An inverse correlation between colonization of the human nasopharynx by Streptococcus pneumoniae and Haemophilus influenzae , both common upper respiratory pathogens, has been reported. Coculture of S. pneumoniae and H. influenzae led to rapid killing of H. influenzae . Exogenous catalase prevented killing of H. influenzae , suggesting that hydrogen peroxide may be responsible for this bactericidal activity. S. pneumoniae which did not produce hydrogen peroxide were unable to kill H. influenzae . H 2 O 2 produced by S. pneumoniae also inhibited the growth of the respiratory tract pathogens Moraxella catarrhalis and Neisseria meningitidis .
Loss of function mutations were detected in seven genes, pspA, spxB, xba, licD2, lytA, nanA and atpC . Factors associated with these mutations included: (1) frameshifts caused by reversible gain and loss of single bases within homopolymeric repeats as short as six bases, (2) deletions caused by recombinational events between non-tandem direct repeats as short as eight bases, and (3) substitutions of guanine residues caused at an increased frequency by endogenous hydrogen peroxide production. The latter accounted for a spontaneous mutation rate as high as 2.8 × 10 -6 for resistance to optochin but was 10 to 200-fold lower in the absence of detectable H 2 O 2 .
Hydrogen peroxide is produced in S. pneumoniae by pyruvate oxidase (SpxB), with concentrations in the media often exceeding 1mM. SpxB mutants were found to exhibit 10 2 -10 3 -fold decreased survival in 20mM H 2 O 2 . SpxB activity was required, rather than a peroxide-inducible pathway. SpxB mutants had decreased levels of acetyl-phosphate, a potential source of ATP. During exposure to H 2 O 2 , ATP levels decreased more rapidly in SpxB mutants than in wild-type cells, suggesting their increased killing was due to more rapid ATP depletion. These data support the hypothesis that pyruvate oxidase maintains a reserve energy source that helps to maintain viability during oxidative stress. Although more H 2 O 2 -resistant than E. coli, S. pneumoniae contained similar amounts of free iron, suggesting that S. pneumoniae prevents iron from participating with H 2 O 2 in the production of hydroxy radicals or somehow avoids the effects of these radicals. The ability of S. pneumoniae to produce and survive high concentrations of H 2 O 2 may be important in colonization or disease.
