Effect of xylitol and other carbon sources on Streptococcus pneumoniae biofilm formation and gene expression in vitro.

Xylitol inhibits the growth of Streptococcus pneumoniae. In clinical trials, xylitol decreased the occurrence of acute otitis media in day-care children, but did not decrease nasopharyngeal carriage of pneumococci. We hypothesized that xylitol inhibits biofilm formation of pneumococci, and measured biofilm formation and gene expression levels of the capsule gene cpsB and two other genes: autolysin encoding gene lytA and competence gene comA in different growth media in vitro. Twenty pneumococcal isolates were grown on polystyrene plates for 18 h in test media containing 0.5% xylitol, 0.5% glucose, 0.5% xylitol and 0.5% glucose, 0.5% fructose, 0.5% xylitol and 0.5% fructose or brain heart infusion (BHI) medium supplemented with 10% horse serum. Gene expression levels were measured after 5 h of growth using a relative quantification method with calibrator normalization. Exposure to xylitol lowered OD values, which were used as an indication of biofilm, compared with BHI medium, but when the medium was supplemented with glucose or fructose, biofilm formation was enhanced and the inhibitory effect of xylitol on biofilm formation was not observed. Xylitol also lowered lytA expression levels. Changes in biofilm formation in response to different sugar compounds may partly explain the efficacy of xylitol to prevent acute otitis media in previous clinical trials.

Presence of capsular locus genes in immunochemically identified encapsulated and unencapsulated Streptococcus pneumoniae sputum isolates obtained from elderly patients with acute lower respiratory tract infection.

The principal virulence factor of Streptococcus pneumoniae is capsular polysaccharide, and encapsulated pneumococci are more common causes of disease than unencapsulated strains. This study analysed the presence of capsular genes in 59 pneumococcal isolates using two PCR methods targeted at the cpsA and cpsB genes of the capsular biosynthesis locus. The PCR method targeted at the cpsB gene, reported to be essential for encapsulation, was developed in this study. Of 59 pneumococcal isolates, 49 (83 %) were obtained from the sputum samples of elderly patients (≥65 years) with community-acquired pneumonia (CAP) and 10 (17 %) were from those with other acute lower respiratory tract infections (ARIs). Forty (82 %) of the CAP isolates and two (20 %) of the ARI isolates were encapsulated, as assessed by conventional immunochemical methods. Forty-one (98 %) of the 42 encapsulated strains had the cpsB gene present, and in 38 strains the cpsA gene was also detected. One of the unencapsulated isolates gave a positive result for the cpsB gene, and neither of the capsular locus genes were present in all the other unencapsulated strains. The distribution of encapsulated and unencapsulated isolates differed significantly between the two patient groups regardless of whether the presence of capsule was determined immunochemically (P<0.001) or by cpsB PCR (P=0.002). The cpsB PCR developed here was found to be a rapid and reliable method to detect the pneumococcal capsule locus and may have potential in sputum diagnostics when investigating the pneumococcal aetiology of CAP.

Xylitol and capsular gene expression in Streptococcus pneumoniae.

Xylitol is a sugar alcohol that inhibits the growth and adherence of Streptococcus pneumoniae. In clinical trials, xylitol has been shown to decrease the occurrence of acute otitis media in day-care children but did not decrease nasopharyngeal carriage of the pneumococci. It has also been shown that xylitol affects the ultrastructure of the pneumococcal capsule. Here, it was hypothesized that xylitol might affect the expression of pneumococcal capsular genes. Capsule gene expression levels were studied in 24 clinical pneumococcal isolates and one ATCC strain (49619) by using a real-time RT-PCR method targeting the mRNA of the second gene of the pneumococcal capsular locus, the cpsB gene. The isolates were exposed to 5 % glucose, 5 % xylitol and control medium (brain heart infusion medium containing 10 % fetal bovine serum) for 2 h. cpsB gene expression levels were measured by using a relative quantification method with calibrator normalization where the 16S rRNA gene of pneumococcus was used as a reference. Exposure to xylitol lowered cpsB gene expression levels significantly compared with those in the control (P=0.035) and glucose (P=0.011) media. This finding supports previous results where exposure to xylitol changed the ultrastructure of the pneumococcal capsule and could explain further the high clinical efficacy of xylitol in preventing otitis media.