Metatranscriptomic Analysis Reveals Active Bacterial Communities in Diabetic Foot Infections

Abstract

Despite the extended view of the composition of diabetic foot infections (DFIs), little is known about which transcriptionally active bacterial communities are pertinent to infection, and if any differences are associated with increased infection severity. We applied a RNA sequencing approach to analyze the composition, function, and pathogenicity of the active bacterial communities in DFIs. Taxonomic profiling of bacterial transcripts revealed the presence of 14 bacterial phyla in DFIs. The abundance of the Spiroplasma, Vibrio, and Mycoplasma were significantly different in different infection severities (P < 0.05). Mild and severe stages of infections were dominated by Staphylococcus aureus and Porphyromonas asaccharolytica, respectively. A total of 132 metabolic pathways were identified of which ribosome and thiamin being among the most highly transcribed pathways. Moreover, a total of 131 antibiotic resistance genes, primarily involved in the multidrug efflux pumps/exporters, were identified. Furthermore, iron acquisition systems (synthesize and regulation of siderophores) and pathways involved in the synthesis and regulation of cell-surface components associated with adhesion, colonization, and movement of bacterial cells were the most common virulence factors. These virulence factors may help bacteria compete for scares resources and survive the host wound proteases. Characterization of transcriptionally active bacterial communities can help to provide an understanding of the role of key pathogens in the development of DFIs. Such information can be clinically useful allowing replacement of DFIs empirical therapy with targeted treatment.

Evaluation of Host Immune Response in Diabetic Foot Infection Tissues Using an RNA Sequencing-Based Approach

“…Our investigation identified the highest difference and the most interesting findings between samples with a low number of virulence factors (control) and samples with a high number of virulence factors (perturbation) which were defined previously ( Heravi et al, 2020b ). Differential gene expression analysis between control (DFI121, DFI109, DFI111, and DFI167) and perturbation groups (DFI166, DFI161, and DFI126) showed more than 500 differentially expressed genes (DEGs).…”

“…A detailed description of initial steps regarding sample preparation, RNA extraction, and sequencing process can be found in the previously described study ( Heravi et al, 2020b ).…”

Adaptation of Staphylococcus aureus in a Medium Mimicking a Diabetic Foot Environment

“…Real-time PCR assays were performed in a LightCycler ® 480 device using the LightCycler FastStart DNA Master PLUS SYBRGreen I kit (Roche, Meylan, France) with 100 ng of cDNA and 10 pmol of target primers ( Supplementary Materials Table S5 ) [ 18 , 69 , 70 , 71 , 72 , 73 , 74 ]. The specificity of the PCR products was tested by melting point analysis.…”

“…During DFI, S. aureus has been shown to activate its virulence factors to invade the tissue [ 18 ]. However, this infection is chronic and complex, with a different clinical presentation to SSTIs, notably with the mitigated impact of toxinogenic strains [ 6 ].…”

Metabolic Modeling to Interrogate Microbial Disease: A Tale for Experimentalists

“…Given that multiple chronic diseases such as periodontitis, CF and diabetic foot ulcers are driven by the presence of polymicrobial communities that exhibit recalcitrance to antimicrobials ( Rams et al, 2014 ; O’Toole, 2018 ; Orazi and O'Toole, 2019 ; Heravi et al, 2020 ), the field is poised to apply combined modeling and experimental approaches to better understand how to effectively treat such polymicrobial infections. To date however, little or no efforts have been directed towards modeling these complicated, often chronic polymicrobial infections and their predicted responses to antibiotic treatment.…”