Defining Microbial Community Functions in Chronic Human Infection with Metatranscriptomics

Chronic polymicrobial infections (cPMIs) harbor complex bacterial communities with diverse metabolic capacities, leading to competitive and cooperative interactions. Although the microbes present in cPMIs have been established through culture-dependent and-independent methods, the key functions that drive different cPMIs and the metabolic activities of these complex communities remain unknown. To address this knowledge gap, we analyzed 102 published metatranscriptomes collected from cystic fibrosis sputum (CF) and chronic wound infections (CW) to identify key bacterial members and functions in cPMIs. Community composition analysis identified a high prevalence of pathogens, particularly Staphylococcus and Pseudomonas, and anaerobic members of the microbiota, including Porphyromonas, Anaerococcus, and Prevotella. Functional profiling with HUMANn3 and SAMSA2 revealed that while functions involved in bacterial competition, oxidative stress response, and virulence were conserved across both chronic infection types, >40% of the functions were differentially expressed (padj < 0.05, fold-change >2). Higher expression of antibiotic resistance and biofilm functions were observed in CF, while tissue destructive enzymes and oxidative stress response functions were highly expressed in CW samples. Of note, strict anaerobes had negative correlations with traditional pathogens in both CW (P = -0.43) and CF (P = -0.27) samples and they significantly contributed to the expression of these functions. Additionally, we show microbial communities have unique expression patterns and distinct organisms fulfill the expression of key functions in each site, indicating the infection environment strongly influences bacterial physiology and that community structure influences function. Collectively, our findings indicate that community composition and function should guide treatment strategies for cPMIs.