Inhibitory and bactericidal effects of hydrogen peroxide production by Streptococcus pneumoniae on other inhabitants of the upper respiratory tract

Abstract

An inverse correlation between colonization of the human nasopharynx by Streptococcus pneumoniae and Haemophilus influenzae, both common upper respiratory pathogens, has been reported. Studies were undertaken to determine if either of these organisms produces substances which inhibit growth of the other. Culture supernatants from S. pneumoniae inhibited growth of H. influenzae, whereas culture supernatants from H. influenzae had no effect on the growth of S. pneumoniae. Moreover, coculture of S. pneumoniae and H. influenzae led to a rapid decrease in viable counts of H. influenzae. The addition of purified catalase prevented killing of H. influenzae in coculture experiments, suggesting that hydrogen peroxide may be responsible for this bactericidal activity. H. influenzae was killed by concentrations of hydrogen peroxide similar to that produced by S. pneumoniae. Hydrogen peroxide is produced by the pneumococcus through the action of pyruvate oxidase (SpxB) under conditions of aerobic growth. Both an spxB mutant and a naturally occurring variant of S. pneumoniae, which is downregulated in SpxB expression, were unable to kill H. influenzae. A catalase-reversible inhibitory effect of S. pneumoniae on the growth of the respiratory tract pathogens Moraxella catarrhalis and Neisseria meningitidis was also observed. Elevated hydrogen peroxide production, therefore, may be a means by which S. pneumoniae is able to inhibit a variety of competing organisms in the aerobic environment of the upper respiratory tract.

FIG. 1

Effect of coculture of S. pneumoniae P394 and H. influenzae Rd. Following growth to mid-log phase, H. influenzae was washed and incubated in sBHI containing heat-inactivated catalase either with (□) or without (○) S. pneumoniae for the time indicated, and viable counts were determined in duplicate on selective media. Viable counts of S. pneumoniae incubated in coculture with (■) or without (●) H. influenzae were determined in duplicate by plating on selective media. The same amount of active catalase (1,000 U/ml) was included during coculture of S. pneumoniae (▴) and H. influenzae (▵). Values represent the average of three independent determinations in duplicate, and the error bars represent the standard deviations.

FIG. 2

Dose-dependent killing of H. influenzae Rd by S. pneumoniae P394. Following growth to mid-log phase, H. influenzae was washed and cultured alone (triangles) or with 10⁵ (squares), 10⁶ (circles), or 10⁷ (diamonds) CFU of S. pneumoniae per ml and incubated in sBHI for the times indicated; viable counts were determined on selective media. Values represent the average of two independent determinations in duplicate.

FIG. 3

Effect of coculture of H. influenzae Rd with S. pneumoniae D39 and its spxB mutant, P878. Following growth to mid-log phase, H. influenzae was washed and incubated in sBHI alone (○), with D39 (□), or with P878 (▵) for the times indicated, and viable counts were determined in duplicate on selective media. Viable counts of D39 (■) or P878 (▴) incubated in coculture with H. influenzae were determined in duplicate by plating on selective media. Values represent the average of three independent determinations in duplicate, and the error bars represent the standard deviations.

FIG. 4

Effect of coculture of S. pneumoniae P394 with either M. catarrhalis (Bc1) or N. meningitidis (MC58C3). Following growth to mid-log phase, S. pneumoniae (P394) was washed and incubated in BHI alone, with N. meningitidis (MC58C3) for 1.5 h, or with M. catarrhalis (Bc1) for 3 h. Viable counts of N. meningitidis (stippled bar) or M. catarrhalis (hatched bar) incubated in coculture with S. pneumoniae were determined in duplicate by plating on selective media. Viable counts of S. pneumoniae in coculture with N. meningitidis (black bar) or M. catarrhalis (white bar) were determined in duplicate on selective media. Values represent the change in viable count expressed as a percentage of a control culture containing that organism alone. Values are the average of three experiments, and error bars represent the standard deviations.

FIG. 5

Effect of H₂O₂ on the survival of S. pneumoniae (P394), M. catarrhalis (Bc1), N. meningitidis (MC58C3), and H. influenzae (Rd). Following growth to mid-log phase, S. pneumoniae (black bars), M. catarrhalis (white bars), N. meningitidis (stippled bars), or H. influenzae (hatched bars) were washed and incubated at 37°C in BHI or sBHI containing the indicated concentration of H₂O₂. After 30 min, viable counts were determined on BHI or sBHI plates containing 200 U of catalase per ml. Values represent the average of three independent determinations in duplicate, and the error bars represent the standard deviations. ∗, Below the limit of detection.

FIG. 6

Effect of coculture of H. influenzae Rd with S. pneumoniae opaque (P62) or transparent (P64) variants of a type 9V isolate. Following growth to mid-log phase, H. influenzae was washed and incubated in sBHI either alone (○), with P62 (▵), or with P64 (□) for the times indicated, and viable counts were determined in duplicate on selective media. Viable counts of P62 (▴) or P64 (■) incubated in coculture with H. influenzae were determined in duplicate by plating on selective media. Values represent the average of three independent determinations in duplicate, and the error bars represent the standard deviations. (Inset) Relative expression of SpxB in S. pneumoniae variants P62 (black bar) and P64 (hatched bar) as determined by two-dimensional gel electrophoresis followed by mass spectrometric analysis. Results represent the average of four independent experiments, with error bars representing the standard deviations.

FIG. 7

Western blot showing the effect of environmental oxygen and carbon dioxide tension on pyruvate oxidase (SpxB) expression in S. pneumoniae P878, which contains an in-frame fusion to PhoA. Cell lysates of spxB::phoA mutant (P878) grown under 20% O₂–0.03% CO₂ (lane 1), 17% O₂–3% CO₂ (lane 2), or <0.01% O₂–10% CO₂ (lane 3) were electrophoresed on an SDS–10% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and immunoblotted with an antibody to PhoA. As a negative control, cell lysates from the parent strain (D39) grown under 17% O₂–3% CO₂ (lane 4) were included. Size markers are in kilodaltons.