Association between opaque variants and serotypes in invasive isolates of Streptococcus pneumoniae

Abstract number: 1733_1294

Serrano I., Melo-Cristino J., Ramirez M.

Objectives: Determine the opacity phenotype of Streptococcus pneumoniae responsible for invasive human infections and probe the relationship between serotype and opacity phenotype.

Methods: The phenotype of 304 invasive isolates from Portugal (1999–2002) analysed previously and representing the 10 most prevalent serotypes was determined. Three possible phenotypes were considered, opaque (O), transparent (T) and intermediate (I). For the scoring of the dominant phenotype, 10⁴ cfu were plated on a transparent medium and incubated at 37°C in a candle extinction jar for 16 h. The colony morphology was determined using a stereomicroscope and oblique transmitted illumination.

Results: The visual inspection of the plates allowed unambiguous identification of the phenotype of the strain, which was considered to be the phenotype exhibited by >70% of the colonies. 18 strains could not be classified according to this criterium. The majority of the isolates classified (52%, n = 158) were O variants and only 26% (n = 79) were T variants while the remaining 16% (n = 49) were I phenotype. We found a striking difference in prevalence of each phenotype among serotypes, therefore serotype specific empirical odds ratio (OR) and 95% confidence intervals (CI) were calculated by reference to all other serotypes. In this analysis strains classified as I variants were excluded. Serotypes 1, 4 and 23F presented a higher association with the opaque phenotype than expected by chance (OR > 1), whereas serotypes 3 and 14 were more associated with the T phenotype than expected (OR < 1). The serotypes 9V, 8, 19A and 7F were not statistically associated with the O phenotype and the calculation of OR was not applicable to serotype 12B. We found no relationship between the O phenotype and the different genetic backgrounds of the isolates as determined by PFGE and MLST.

Conclusion: The O phenotype dominated on a 2:1 ratio over T phenotype among our collection of invasive isolates representing 10 serotypes, suggesting a role for phase variation in pneumococcal human infections. This observation highlights the heterogeneity of opacity phenotypes in invasive isolates and lends further support to the proposal that other factors, in addition to the site of isolation, determine the opacity phenotype of a given isolate. The association between serotype and colonial opacity could help explain epidemiological differences observed among pneumococcal serotypes such as a higher invasive disease potential.