Streptococcus pneumoniae Modulates Staphylococcus aureus Biofilm Dispersion and the Transition from Colonization to Invasive Disease

Abstract

Streptococcus pneumoniae and Staphylococcus aureus are ubiquitous upper respiratory opportunistic pathogens. Individually, these Gram-positive microbes are two of the most common causative agents of secondary bacterial pneumonia following influenza A virus infection, and they constitute a significant source of morbidity and mortality. Since the introduction of the pneumococcal conjugate vaccine, rates of cocolonization with both of these bacterial species have increased, despite the traditional view that they are antagonistic and mutually exclusive. The interactions between S. pneumoniae and S. aureus in the context of colonization and the transition to invasive disease have not been characterized. In this report, we show that S. pneumoniae and S. aureus form stable dual-species biofilms on epithelial cells in vitro When these biofilms are exposed to physiological changes associated with viral infection, S. pneumoniae disperses from the biofilm, whereas S. aureus dispersal is inhibited. These findings were supported by results of an in vivo study in which we used a novel mouse cocolonization model. In these experiments, mice cocolonized in the nares with both bacterial species were subsequently infected with influenza A virus. The coinfected mice almost exclusively developed pneumococcal pneumonia. These results indicate that despite our previous report that S. aureus disseminates into the lungs of mice stably colonized with these bacteria following influenza A virus infection, cocolonization with S. pneumoniae in vitro and in vivo inhibits S. aureus dispersal and transition to disease. This study provides novel insight into both the interactions between S. pneumoniae and S. aureus during carriage and the transition from colonization to secondary bacterial pneumonia.IMPORTANCE In this study, we demonstrate that Streptococcus pneumoniae can modulate the pathogenic potential of Staphylococcus aureus in a model of secondary bacterial pneumonia. We report that host physiological signals related to viral infection cease to elicit a dispersal response from S. aureus while in a dual-species setting with S. pneumoniae, in direct contrast to results of previous studies with each species individually. This study underscores the importance of studying polymicrobial communities and their implications in disease states.

Keywords: Polymicrobial infections; Staphylococcus aureus; Streptococcus pneumoniae; secondary bacterial pneumonia.

FIG 1

S. pneumoniae EF3030 and S. aureus UAMS-1 form dual-species biofilms in vitro. Biofilm-associated EF3030 and UAMS-1 were enumerated from cocultured biofilms after 48 h (n = 8). Both species of bacteria were recovered from each well. The dashed line indicates the limit of detection. Solid bars are means ± standard deviations.

FIG 2

CLSM images of dual-species biofilms formed by S. pneumoniae EF3030-GFP (green) and S. aureus UAMS1-dsRED (red) following 48 h of coculture. The image shows the average intensity projections through the confocal image stack, with the maximum-intensity x-z and y-z projections shown along the bottom and side of the image; a representative image of a single slice from the central region of the biofilm is shown. Images were produced using ImageJ.

FIG 3

In vitro dispersal of 48 h S. pneumoniae EF3030 (A) and S. aureus UAMS-1 (B) single-species and dual-species biofilms following 4 h of heat treatment (38.5°C). Dispersal values are presented as the ratio of supernatant to biofilm from a minimum of four independent experiments (means ± standard errors of the means). Statistical analysis of the effect heat dispersal compared to controls was performed with the unpaired Student t test. *, P < 0.05.

FIG 4

In vitro dispersal of S. aureus UAMS-1 from 48-h single-species biofilms compared to dual-species biofilms with S. mitis NS5S following 4 h of heat treatment (38.5°C). Dispersal values are presented as the ratio of supernatant to biofilm from a minimum of four independent experiments (mean result ± standard deviation). Statistical analysis on heat dispersal versus that in controls was performed with the unpaired Student t test. *, P < 0.05.

FIG 5

Dual-species colonization of the murine nasal mucosa. Samples were harvested 48 h after colonization; each point presents the CFU recovered from individual mice (n = 12). Blue circles represent S. pneumoniae EF3030, and the black squares represent S. aureus UAMS-1. The dashed line indicates the limit of detection. Bars indicate standard deviations.

FIG 6

Dissemination following IAV coinfection. Forty-eight hours post-bacterial colonization, IAV cohorts received intranasal virus inoculations while control cohorts (CTRL) received only saline, and the infection was allowed to progress an additional 48 h prior to harvest. Each point represents the CFU recovered from nasal tissues (NT) and lung tissues (LT) harvested from individual mice (n = 16) 96 h after cocolonization with S. pneumoniae EF3030 (blue circles) and S. aureus UAMS-1 (black squares). The dashed line indicates the limit of CFU detection. Solid bars indicate the standard deviations. Statistical analysis was performed with the nonparametric Mann-Whitney test. *, P < 0.05; ***, P < 0.001.