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Protective mechanisms of respiratory tract Streptococci against Streptococcus pyogenes biofilm formation and epithelial cell infection.

ABSTRACT: Streptococcus pyogenes (group A streptococci [GAS]) encounter many streptococcal species of the physiological microbial biome when entering the upper respiratory tract of humans, leading to the question how GAS interact with these bacteria in order to establish themselves at this anatomic site and initiate infection. Here we show that S. oralis and S. salivarius in direct contact assays inhibit growth of GAS in a strain-specific manner and that S. salivarius, most likely via bacteriocin secretion, also exerts this effect in transwell experiments. Utilizing scanning electron microscopy documentation, we identified the tested strains as potent biofilm producers except for GAS M49. In mixed-species biofilms, S. salivarius dominated the GAS strains, while S. oralis acted as initial colonizer, building the bottom layer in mixed biofilms and thereby allowing even GAS M49 to form substantial biofilms on top. With the exception of S. oralis, artificial saliva reduced single-species biofilms and allowed GAS to dominate in mixed biofilms, although the overall two-layer structure was unchanged. When covered by S. oralis and S. salivarius biofilms, epithelial cells were protected from GAS adherence, internalization, and cytotoxic effects. Apparently, these species can have probiotic effects. The use of Affymetrix array technology to assess HEp-2 cell transcription levels revealed modest changes after exposure to S. oralis and S. salivarius biofilms which could explain some of the protective effects against GAS attack. In summary, our study revealed a protection effect of respiratory tract bacteria against an important airway pathogen and allowed a first in vitro insight into local environmental processes after GAS enter the respiratory tract.

SUBMITTER: Fiedler T

PROVIDER: S-EPMC3568619 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Protective mechanisms of respiratory tract Streptococci against Streptococcus pyogenes biofilm formation and epithelial cell infection.

Fiedler Tomas T Riani Catur C Koczan Dirk D Standar Kerstin K Kreikemeyer Bernd B Podbielski Andreas A

Applied and environmental microbiology 20121214 4

Streptococcus pyogenes (group A streptococci [GAS]) encounter many streptococcal species of the physiological microbial biome when entering the upper respiratory tract of humans, leading to the question how GAS interact with these bacteria in order to establish themselves at this anatomic site and initiate infection. Here we show that S. oralis and S. salivarius in direct contact assays inhibit growth of GAS in a strain-specific manner and that S. salivarius, most likely via bacteriocin secretio ...[more]

PMID: 23241973

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Project description:Biofilms play an important role in the pathogenesis of group A streptococcus (GAS), a Gram-positive pathogen responsible for a wide range of infections and with a significant public health impact. Although most GAS serotypes are able to form biofilms, there is a large amount of heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes the initial adhesion of bacterial cells to a surface followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional (3D) biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or nonreplicating late-exponential- or early-stationary-phase planktonic cells via sedimentation and fixation of GAS chains. In addition, we show that the GAS capsule specifically contributes to the alternative sedimentation-initiated biofilms. Microcolony-independent sedimentation biofilms are similar in morphology and 3D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate noncanonical mechanism that does not require transition from microcolony formation to biofilm maturation and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes.IMPORTANCE The static biofilm assay is a common tool for easy biomass quantification of biofilm-forming bacteria. However, Streptococcus pyogenes biofilm formation as measured by the static assay is strain dependent and yields heterogeneous results for different strains of the same serotype. In this study, we show that two independent mechanisms, for which the protective capsule contributes opposing functions, may contribute to static biofilm formation. We propose that separation of these mechanisms for biofilm formation might uncover previously unappreciated biofilm phenotypes that may otherwise be masked in the classic static assay.

Project description:BACKGROUND:Infections of the ears, paranasal sinuses, nose and throat are very common and represent a serious issue for the healthcare system. Bacterial biofilms have been linked to upper respiratory tract infections and antibiotic resistance, raising serious concerns regarding the therapeutic management of such infections. In this context, novel strategies able to fight biofilms may be therapeutically beneficial and offer a valid alternative to conventional antimicrobials. Biofilms consist of mixed microbial communities, which interact with other species in the surroundings and communicate through signaling molecules. These interactions may result in antagonistic effects, which can be exploited in the fight against infections in a sort of "bacteria therapy". Streptococcus salivarius and Streptococcus oralis are α-hemolytic streptococci isolated from the human pharynx of healthy individuals. Several studies on otitis-prone children demonstrated that their intranasal administration is safe and well tolerated and is able to reduce the risk of acute otitis media. The aim of this research is to assess S. salivarius 24SMB and S. oralis 89a for the ability to interfere with biofilm of typical upper respiratory tract pathogens. METHODS:To investigate if soluble substances secreted by the two streptococci could inhibit biofilm development of the selected pathogenic strains, co-cultures were performed with the use of transwell inserts. Mixed-species biofilms were also produced, in order to evaluate if the inhibition of biofilm formation might require direct contact. Biofilm production was investigated by means of a spectrophotometric assay and by confocal laser scanning microscopy. RESULTS:We observed that S. salivarius 24SMB and S. oralis 89a are able to inhibit the biofilm formation capacity of selected pathogens and even to disperse their pre-formed biofilms. Diffusible molecules secreted by the two streptococci and lowered pH of the medium revealed to be implied in the mechanisms of anti-biofilm activity. CONCLUSIONS:S. salivarius 24SMB and S. oralis 89a possess desirable characteristics as probiotic for the treatment and prevention of infections of the upper airways. However, the nature of the inhibition appear to be multifactorial and additional studies are required to get further insights.

Project description:The sequence of the fibronectin-binding domain of the fibronectin-binding protein of Streptococcus pyogenes (Sfb protein) was determined, and its role in streptococcal adherence was investigated by use of an Sfb fusion protein in adherence studies. A 1-kb DNA fragment coding for the binding domain of Sfb protein was cloned into the expression vector pEX31 to produce an Sfb fusion protein consisting of the N-terminal part of MS2 polymerase and a C-terminal fragment of the streptococcal protein. Induction of the vector promoter resulted in hyperexpression of fibronectin-binding fusion protein in the cytoplasm of the recombinant Escherichia coli cells. Sequence determination of the cloned 1-kb fragment revealed an in-frame reading frame for a 268-amino-acid peptide composed of a 37-amino-acid sequence which is completely repeated three times and incompletely repeated a fourth time. Cloning of one repeat into pEX31 resulted in expression of small fusion peptides that show fibronectin-binding activity, indicating that one repeat contains at least one binding domain. Each repeat exhibits two charged domains and shows high homology with the 38-amino-acid D3 repeat of the fibronectin-binding protein of Staphylococcus aureus. Sequence comparison with other streptococcal ligand-binding surface proteins, including M protein, failed to reveal significant homology, which suggests that Sfb protein represents a novel type of functional protein in S. pyogenes. The Sfb fusion protein isolated from the cytoplasm of recombinant cells was purified by fast protein liquid chromatography. It showed a strong competitive inhibition of fibronectin binding to S. pyogenes and of the adherence of bacteria to cultured epithelial cells. In contrast, purified streptococcal lipoteichoic acid showed only a weak inhibition of fibronectin binding and streptococcal adherence. These results demonstrate that Sfb protein is directly involved in the fibronectin-mediated adherence of S. pyogenes to epithelial cells.

Dataset Information

Protective mechanisms of respiratory tract Streptococci against Streptococcus pyogenes biofilm formation and epithelial cell infection.

Publications

Protective mechanisms of respiratory tract Streptococci against Streptococcus pyogenes biofilm formation and epithelial cell infection.

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