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Screening of Escherichia coli species biodiversity reveals new biofilm-associated antiadhesion polysaccharides.

Rendueles O, Travier L, Latour-Lambert P, Fontaine T, Magnus J, Denamur E, Ghigo JM - MBio (2011)

Bottom Line: Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.The use of natural strains representative of Escherichia coli species biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm).Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Génétique des Biofilms, Paris, France.

ABSTRACT

Unlabelled: Bacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted from in vitro mature biofilms formed by 122 natural Escherichia coli isolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animal E. coli strain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion of Staphylococcus aureus from mixed E. coli and S. aureus biofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.

Importance: We sought to demonstrate that bacterial biofilms are reservoirs for unknown molecules that antagonize bacterial adhesion. The use of natural strains representative of Escherichia coli species biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm). Release of an antiadhesion polysaccharide confers a competitive advantage upon the producing strain against clinically relevant pathogens such as Staphylococcus aureus. Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

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Production of antiadhesion E. coli Ec300 polysaccharide is regulated by rfaH. (A) Effect of nonconcentrated biofilm extracts from indicated strains on S. aureus biofilm formation. M63B1, control in which only M63B1 minimal medium was added. (B) Beta-galactosidase activity measurements of lacZ transcriptional fusions in genes located in contigs essential for production of E. coli Ec300 antiadhesion polysaccharide in a wild-type and ∆rfaH background. ∆1680, control; region not involved in Ec300p synthesis. Experiments were performed in triplicate; error bars represent standard deviations of the means. Statistical t tests were used to evaluate the significance of biofilm inhibition from various extracts compared to that of addition of M63B1. *, P < 0.05; ***, P < 0.001.
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f4: Production of antiadhesion E. coli Ec300 polysaccharide is regulated by rfaH. (A) Effect of nonconcentrated biofilm extracts from indicated strains on S. aureus biofilm formation. M63B1, control in which only M63B1 minimal medium was added. (B) Beta-galactosidase activity measurements of lacZ transcriptional fusions in genes located in contigs essential for production of E. coli Ec300 antiadhesion polysaccharide in a wild-type and ∆rfaH background. ∆1680, control; region not involved in Ec300p synthesis. Experiments were performed in triplicate; error bars represent standard deviations of the means. Statistical t tests were used to evaluate the significance of biofilm inhibition from various extracts compared to that of addition of M63B1. *, P < 0.05; ***, P < 0.001.

Mentions: In order to study the regulation of Ec300p production, we analyzed the antiadhesion activities of biofilm extracts obtained from strains carrying mutations corresponding to regulators previously associated with biofilm and polysaccharide regulation, including rscB, luxS, rpoS, oxyR, crr, crp, rpoN, phoP, hns, and rfaH (23). E. coli Ec300 rfaH was the only mutant to impair Ec300 antiadhesion activity against S. aureus biofilm (Fig. 4A). Consistently, introduction of a wild-type rfaH allele in trans at the lambda attachment site restored biofilm-associated antiadhesion activity in Ec300∆rfaH λrfaH (Fig. 4A). RfaH is an antiterminator that positively controls expression of long operons coding for extracellular components such as secreted virulence factors, lipopolysaccharide, and conjugation proteins. Although RfaH binds 12-bp JUMPstart motifs (5′-RGGCGGTAGYNT-3′) often located in the promoter-proximal 5′ region, analysis of the DNA region located in the E. coli Ec300 galF-hisE region corresponding to contigs 6759, 12208, and 1040 did not reveal any convincing JUMPstart motifs.


Screening of Escherichia coli species biodiversity reveals new biofilm-associated antiadhesion polysaccharides.

Rendueles O, Travier L, Latour-Lambert P, Fontaine T, Magnus J, Denamur E, Ghigo JM - MBio (2011)

Production of antiadhesion E. coli Ec300 polysaccharide is regulated by rfaH. (A) Effect of nonconcentrated biofilm extracts from indicated strains on S. aureus biofilm formation. M63B1, control in which only M63B1 minimal medium was added. (B) Beta-galactosidase activity measurements of lacZ transcriptional fusions in genes located in contigs essential for production of E. coli Ec300 antiadhesion polysaccharide in a wild-type and ∆rfaH background. ∆1680, control; region not involved in Ec300p synthesis. Experiments were performed in triplicate; error bars represent standard deviations of the means. Statistical t tests were used to evaluate the significance of biofilm inhibition from various extracts compared to that of addition of M63B1. *, P < 0.05; ***, P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3101779&req=5

f4: Production of antiadhesion E. coli Ec300 polysaccharide is regulated by rfaH. (A) Effect of nonconcentrated biofilm extracts from indicated strains on S. aureus biofilm formation. M63B1, control in which only M63B1 minimal medium was added. (B) Beta-galactosidase activity measurements of lacZ transcriptional fusions in genes located in contigs essential for production of E. coli Ec300 antiadhesion polysaccharide in a wild-type and ∆rfaH background. ∆1680, control; region not involved in Ec300p synthesis. Experiments were performed in triplicate; error bars represent standard deviations of the means. Statistical t tests were used to evaluate the significance of biofilm inhibition from various extracts compared to that of addition of M63B1. *, P < 0.05; ***, P < 0.001.
Mentions: In order to study the regulation of Ec300p production, we analyzed the antiadhesion activities of biofilm extracts obtained from strains carrying mutations corresponding to regulators previously associated with biofilm and polysaccharide regulation, including rscB, luxS, rpoS, oxyR, crr, crp, rpoN, phoP, hns, and rfaH (23). E. coli Ec300 rfaH was the only mutant to impair Ec300 antiadhesion activity against S. aureus biofilm (Fig. 4A). Consistently, introduction of a wild-type rfaH allele in trans at the lambda attachment site restored biofilm-associated antiadhesion activity in Ec300∆rfaH λrfaH (Fig. 4A). RfaH is an antiterminator that positively controls expression of long operons coding for extracellular components such as secreted virulence factors, lipopolysaccharide, and conjugation proteins. Although RfaH binds 12-bp JUMPstart motifs (5′-RGGCGGTAGYNT-3′) often located in the promoter-proximal 5′ region, analysis of the DNA region located in the E. coli Ec300 galF-hisE region corresponding to contigs 6759, 12208, and 1040 did not reveal any convincing JUMPstart motifs.

Bottom Line: Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.The use of natural strains representative of Escherichia coli species biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm).Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, Unité de Génétique des Biofilms, Paris, France.

ABSTRACT

Unlabelled: Bacterial biofilms often form multispecies communities in which complex but ill-understood competition and cooperation interactions occur. In light of the profound physiological modifications associated with this lifestyle, we hypothesized that the biofilm environment might represent an untapped source of natural bioactive molecules interfering with bacterial adhesion or biofilm formation. We produced cell-free solutions extracted from in vitro mature biofilms formed by 122 natural Escherichia coli isolates, and we screened these biofilm extracts for antiadhesion molecules active on a panel of Gram-positive and Gram-negative bacteria. Using this approach, we showed that 20% of the tested biofilm extracts contained molecules that antagonize bacterial growth or adhesion. We characterized a compound, produced by a commensal animal E. coli strain, for which activity is detected only in biofilm extract. Biochemical and genetic analyses showed that this compound corresponds to a new type of released high-molecular-weight polysaccharide whose biofilm-associated production is regulated by the RfaH protein. We demonstrated that the antiadhesion activity of this polysaccharide was restricted to Gram-positive bacteria and that its production reduced susceptibility to invasion and provided rapid exclusion of Staphylococcus aureus from mixed E. coli and S. aureus biofilms. Our results therefore demonstrate that biofilms contain molecules that contribute to the dynamics of mixed bacterial communities and that are not or only poorly detected in unconcentrated planktonic supernatants. Systematic identification of these compounds could lead to strategies that limit pathogen surface colonization and reduce the burden associated with the development of bacterial biofilms on medical devices.

Importance: We sought to demonstrate that bacterial biofilms are reservoirs for unknown molecules that antagonize bacterial adhesion. The use of natural strains representative of Escherichia coli species biodiversity showed that nonbiocidal antiadhesion polysaccharides are frequently found in mature biofilm extracts (bacterium-free suspensions which contain soluble molecules produced within the biofilm). Release of an antiadhesion polysaccharide confers a competitive advantage upon the producing strain against clinically relevant pathogens such as Staphylococcus aureus. Hence, exploring the biofilm environment provides a better understanding of bacterial interactions within complex communities and could lead to improved control of pathogen colonization.

Show MeSH
Related in: MedlinePlus