Investigation of cultivation conditions for capsular polysaccharide production by Streptococcus pneumoniae serotype 14

Streptococcus pneumoniae (pneumococcus) is among the most significant causes of bacterial disease in humans. Capsular polysaccharide (CPS) production is essential for pneumococcal virulence. Pneumococcal CPS has been widely used as vaccine antigen. This study is focused on the influence of culture conditions of Streptococcus pneumoniae serotype 14 as for developing an industrial method for polysaccharide production. The pH proved to be a highly important variable in batchwise culture. Using the pH control all glucose added was consumed resulting in a four-fold increase in polysaccharide productivity relative to cultivation without pH control. S. pneumoniae is a lactic acid bacterium, so named for its primary metabolic byproduct (lactate), which has an inhibitory effect on cell growth in concentrations ranging from 4 to 5 g/L. An increase of 30 percent in polysaccharide productivity was observed using glucose pulses with 5.5 hrs of growth, resulting in a maximum polysaccharide concentration of 185.2 mg/L. Our data suggest the possibility of using a medium of non-animal origin and employing pH control for the cultivation of pneumococcus to produce a polysaccharide vaccine.

Characterization of Streptococcus pneumoniae ophthalmic, systemic & commensal isolates by pulsed-field gel electrophoresis & ribotyping.

BACKGROUND & OBJECTIVE: Streptococcus pneumoniae is common in ocular and systemic infections and is a part of normal nasopharyngeal flora. Very few studies regarding genetic analysis of S. pneumoniae isolates causing eye infections are available. This study was undertaken to do pulse field gel electrophoresis (PFGE) analysis and ribotyping of S. pneumoniae isolates obtained from eye infections, systemic infections and nasopharyngeal flora. METHODS: Sixty one well characterized S. pneumoniae isolates (38 from ophthalmic infections, 9 from systemic infections and 14 commensals) were characterized using PFGE of the whole genome after SmaI, restriction enzyme digestion and conventional ribotyping using Escherichia coli rRNA operon as the probe. Phylogenetic tree was drawn using unweighted pair group method analysis (UPGMA). RESULTS: The 38 S. pneumoniae isolates from eye infections belonging to 15 serotypes were placed in to 11 PFGE types and 15 ribotypes. The 9 systemic isolates (7 seotypes) were distributed in 7 PFGE types and 6 ribotypes. The 14 commensal isolates were placed in 11 serotypes, 5 PFGE types and 6 ribotypes. Most of the PFGE types and ribotypes consisting of ocular isolates also contained systemic and commensal isolates. INTERPRETATION & CONCLUSION: Considerable genetic similarity was observed between the isolates from ocular and systemic infections and those colonized in nasopharynx. PFGE analysis could differentiate majority of the isolates according to site of infections. There was a considerable DNA polymorphism within the studied bacterial population.

Interaction of human factor H with PspC of Streptococcus pneumoniae.

BACKGROUND & OBJECTIVES: Streptococcus pneumoniae has acquired virulence factors such as the polysaccharide capsule and various surface proteins, which prevent opsonization mediated by the complement system. PspC is one of the multi-functional pneumococcal surface proteins capable of eliciting an antibody response in mice. Our study further explores the role of pneumococcal surface proteins in resistance to complement mediated opsonophagocytosis by providing evidence that PspC binds human Factor H (FH), a regulatory protein of the alternative complement pathway. The present study was carried out to map the binding regions on PspC and FH, and to assess the functional activity of FH upon binding to PspC. METHODS: FH binding to D39 and other pneumococcal strains was observed by flow cytometry. A series of FH truncated and deletion mutants and PspC mutants were used to localize binding regions within these molecules. The functional activity of FH upon binding to PspC was measured by a haemolysis assay. RESULTS: FH binding to D39 and not to TRE108 (PspC-) cells was demonstrated by flow cytometry. Pneumococcal isolates of 14 different strains varied in their ability to bind FH. The binding region of FH within PspC to the first 225 amino acids of the alpha-helical domain was localized. The corresponding binding site for PspC is located within the SCR 6-10 region of FH. Haemolysis of rabbit red blood cells was inhibited by FH even in the presence of PspC. INTERPRETATION & CONCLUSION: FH binding is specific to PspC on the pneumococcal cell surface. The binding region on PspC mapped to the non-conserved N-terminal region of the alpha-helical domain. The binding site on FH to PspC is different from the active site that functions in degradation of C3b. A haemolysis assay provided evidence that the functional activity of FH was maintained upon binding to PspC. Thus, binding of FH to PspC might be an important mechanism by which S. pneumoniae resist complement activation and opsonophagocytosis.

Adherence of Streptococcus pneumoniae to buccal epithelial cells of smokers & non-smokers.

In vitro adherence of Streptococcus pneumoniae to buccal epithelial cells was assessed among smokers (51.9 +/- 20.8) and non-smokers (24.7 +/- 9.6) and found to be significantly higher (P less than 0.001) in smokers. The higher incidence of respiratory tract infection in smokers may be related to the enhanced adherence and better colonization of this bacterium in the throat.