Anaerobic Microbiota Derived from the Upper Airways Impact Staphylococcus aureus Physiology

Abstract

Staphylococcus aureus is associated with the development of persistent and severe inflammatory diseases of the upper airways. Yet, S. aureus is also carried asymptomatically in the sinonasal cavity of ∼50% of healthy adults. The causes of this duality and host and microbial factors that tip the balance between S. aureus pathogenesis and commensalism are poorly understood. We have shown that by degrading mucins, anaerobic microbiota support the growth of airway pathogens by liberating metabolites that are otherwise unavailable. Given the widely reported culture-based detection of anaerobes from individuals with chronic rhinosinusitis (CRS), here we tested our hypothesis that CRS microbiota is characterized by a mucin-degrading phenotype that alters S. aureus physiology. Using 16S rRNA gene sequencing, we indeed observed an increased prevalence and abundance of anaerobes in CRS relative to non-CRS controls. PICRUSt2-based functional predictions suggested increased mucin degradation potential among CRS microbiota that was confirmed by direct enrichment culture. Prevotella, Fusobacterium, and Streptococcus comprised a core mucin-degrading community across CRS subjects that generated a nutrient pool that augmented S. aureus growth on mucin as a carbon source. Finally, using transcriptome sequencing (RNA-seq), we observed that S. aureus transcription is profoundly altered in the presence of mucin-derived metabolites, though expression of several key metabolism- and virulence-associated pathways varied between CRS-derived bacterial communities. Together, these data support a model in which S. aureus metabolism and virulence in the upper airways are dependent upon the composition of cocolonizing microbiota and the metabolites they exchange.

Keywords: Staphylococcus aureus; anaerobes; cross-feeding; mucin; sinusitis.

FIG 1

Bacterial community compositions differ between CRS and non-CRS. (a) DPCoA ordination biplot shows dissimilarity in presence and abundance of bacteria in CRS and non-CRS samples. (b) Percentage of abundance of actinobacteria is associated with non-CRS status (Wilcoxon, ***, P < 0.001; CRS, n = 51; non-CRS, n = 18). (c) Among CRS samples, the percentage of abundance of Actinobacteria is associated with no prior FESS treatment (Wilcoxon, ***, P < 0.001; Yes, n = 35; No, n = 16). (d) Spearman’s correlation of percentage of abundance of Actinobacteria negatively correlates with percentages of abundance of both Bacteroidetes and Fusobacteria. (e) Heat map representation of the top 15 genera in each sample group (CRS and non-CRS). Closed boxes (■) represent obligate anaerobic genera, and open boxes (□) denote genera with obligate anaerobic species. Colors throughout the figure match the color key for panel a.

FIG 2

PICRUSt2-predicted metagenomes reveal increased mucin-degrading capacity of CRS microbiota relative to non-CRS. Enyzme classes (ECs) and their corresponding CAZy database classifications (auxiliary activity [AA], carbohydrate esterase [CE], glycoside hydrolases [GH], glycoside transferase [GT], and polysaccharides lyase [PL]) were identified in PICRUSt2-predicted metagenomes. The average relative functional abundances of each CAZy enzyme class contributed by the top 10 taxa in (a) CRS and (b) non-CRS samples are shown. Data are sorted by median relative functional abundance.

FIG 3

Anaerobic enrichment culturing of sinus mucus reveals a core mucin-degrading community. (a) Relative abundance of ASVs grouped at the genus level for each original sample and enrichment culture. FESS samples for D and F fell below 2,000 sequences but are included here for comparison. (b) Mean relative abundances for the top 20 genera in original sinus mucus samples and enrichment cultures. (c) DPCoA biplot comparing proportional data from original samples and enrichment cultures with associated phyla. (d) FPLC chromatogram of high-molecular-weight mucin proteins in MMM and CFS from 48-h CRS mucin enrichment cultures. Closed boxes (■) represent obligate anaerobic genera, and open boxes (□) denote genera with obligate anaerobic species.

FIG 4

S. aureus growth is limited on mucin alone but restored by mucin-derived metabolites. (a) S. aureus LAC and three CRS-derived clinical isolates grown in minimal mucin medium (MMM), amended with 0.5% Casamino Acids (MMMC), 0.5% lactate (MMML), 0.25% glucose (MMMG), or a combination of these (MMMLC and MMMGC). Growth curves were measured under aerobic conditions; error bars represent the mean and standard deviation (SD) for n = 3. (b and c) Growth of LAC measured at OD₆₀₀ after 24 h of growth under (b) anaerobic conditions and (c) anaerobic conditions with 3 mM nitrate. The data shown are the mean of n = 6 (MMM, MMMC, MMML, and MMMLC) and n = 3 (MMMG and MMMGC) biological replicates. Error bars are SD. Significance relative to MMM was determined by t test with Holm-Bonferroni adjustment. ***, P < 0.001.

FIG 5

S. aureus growth on cell-free supernatants from CRS mucin-degrading communities. (a) S. aureus LAC aerobic growth on CFS from CRS-derived mucin-degrading communities relative to intact mucin alone (MMM [black]). (b) S. aureus anaerobic growth on CFS with and without sodium nitrate (3 mM). (c) HPLC quantification of mixed-acid fermentation metabolite concentrations (mM) in CFS. x, not detectable; ***, P < 0.001; **, P < 0.01; *, P < 0.05.

FIG 6

S. aureus transcription is altered by mucin degradation and is dependent on cocolonizing microbiota. (a) MA plot shows differential expression of genes (79 upregulated and 14 downregulated) in cell-free supernatant conditions from communities D, F, and H compared to MMMG controls. The Wald test was used to assign significance for genes with a fold change (|FC|) of >2, and false-discovery rate (FDR)-adjusted P value of <0.05. (b and c) Transcripts involved in sialic acid degradation and central metabolism were differentially increased in expression under supernatant conditions compared to glucose. (d) Heat map of select genes with significant differences between each cell-free supernatant condition. Significance was determined using the likelihood ratio test, keeping only genes with a log fold change (|LFC|) of >2 and FDR-adjusted P value of <1 × 10⁻¹⁰. The data presented are differences from the mean for each gene calculated from regularized log-transformed counts. All data are representative of three biological replicates per condition. Panel b was modified from reference .