sodium-dodecyl-sulfate and Otitis-Media

The mechanisms that control Streptococcus pneumoniae's ability to colonize the nasopharynx or to invade the middle ear and cause acute otitis media are not understood. Focused study of these mechanisms requires efficient methods for the extraction of microbial RNA from minute clinical samples. Several lysis/extraction methods were tested and compared to determine the optimal conditions for isolating intact total RNA from pneumococcal cells. The sensitivity and efficiency of the extractions were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Compared to other methods, mechanical homogenization in TRIZOL was the most efficient for releasing microbial RNA, and addition of polyinosinic acid (Poly I) as an RNA carrier increased the assay sensitivity to 10(2) colony forming units when detected by RT-PCR amplification of 16S ribosomal RNA or messenger RNA for penicillin binding protein 2b. Quantitative results were confirmed using a ribonuclease protection assay. Penicillin binding protein 2b was also detected in rat middle ear mucosa recovered 5 weeks after middle ear challenge with S. pneumoniae. This study describes a useful core methodology for use in identifying pneumococcal virulence genes from small titer samples and has promising applications in clinical studies of pneumococcal nasopharyngeal colonization and otitis media pathogenesis.

Topics: Animals; Endopeptidase K; Endopeptidases; Guanidines; Humans; Otitis Media; Phenols; Pneumococcal Infections; Poly I; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Bacterial; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Sodium Dodecyl Sulfate; Streptococcus pneumoniae

To study serological antibody response to pneumococcal protein antigens, experimental pneumococcal (type 3) acute otitis media (AOM) was induced in 6 rats, sera being analysed with the Western blot technique at different intervals after bacterial challenge. The most striking finding was a distinct antibody response to a protein of about 35 kDa visible in 5 of the rat sera within 14 days, and persisting throughout the remainder of the study (i.e., 56 days in all). Moreover, appear to be a phenomenon restricted to type 3 pneumococci. Both the pathogenetic importance of this protein and the immunological response it evokes are still unclear. However, as antibodies to protein antigens may contribute to inflammatory reactions in the middle ear (e.g., otitis media with effusion), this 35-kDa protein might be important for the development of sequelae to AOM.

Topics: Acute Disease; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Blotting, Western; Electrophoresis, Polyacrylamide Gel; Male; Otitis Media; Polysaccharides, Bacterial; Rats; Rats, Sprague-Dawley; Sodium Dodecyl Sulfate; Streptococcus pneumoniae

Topics: Demeclocycline; Erythromycin; Humans; Infant; Otitis Media; Respiratory Tract Infections; Sodium Dodecyl Sulfate