Biofilm growth and colony variance in Streptococcus pneumoniae serotypes

Streptococcus pneumoniae is causatively attributed to both invasive and chronic diseases. The pneumococcus has been shown to form inherently resistant and surface-associated communities known as biofilms. However, little is known about the developmental process, the architecture and the transitional changes that occur upon biofilm development. In this study, biofilm formation was found to occur in three distinct stages, including initial attachment, cluster formation, and biofilm maturation. Proteomic analysis further confirmed the presence of distinct biofilm developmental stages by the detection of multiple phenotypes over the course of biofilm development. Using CLSM we have shown that mature biofilm architecture differed among various capsular serotypes resulting in three biofilm groups. Further analysis of two serotypes, serotype 3 and 19, representatives of group I and III, respectively, revealed the emergence of mucoid colony variants and a small non-mucoid phenotype (SCV) during biofilm formation. The colony variants differed in colony size and their mucoid appearance on blood agar. The SCV phenotype emerged and dominated during the initial attachment stage of biofilm formation while mucoid variants appeared to predominate during later biofilm developmental stages. Challenge with antimicrobial agents significantly decreased the SCV population. Unlike the mucoid phenotype, SCVs exhibited biofilm specific phenotypes including autoaggregation and hyperadherence to abiotic and biotic surfaces, such as lung epithelial cells. Hyperadherence was probably due to pili-like structures exclusively present in the SCV population. Interestingly, serotype 3 SCVs were present only in the nasopharynx of mice infected with pneumonia while WT and mucoid variants were detected in the nasopharynx and lung. Analysis of the serotype 3 capsule specific biosynthetic operon indicated that the emergence of non-mucoid variants was due to a >7 kb deletion comprising cps3DSU. In contrast to the stable serotype 3 SCVs, serotype 19 SCVs underwent reversion to a small mucoid phenotype at high frequency. Emergence of serotype 19 SCVs was found to coincide with the absence of cps19F transcript as well as base pair mutations within the same gene. Although further investigation is required to understand the regulation underlying S. pneumoniae colony variance, the emergence of phenotypes with distinct biofilm characteristics may promote adherence during nasopharyngeal colonization.