Pseudomonas aeruginosa Production of Hydrogen Cyanide Leads to Airborne Control of Staphylococcus aureus Growth in Biofilm and In Vivo Lung Environments

Abstract

Diverse bacterial volatile compounds alter bacterial stress responses and physiology, but their contribution to population dynamics in polymicrobial communities is not well known. In this study, we showed that airborne volatile hydrogen cyanide (HCN) produced by a wide range of Pseudomonas aeruginosa clinical strains leads to at-a-distance in vitro inhibition of the growth of a wide array of Staphylococcus aureus strains. We determined that low-oxygen environments not only enhance P. aeruginosa HCN production but also increase S. aureus sensitivity to HCN, which impacts P. aeruginosa-S. aureus competition in microaerobic in vitro mixed biofilms as well as in an in vitro cystic fibrosis lung sputum medium. Consistently, we demonstrated that production of HCN by P. aeruginosa controls S. aureus growth in a mouse model of airways coinfected by P. aeruginosa and S. aureus. Our study therefore demonstrates that P. aeruginosa HCN contributes to local and distant airborne competition against S. aureus and potentially other HCN-sensitive bacteria in contexts relevant to cystic fibrosis and other polymicrobial infectious diseases. IMPORTANCE Airborne volatile compounds produced by bacteria are often only considered attractive or repulsive scents, but they also directly contribute to bacterial physiology. Here, we showed that volatile hydrogen cyanide (HCN) released by a wide range of Pseudomonas aeruginosa strains controls Staphylococcus aureus growth in low-oxygen in vitro biofilms or aggregates and in vivo lung environments. These results are of pathophysiological relevance, since lungs of cystic fibrosis patients are known to present microaerobic areas and to be commonly associated with the presence of S. aureus and P. aeruginosa in polymicrobial communities. Our study therefore provides insights into how a bacterial volatile compound can contribute to the exclusion of S. aureus and other HCN-sensitive competitors from P. aeruginosa ecological niches. It opens new perspectives for the management or monitoring of P. aeruginosa infections in lower-lung airway infections and other polymicrobial disease contexts.

Keywords: Pseudomonas aeruginosa; Staphylococcus aureus; bacterial coinfection; hydrogen cyanide; volatile compounds.

FIG 1

Semiquantitative detection of volatile HCN emitted from P. aeruginosa cultures under various conditions. Semiquantitative HCN detection upon HCN reaction with copper(II) ethylacetoacetate and 4,4′-methylenebis-(N,N-dimethylaniline). (A) Semiquantitative detection of volatile HCN emitted from P. aeruginosa WT and PAO1ΔhcnB, in LB or SCFM2 medium supplemented or not with 0.4% (wt/vol) glycine, after 24 h of incubation at 37°C under aerobic conditions. (B) Semiquantitative detection of volatile HCN from P. aeruginosa WT and PAO1ΔhcnB, in LB or SCFM2 medium supplemented or not with 0.4% (wt/vol) glycine, after 24 h of incubation at 37°C under microaerobic conditions. Pictures were taken after 24 h of incubation at 37°C using the 2-petri-dish assay described in Fig. S1. Each experiment was performed at least three times. (C) Semiquantitative detection of volatile HCN showed an increased production of HCN by P. aeruginosa PAO1 at different stages of growth in LB medium. Each experiment was performed at least three times.

FIG 2

P. aeruginosa production of HCN in LB leads to airborne inhibition of S. aureus growth. (A) Quantification of the effect of exposure to P. aeruginosa HCN on S. aureus MW2 growth in LB aerobic conditions. Bacteria were grown on 10⁻⁴ to 10⁻⁸ dilution spots (see Fig. S1 for setup) and exposed to P. aeruginosa HCN or not. Each spot was punched out from the LB agar plate and resuspended in PBS, and the corresponding OD₆₀₀ was determined. The fold differences observed between different conditions at comparable dilution were calculated based on the ratio of the means of 4 independent quantifications at each dilution. (B) Serial dilution of S. aureus MW2 exposed to P. aeruginosa WT or PAO1ΔhcnB cultures in LB supplemented or not with 0.4% (wt/vol) glycine in the 2-petri-dish assay (Fig. S1). No inhibition of S. aureus MW2 growth was observed when the middle plate containing P. aeruginosa culture was covered and sealed with Parafilm. Pictures were taken after 24 h of incubation at 37°C under aerobic conditions. Each experiment was performed at least three times. (C and D) As for panels A and B, except that the experiments were performed under microaerobic conditions. Statistics correspond to a two-tailed unpaired t test with Welch correction. **, P ≤ 0.01; ****, P ≤ 0.0001.

FIG 3

Production of HCN controls S. aureus growth in in vitro mixed biofilms. (A) Number of CFU in single-species biofilms grown in LB medium in continuous-flow microfermentors for 48 h at 37°C. (B) Number of CFU in two-species mixed biofilms. Each S. aureus strain was mixed with either WT P. aeruginosa PAO1 or its ΔhcnB mutant at a 1:1 ratio. The biofilms were grown in LB medium in continuous-flow microfermentors for 48 h at 37°C. Statistics correspond to a two-tailed unpaired t test with Welch correction. N.S., not significant; **, P ≤ 0.01; ***, P ≤ 0.001.

FIG 4

P. aeruginosa production of HCN controls S. aureus growth in CF-relevant conditions. (A) Total biomass of S. aureus aggregates as monoculture or in coinfection with the PAO1 wild type and/or the hcnB mutant. Isolates were cultured in SCFM2 and imaged using confocal microscopy at 3, 8, and 22 h. Statistics correspond to a two-tailed unpaired t test with Welch correction. N.S., not significant; *, P ≤ 0.05. (B) Representative rendered confocal micrograph of S. aureus and P. aeruginosa coinfection in SCFM2. (Top) Wild-type P. aeruginosa in green and S. aureus in red. (Bottom) P. aeruginosa ΔhcnB mutant in green and S. aureus in red. (C) In vivo competition experiments in mouse lungs. Monoinoculation and mixed (1:2) coinoculations of S. aureus Xen36, P. aeruginosa PAO1 WT, or the ΔhcnB mutant were used to infect animals. CFU were counted in the lung homogenates of mice 24 h after infection. Noninfected SOPF mice showed minimal lung bacterial contamination, with <100 CFU (horizontal dotted line). Statistics correspond to a two-tailed unpaired t test with Welch correction. N.S., not significant; ****, P ≤ 0.0001.