At least one-quarter of people alive with diabetes will develop a diabetic foot ulcer (DFU) (1), and about half of these will become clinically infected (2). Once this occurs, 20–30% of infected DFUs lead to lower-extremity amputation, with its dire consequences (3). Not surprisingly, the direct costs of the treatment of DFUs now exceed those of the five most costly cancers in the U.S. (4).
All DFUs are colonized by microbes. This colonization is comprised of a complex mélange of species, with Staphylococcus aureus generally being the most prevalent (5,6). However, it is unclear why some wounds harboring this potentially virulent pathogen remain colonized, while others become infected. Defining infection in an open wound has been a persistent problem. While most authorities define it on the presence of local (and less often systemic) signs of inflammatory response (6), some believe that either the presence of specific species or a high number of colonies is pathogenic. This belief has led many to unnecessarily use antimicrobials to treat clinically noninfected wounds (6,7). In this issue of Diabetes, we read with interest the study by Messad et al. (8), which tantalizingly suggests it may be possible to differentiate a “colonizing” from a more microbiologically nefarious phenotype of S. aureus, the “prince of pedal pathogens.” Even more promising is that this work may identify an approach to “stun” a pathogenic colony into one that seeks peaceful coexistence (8).
Bacteria and their viruses (bacteriophages) may seem unlikely allies in the battle against the human immune system. Bacterial lysis releases a potpourri of cellular debris (proteins, lipids, and nucleic acids) that are recognized by the human immune system as pathogen-associated molecular patterns, activating inflammatory signaling cascades. Thus, by entering a lysogenic rather than a lytic state (through incorporation in the bacterial host genome as a prophage), phages and their bacterial hosts can remain undetected by the human immune system. By this mechanism, S. aureus, with its great potential for pathogenicity, may masquerade as a harmless bacterium when colonizing a chronic wound. Messad et al. (8) demonstrate that one mechanism for turning a pathogen into a pal is to promote biofilm formation. Another may be that bacterial cells with an integrated prophage are immune to superinfection and have a considerably smaller chance (10−5) of being lysed by the infecting prophage (9). In polymicrobial wounds where bacterial species compete for space and nutrients, prophages may provide a selective advantage.
Recent data are shedding light on the interaction among the viral component of the human microbiome (virome), bacteria, and human host physiology (10,11). Studies have shown that the human virome consists of a remarkable diversity of bacteriophages, as well as eukaryotic viruses (12,13). Viruses can horizontally transfer genetic material among their bacterial hosts, including genes related to pathogenesis (such as pertussis, shiga, and cholera toxins) and antibiotic resistance, thereby perhaps helping to drive genetic diversity in the human microbiome (14,15). Importantly, as described in Messad et al., prophages inserted into the S. aureus host genome in a DFU may also act to protect both the bacteria and the human host. Furthermore, they may speed up bacterial evolutionary processes by extracting and reinserting themselves into new hosts and constantly shuffling genetic diversity.
Despite recent genomic advances, studying the functional capacity of viromes (and prophages detected in bacterial genomes, as shown by Messad et al.) remains elusive; the majority of DNA sequences, or “contigs,” are not yet represented in genomic reference databases (12,16,17). This “viral dark matter” remains uncharacterized due to technological limitations, despite decades of viral cultivation and subsequent genomic sequencing. Thus, as we continue on this journey to understand the stunning effects of phages on their bacterial hosts and the human immune response, researchers must boldly venture from cultivated model systems to new experimental and informatics methods to investigate the new “known unknown” (17).
Functional analyses of phage genomes and viromes from various ecosystems have shown that phages contain bacterial host genes (called auxiliary metabolic genes) that when expressed bolster host fitness during infection (18). Specifically, marine phages have been shown to modulate bacterial host metabolism and energy production by harboring genes that are specifically adapted to their environmental niche (e.g., surface vs. deep ocean) (17). Given the new data presented by Messad et al. (8), the ROSA-like prophage may provide a similar beneficial role to its bacterial host by increasing biofilm formation and promoting colonization within the DFU. In light of the continuing problem of defining infection in a chronic wound, gaining a better understanding of the virome may open a new approach to selecting treatments and improving outcomes (Fig. 1). More speculatively, changes in the human microbiome may be driven by changes in the virome, as through the emergence of bacteria containing temperate bacteriophages (as discussed by Messad et al.) or new bacteriophages that arise from the environment or human contact. The interesting issues around the 100-year-old remedy of bacteriophages—both in how they may benefit their bacterial hosts, as shown in the work of Messad et al., or in how they control bacterial population size—may prove to be fundamental in our understanding of wound ecology (19).
Illuminating viral dark matter. A: The known protein universe. Bacterial genomes are produced by cultivating bacterial isolates, extracting and sequencing DNA to produce reads, assembling the reads into contigs, and scaffolding the contigs together to produce genomes. Gene-finding algorithms identify open reading frames (or genes) that are compared with known proteins to derive functional annotation. Bacteriophage genomes are produced similarly, except that pure phages are isolated and further purified from plaques in susceptible host cultures. Phages can also integrate in their host genomes as prophages. B: Into the unknown protein universe. New metagenomic techniques to sequence DNA directly from a sample offer a glimpse into uncultured bacteria and their phages. DNA is isolated from microbial communities and enriched for either bacteria or viral-like particles. Reads are assembled as in A; however, contigs are fragmentary, generally do not represent whole genomes, and may be misassembled with reads from closely related species. Single amplified genomes provide an alternative where community samples are enriched for a single bacterial species, making uncultured genomes obtainable. Likewise, bacterial sequence inserts can be maintained in fosmid libraries and sequenced to obtain large single-species uncultured bacterial genome fragments. Phage DNA can be discovered in community metagenomes enriched for bacteria as a result of prophage or phage actively infecting bacteria. Proteins from open reading frames on assembled contigs or reads are clustered and compared with known proteins to define known and unknown protein clusters (or the “known unknown”). Reads from unassembled metagenomes can also be compared using k-mers and evaluated using social network analyses to link clinical factors with community structure.
Illuminating viral dark matter. A: The known protein universe. Bacterial genomes are produced by cultivating bacterial isolates, extracting and sequencing DNA to produce reads, assembling the reads into contigs, and scaffolding the contigs together to produce genomes. Gene-finding algorithms identify open reading frames (or genes) that are compared with known proteins to derive functional annotation. Bacteriophage genomes are produced similarly, except that pure phages are isolated and further purified from plaques in susceptible host cultures. Phages can also integrate in their host genomes as prophages. B: Into the unknown protein universe. New metagenomic techniques to sequence DNA directly from a sample offer a glimpse into uncultured bacteria and their phages. DNA is isolated from microbial communities and enriched for either bacteria or viral-like particles. Reads are assembled as in A; however, contigs are fragmentary, generally do not represent whole genomes, and may be misassembled with reads from closely related species. Single amplified genomes provide an alternative where community samples are enriched for a single bacterial species, making uncultured genomes obtainable. Likewise, bacterial sequence inserts can be maintained in fosmid libraries and sequenced to obtain large single-species uncultured bacterial genome fragments. Phage DNA can be discovered in community metagenomes enriched for bacteria as a result of prophage or phage actively infecting bacteria. Proteins from open reading frames on assembled contigs or reads are clustered and compared with known proteins to define known and unknown protein clusters (or the “known unknown”). Reads from unassembled metagenomes can also be compared using k-mers and evaluated using social network analyses to link clinical factors with community structure.
Infections related to DFUs exact a heavy price on patients, their families, and collective health care systems. The fascinating findings of Messad et al. (8) suggest that we might be able to practically (and rapidly) detect phages present within chronic wounds to identify ones that might either increase or decrease the degree of bacterial virulence. If so, perhaps we can use this new information to help decide which patients need antimicrobial therapy and which do not—a major issue in this era of rising antibiotic resistance. Both measuring (20,21) and manipulating the microbiome by focusing on its microbial ringleaders (such as S. aureus) might offer a way to improve clinical decision making in this key area of infected DFUs. In this way, we may help our patients to live longer and prosper.
See accompanying article, p. .
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Duality of Interest. No potential conflicts of interest relevant to this article were reported.
