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Identification of ypqP as a New Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities.

ABSTRACT: In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SP? prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SP? prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities.

SUBMITTER: Sanchez-Vizuete P

PROVIDER: S-EPMC4272715 | biostudies-literature | 2015 Jan

REPOSITORIES: biostudies-literature

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Identification of ypqP as a New Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities.

Sanchez-Vizuete Pilar P Le Coq Dominique D Bridier Arnaud A Bridier Arnaud A Herry Jean-Marie JM Aymerich Stéphane S Briandet Romain R

Applied and environmental microbiology 20141017 1

In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species ("public goods"), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action ...[more]

PMID: 25326298

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Project description:Biofilm-associated polymicrobial infections, particularly those involving fungi and bacteria, are responsible for significant morbidity and mortality and tend to be challenging to treat. Candida albicans and Staphylococcus aureus specifically are considered leading opportunistic fungal and bacterial pathogens, respectively, mainly due to their ability to form biofilms on catheters and indwelling medical devices. However, the impact of mixed-species biofilm growth on therapy remains largely understudied. In this study, we investigated the influence of C. albicans secreted cell wall polysaccharides on the response of S. aureus to antibacterial agents in biofilm. Results demonstrated significantly enhanced tolerance for S. aureus to drugs in the presence of C. albicans or its secreted cell wall polysaccharide material. Fluorescence confocal time-lapse microscopy revealed impairment of drug diffusion through the mixed biofilm matrix. Using C. albicans mutant strains with modulated cell wall polysaccharide expression, exogenous supplementation, and enzymatic degradation, the C. albicans-secreted ?-1,3-glucan cell wall component was identified as the key matrix constituent providing the bacteria with enhanced drug tolerance. Further, antibody labeling demonstrated rapid coating of the bacteria by the C. albicans matrix material. Importantly, via its effect on the fungal biofilm matrix, the antifungal caspofungin sensitized the bacteria to the drugs. Understanding such symbiotic interactions with clinical relevance between microbial species in biofilms will greatly aid in overcoming the limitations of current therapies and in defining potential new targets for treating polymicrobial infections.The fungus Candida albicans and the bacterium Staphylococcus aureus are important microbial pathogens responsible for the majority of infections in hospitalized patients and are often coisolated from a host. In this study, we demonstrated that when grown together, the fungus provides the bacterium with enhanced tolerance to antimicrobial drugs. This process was mediated by polysaccharides secreted by the fungal cell into the environment. The biofilm matrix formed by these polysaccharides prevented penetration by the drugs and provided the bacteria with protection. Importantly, we show that by inhibiting the production of the fungal polysaccharides, a specific antifungal agent indirectly sensitized the bacteria to antimicrobials. Understanding the therapeutic implications of the interactions between these two diverse microbial species will aid in overcoming the limitations of current therapies and in defining new targets for treating complex polymicrobial infections.

Project description:BackgroundMethicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms.MethodsUSA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®).ResultsComparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001).ConclusionsThis sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.

Dataset Information

Identification of ypqP as a New Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities.

Publications

Identification of ypqP as a New Bacillus subtilis biofilm determinant that mediates the protection of Staphylococcus aureus against antimicrobial agents in mixed-species communities.

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