Mixed biofilms formed by Haemophilus influenzae and Streptococcus pneumoniae and expression of certain biofilm-related genes

Abstract number: P1800

Kulmuni E., Asumaniemi P., Kaijalainen T., Tapiainen T., Uhari M., Leinonen M., Saukkoriipi A.

Objectives: Nontypeable Haemophilus influenzae (NTHi) and Streptococcus pneumoniae (Pnc) cause otitis media, which has been suggested to be caused by bacteria residing in biofilms. Both NTHi and Pnc have been shown to be able to form biofilms. We studied biofilm formation of mixed cultures of Hi and Pnc in vitro and the expression of certain genes that have been associated to the biofilm form of growth in these bacteria.

Methods: Four H. influenzae (Hi; three NTHi and one type b) and 4 Pnc isolates (two unencapsulated, two encapsulated) were grown separately and combined on polystyrene microwell plates for 5, 18, and 22 h. For each time point there was one plate for crystal violet staining and another for RNA extraction. All strains and combinations were tested in triplicate. The expression levels of peroxiredoxin-glutaredoxin (pdgX) and outer membrane protein P6 genes of Hi and autolysin (lytA) and penicillin-binding protein 2x (pbp2x) genes of Pnc were studied by relative quantitative PCR where the 16 s rRNA genes of Hi and Pnc were used as reference genes.

Results: At 18 h, the unencapsulated Pnc isolates formed biofilms detectable by crystal violet staining whereas the Hi isolates did not. When an unencapsulated Pnc was grown together with an NTHi and Hib isolate, the OD540 value of the biofilm decreased from 0.31 to 0.07 and 0.09. However, when the unencapsulated Pnc was grown together with a capsulated Pnc, no difference was seen. Differences in gene expression were seen at 5 h. The mean expression levels of P6 were 3.79 and 3.32 in two of the nontypeable Hi isolates and 0.20 in the Hib isolate. When an unencapsulated Pnc isolate was grown with the NTHi and Hib, the mean expression level of P6 was 5.98 and 3.26, respectively. When the NTHi and Hib isolates were grown together, no increase in the expression levels were seen as compared to the NTHi alone (3.94 vs. 3.79). The expression of lytA and pbp2x at 5 h in one of the unencapsulated Pnc isolates were 1.38 and 0.59, respectively. When an NTHI or Hib was grown together with this isolate, the expression of lytA decreased to 0.41 and 0.37, respectively, and pbp2x to 0.13 and 0.26, respectively. Expression of these two genes in the other Pnc isolates was below 0.5 when grown separately and together with Hi.

Conclusion: Unencapsulated Pnc and Hi affect each other's biofilm formation and gene expression in vitro. Mixed biofilms may play a role e.g. in the pathogenesis of otitis media.

Session Details

Date: 16/05/2009
Time: 00:00-00:00
Session name: 19th European Congress of Clinical Microbiology and Infectious Diseases
Location: Helsinki, Finland, 16 - 19 May 2009
Presentation type:
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