Rosario Muñoz

Molecular basis of the optochin-sensitive phenotype of pneumococcus : characterization of the genes encoding the F0 complex of the Streptococcus pneumoniae and Streptococcus oralis H+-ATPases

The gene responsible for the optochin‐sensitive (OptS) phenotype of Streptococcus pneumoniae has been characterized. Sequence comparisons indicated that the genes involved encoded the subunits of the F0 complex of an H+‐ATPase. Sequence analysis and transformation experiments showed that the atpC gene is responsible for the optochin‐sensitive resistant (OptS/OptR) phenotype. Our results also show that natural as well as laboratory OptR isolates have arisen by point mutations that produce different amino acid changes at positions 48, 49 or 50 of the ATPase c subunit. The nucleotide sequence of the F F0 complex of the Streptococcus oralis ATPase has also been determined. In addition, comparison of the sequence of the atpCAB genes of S. pneumoniae R6 (OptS) and M222 (an OptR strain produced by inter‐species recombination between pneumococcus and S. oralis), and S. oralis revealed that, in M222, an interchange of atpC and atpA had occurred. We also demonstrate that optochin specifically inhibited the membrane‐bound ATPase activity of the S. pneumoniae wild‐type (OptS) strains, and found a 100‐fold difference between OptS and OptR strains, both in growth inhibition and in membrane ATPase resistance.

Optochin-resistant variants of Streptococcus pneumoniae

Ten Streptococcus pneumoniae clinical isolates, possessing physiologically typical pneumococcal characteristics, showed optochin-susceptible and optochin-resistant colonies around the optochin disk, when tested for optochin susceptibility. Equivocal optochin disk test results should be confirmed by bile solubility, agglutination tests, or both.

Identification of atypical strains ofStreptococcus pneumoniae by a specific DNA probe

A specific DNA probe containing a 0.65 Kb fragment coding for the amino-terminal region of the major pneumococcal autolysin (amidase) was constructed, deleting the region involved in the specific recognition of cell wall choline residues. The high specificity of this probe was demonstrated in tests withStreptococcus pneumoniae related species includingStreptococcus oralis, which contains choline in its cell wall. The probe was used to characterize pneumococcal isolates showing atypical responses in conventional identification tests. The hybridization obtained with 27 atypical pneumococci and 11 of 17 isolates presumptively identified as viridans streptococci confirmed that the probe is suitable for diagnostic use.

Analysis of protein antigens as a potential marker for Streptococcus pneumoniae.

Protein antigens of the five more commonStreptococcus pneumoniae serotypes in Spain — 1, 3, 4, 5 and 23 — were studied by crossed immunoelectrophoresis and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Most protein antigens defined by crossed immunoelectrophoresis appeared to be common to the five serotypes studied since the degree of cross-reaction was high. Using gel electrophoresis a considerable degree of similarity in the patterns of protein bands was also observed. Thus, protein antigens would not appear to be an epidemiological marker for typing pneumococci.