM Conference on Biofilms was held in Washington D.C. on October 7-11, 2018. This very highly subscribed meeting represented a wide breadth of current research in biofilms, and included over 500 attendees, 12 sessions with 64 oral presentations, and four poster sessions with about 400 posters.

A Putative Type V Pilus Contributes to Bacteroides thetaiotaomicron Biofilm Formation Capacity.

Bacteroides thetaiotaomicron is a prominent anaerobic member of the healthy human gut microbiota. While the majority of functional studies on B. thetaiotaomicron addressed its impact on the immune system and the utilization of diet polysaccharides, B. thetaiotaomicron biofilm capacity and its contribution to intestinal colonization are still poorly characterized. We tested the natural adhesion of 34 B. thetaiotaomicron isolates and showed that although biofilm capacity is widespread among B. thetaiotaomicron strains, this phenotype is masked or repressed in the widely used reference strain VPI 5482. Using transposon mutagenesis followed by a biofilm positive-selection procedure, we identified VPI 5482 mutants with increased biofilm capacity corresponding to an alteration in the C-terminal region of BT3147, encoded by the BT3148-BT3147 locus, which displays homology with Mfa-like type V pili found in many Bacteroidetes We show that BT3147 is exposed on the B. thetaiotaomicron surface and that BT3147-dependent adhesion also requires BT3148, suggesting that BT3148 and BT3147 correspond to the anchor and stalk subunits of a new type V pilus involved in B. thetaiotaomicron adhesion. This study therefore introduces B. thetaiotaomicron as a model to study proteinaceous adhesins and biofilm-related phenotypes in this important intestinal symbiont.IMPORTANCE Although the gut anaerobe Bacteroides thetaiotaomicron is a prominent member of the healthy human gut microbiota, little is known about its capacity to adhere to surfaces and form biofilms. Here, we identify that alteration of a surface-exposed protein corresponding to a type of pili found in many Bacteroidetes increases B. thetaiotaomicron biofilm formation. This study lays the ground for establishing this bacterium as a model organism for in vitro and in vivo studies of biofilm-related phenotypes in gut anaerobes.

Zinc Acetate Potentiates the Action of Tosufloxacin against Escherichia coli Biofilm Persisters.

Formation of bacterial biofilms is a major health threat due to their high levels of tolerance to multiple antibiotics and the presence of persisters responsible for infection relapses. We previously showed that a combination of starvation and induction of SOS response in biofilm led to increased levels of persisters and biofilm tolerance to fluoroquinolones. In this study, we hypothesized that inhibition of the SOS response may be an effective strategy to target biofilms and fluoroquinolone persister cells. We tested the survival of Escherichia coli biofilms to different classes of antibiotics in starved and nonstarved conditions and in the presence of zinc acetate, a SOS response inhibitor. We showed that zinc acetate potentiates, albeit moderately, the activity of fluoroquinolones against E. coli persisters in starved biofilms. The efficacy of zinc acetate to increase fluoroquinolone activity, particularly that of tosufloxacin, suggests that such a combination may be a potential strategy for treating biofilm-related bacterial infections.

Biofilms 2015: Multidisciplinary Approaches Shed Light into Microbial Life on Surfaces.

The 7th ASM Conference on Biofilms was held in Chicago, Illinois, from 24 to 29 October 2015. The conference provided an international forum for biofilm researchers across academic and industry platforms, and from different scientific disciplines, to present and discuss new findings and ideas. The meeting covered a wide range of topics, spanning environmental sciences, applied biology, evolution, ecology, physiology, and molecular biology of the biofilm lifestyle. This report summarizes the presentations with regard to emerging biofilm-related themes.

Systematic Nomenclature for GGDEF and EAL Domain-Containing Cyclic Di-GMP Turnover Proteins of Escherichia coli.

In recent years, Escherichia coli has served as one of a few model bacterial species for studying cyclic di-GMP (c-di-GMP) signaling. The widely used E. coli K-12 laboratory strains possess 29 genes encoding proteins with GGDEF and/or EAL domains, which include 12 diguanylate cyclases (DGC), 13 c-di-GMP-specific phosphodiesterases (PDE), and 4 "degenerate" enzymatically inactive proteins. In addition, six new GGDEF and EAL (GGDEF/EAL) domain-encoding genes, which encode two DGCs and four PDEs, have recently been found in genomic analyses of commensal and pathogenic E. coli strains. As a group of researchers who have been studying the molecular mechanisms and the genomic basis of c-di-GMP signaling in E. coli, we now propose a general and systematic dgc and pde nomenclature for the enzymatically active GGDEF/EAL domain-encoding genes of this model species. This nomenclature is intuitive and easy to memorize, and it can also be applied to additional genes and proteins that might be discovered in various strains of E. coli in future studies.

Functional analysis of Escherichia coli Yad fimbriae reveals their potential role in environmental persistence.

Initial adhesion of bacterial cells to surfaces or host tissues is a key step in colonisation and biofilm formation processes, and is mediated by cell surface appendages. It was previously demonstrated that Escherichia coli K-12 possesses an arsenal of silenced chaperone-usher fimbriae that were functional when constitutively expressed. Among them, production of prevalent Yad fimbriae induces adhesion to abiotic surfaces. Functional characterisation of Yad fimbriae were undertook, and YadN was identified as the most abundant and potential major pilin, and YadC as the potential tip-protein of Yad fimbriae. It was showed that Yad production participates to binding of E. coli K-12 to human eukaryotic cells (Caco-2) and inhibits macrophage phagocytosis, but also enhances E. coli K-12 binding to xylose, a major component of the plant cell wall, through its tip-lectin YadC. Consistently, it was demonstrated that Yad production provides E. coli with a competitive advantage in colonising corn seed rhizospheres. The latter phenotype is correlated with induction of Yad expression at temperatures below 37°C, and under anaerobic conditions, through a complex regulatory network. Taken together, these results suggest that Yad fimbriae are versatile adhesins that beyond potential capacities to modulate host-pathogen interactions might contribute to E. coli environmental persistence.

Starvation, together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin.

High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin-antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.

In vitro activity of gentamicin, vancomycin or amikacin combined with EDTA or l-arginine as lock therapy against a wide spectrum of biofilm-forming clinical strains isolated from catheter-related infections.

OBJECTIVES: Treatment of catheter-related bloodstream infections (CRBSI) is hampered by the characteristic tolerance of bacterial biofilms towards antibiotics. Our objective was to study the effect of the combination of antibiotics and the alkaline amino acid l-arginine or the cation chelator EDTA on the bacterial killing of in vitro biofilms formed by an array of clinical strains responsible for CRBSI and representative of epidemiologically relevant bacterial species. METHODS: Among 32 strains described in a previous clinical study, we focused on the most antibiotic-tolerant strains including CoNS (n = 4), Staphylococcus aureus (n = 4), Enterococcus faecalis (n = 2), Pseudomonas aeruginosa (n = 4) and Enterobacteriaceae (n = 4). We used an in vitro biofilm model (96-well plate assay) to study biofilm tolerance and tested various combinations of antibiotics and non-antibiotic adjuvants. Gentamicin, amikacin or vancomycin was combined with disodium EDTA or l-arginine for 24 h to reproduce the antibiotic lock therapy (ALT) approach. Killing of biofilm bacteria was measured by cfu quantification after a vigorous step of pipetting up and down in order to detach all biofilm bacteria from the surface of the wells. RESULTS: Both of our adjuvant strategies significantly increased the effect of antibiotics against biofilms formed by Gram-positive and Gram-negative bacterial pathogens. The combination of gentamicin + EDTA was active against all tested strains apart from one P. aeruginosa. The combination of gentamicin + l-arginine was active against most of the tested strains with the notable exception of CoNS for which no potentiation was observed. We also demonstrated that amikacin + EDTA was active against Gram-negative bacteria and that vancomycin + EDTA was active against Gram-positive bacteria. CONCLUSIONS: The addition of EDTA enhanced the activity of gentamicin, amikacin and vancomycin against biofilms formed by a wide spectrum of bacterial strains responsible for CRBSI.

ActA promotes Listeria monocytogenes aggregation, intestinal colonization and carriage.

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment.

pH-mediated potentiation of aminoglycosides kills bacterial persisters and eradicates in vivo biofilms.

BACKGROUND: Limitations in treatment of biofilm-associated bacterial infections are often due to subpopulation of persistent bacteria (persisters) tolerant to high concentrations of antibiotics. Based on the increased aminoglycoside efficiency under alkaline conditions, we studied the combination of gentamicin and the clinically compatible basic amino acid L-arginine against planktonic and biofilm bacteria both in vitro and in vivo. METHODS: Using Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli bioluminescent strains, we studied the combination of L-arginine and gentamicin against planktonic persisters through time-kill curves of late stationary-phase cultures. In vitro biofilm tolerance towards gentamicin was assessed using PVC 96 well-plates assays. Efficacy of gentamicin as antibiotic lock treatment (ALT) at 5 mg/mL at different pH was evaluated in vivo using a model of totally implantable venous access port (TIVAP) surgically implanted in rats. RESULTS: We demonstrated that a combination of gentamicin and the clinically compatible basic amino acid L-arginine increases in vitro planktonic and biofilm susceptibility to gentamicin, with 99% mortality amongst clinically relevant pathogens, i.e. S. aureus, E. coli and P. aeruginosa persistent bacteria. Moreover, although gentamicin local treatment alone showed poor efficacy in a clinically relevant in vivo model of catheter-related infection, gentamicin supplemented with L-arginine led to complete, long-lasting eradication of S. aureus and E. coli biofilms, when used locally. CONCLUSION: Given that intravenous administration of L-arginine to human patients is well tolerated, combined use of aminoglycoside and the non-toxic adjuvant L-arginine as catheter lock solution could constitute a new option for the eradication of pathogenic biofilms.

Preventing biofilm formation and associated occlusion by biomimetic glycocalyxlike polymer in central venous catheters.

The use of catheters and other implanted devices is constantly increasing in modern medicine. Although catheters improve patients' healthcare, the hydrophobic nature of their surface material promotes protein adsorption and cell adhesion. Catheters are therefore prone to complications, such as colonization by bacterial and fungal biofilms, associated infections, and thrombosis. Here we describe the in vivo efficacy of biologically inspired glycocalyxlike antiadhesive coatings to inhibit Staphylococcus aureus and Pseudomonas aeruginosa colonization on commercial totally implantable venous access ports (TIVAPs) in a clinically relevant rat model of biofilm infection. Although noncoated TIVAPs implanted in rats were heavily colonized by the 2 biofilm-forming pathogens with a high percentage of occlusion, coating TIVAPs reduced their initial adherence and subsequently led to 4-log reduction in biofilm formation and reduced occlusion. Our antiadhesive approach is a simple and generalizable strategy that could be used to minimize clinical complications associated with the use of implantable medical devices.

In vitro activities of dermaseptins K4S4 and K4K20S4 against Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa planktonic growth and biofilm formation.

The rising number of infections caused by biofilm formation and the difficulties associated with their treatment by conventional antimicrobial therapies have led to an intensive search for novel antibiofilm agents. Dermaseptins are antimicrobial peptides with a number of attractive properties that might offer alternative therapies against resistant microorganisms. In this study, we synthesized a set of dermaseptin-derived peptides and evaluated their activities against Gram-positive and Gram-negative bacterial biofilm formation. All dermaseptin-derived peptides demonstrated concentration-dependent antibiofilm activities at microgram concentrations, and their activities were dependent on the nature of the peptides, with the highest levels of activity being exhibited by highly charged molecules. Fluorescent binding and confocal microscopy demonstrated that dermaseptin K4S4, a substituted derivative of the native molecule S4, significantly decreased the viability of planktonic and surface-attached bacteria and stopped biofilm formation under dynamic flow conditions. Cytotoxicity assays with HeLa cells showed that some of the tested peptides were less cytotoxic than current antibiotics. Overall, these findings indicate that dermaseptin derivatives might constitute new lead structures for the development of potent antibiofilm agents.

A new zebrafish model of oro-intestinal pathogen colonization reveals a key role for adhesion in protection by probiotic bacteria.

The beneficial contribution of commensal bacteria to host health and homeostasis led to the concept that exogenous non-pathogenic bacteria called probiotics could be used to limit disease caused by pathogens. However, despite recent progress using gnotobiotic mammal and invertebrate models, mechanisms underlying protection afforded by commensal and probiotic bacteria against pathogens remain poorly understood. Here we developed a zebrafish model of controlled co-infection in which germ-free zebrafish raised on axenic living protozoa enabled the study of interactions between host and commensal and pathogenic bacteria. We screened enteric fish pathogens and identified Edwardsiella ictaluri as a virulent strain inducing a strong inflammatory response and rapid mortality in zebrafish larvae infected by the natural oro-intestinal route. Using mortality induced by infection as a phenotypic read-out, we pre-colonized zebrafish larvae with 37 potential probiotic bacterial strains and screened for survival upon E. ictaluri infection. We identified 3 robustly protective strains, including Vibrio parahaemolyticus and 2 Escherichia coli strains. We showed that the observed protective effect of E. coli was not correlated with a reduced host inflammatory response, nor with the release of biocidal molecules by protective bacteria, but rather with the presence of specific adhesion factors such as F pili that promote the emergence of probiotic bacteria in zebrafish larvae. Our study therefore provides new insights into the molecular events underlying the probiotic effect and constitutes a potentially high-throughput in vivo approach to the study of the molecular basis of pathogen exclusion in a relevant model of vertebrate oro-intestinal infection.