Streptococcus pyogenes Capsule Promotes Microcolony-Independent Biofilm Formation

Abstract

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.

Cellular chaining influences biofilm formation and structure in Group A Streptococcus

“…Similar to planktonic cultures, wild-type biofilm morphologies were restored in the mur1.2 complemented strain. The lack of a visible matrix was consistent with our previous studies on the same strain, in which no matrix was detected by SEM as well as fluorescent imaging after wheat germ agglutinin (WGA) staining (13). Although we did not observe major defects in cell division even at high magnification using SEM ( Figure 3 ), we noticed during SEM sample preparation that biofilms formed by Δ mur1.2 were more fragile and easily disrupted by handling.…”

“…Biofilms of JS95, and to a smaller extent, MGAS5005 and HSC5, were more stable in the presence of vigorous washing ( Figure 1b ), as compared to control biofilms grown in medium without lysozyme supplementation. Although we did not observe changes for strain JRS4, the biofilm of JRS4 is relatively strong and dense even prior to lysozyme treatment, most likely due to combined effects of strong primary adhesion, tendency to form microcolonies (34), and low levels of capsule production which has been shown to improve static biofilm formation (13, 35, 36). Consequently, the JRS4 biofilm likely represents the upper limit of biofilm formation, which lysozyme is unable to further augment.…”

“…CV assay was performed as described in our previous studies (9, 13) with minor modifications. Briefly, growth medium (THY + 0.5% glucose, referred in text as THY-G) was inoculated 1:100 with overnight culture in THY, transferred to 24-well polystyrene plates and incubated statically for 24 hours at 37°C in a 5% CO 2 atmosphere.…”

“…Furthermore, biofilm formation may play a role in GAS pathogenesis and disease by changing the bacterial transcriptional profile or by modulation of the host response (7-9). Investigations into GAS biofilm formation may yield new therapeutic targets and thus far, numerous bacterial factors, including virulence determinants such as M protein surface adhesin (10), pili (11, 12), capsule (10, 13), and secreted SpeB protease (14) have been implicated in GAS biofilm formation.…”

Singularities of Pyogenic Streptococcal Biofilms – From Formation to Health Implication

“…The IcaA protein is a N-glycosyltransferase – glycosyltransferases are responsible for the production of exopolysaccharides necessary for the production of the EPS matrix and establishment of microcolonies, and are found in S. mutans ( Koo et al, 2010 ). Recently, Matysik and Kline (2019) proposed a microcolony-independent mechanism for the S. pyogenes clinical strain, JS95. In this alternative mechanism, instead of forming microcolonies the cells will sediment and attach to the surface, with the capsule being of crucial importance.…”