Download Set Phages to Stun: Reducing the Virulence of

Diabetes Volume 64, August 2015
2701
David G. Armstrong,1 Bonnie L. Hurwitz,2 and Benjamin A. Lipsky3,4
Set Phages to Stun: Reducing the
Virulence of Staphylococcus aureus in
Diabetic Foot Ulcers
Diabetes 2015;64:2701–2703 | DOI: 10.2337/db15-0543
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 (1025) 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
1Southern Arizona Limb Salvage Alliance, Department of Surgery, The University
of Arizona Health Sciences Center, Tucson, AZ
2Department of Agricultural and Biosystems Engineering, The University of
Arizona, Tucson, AZ
3 Service of Infectious Diseases, Geneva University Hospitals and Department
of Medicine, University of Geneva, Geneva, Switzerland
4 Division of Medical Sciences, Green Templeton College, University of Oxford,
Oxford, U.K.
Corresponding author: David G. Armstrong, [email protected].
© 2015 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit, and
the work is not altered.
See accompanying article, p. 2991.
COMMENTARY
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 pathogenassociated molecular patterns, activating inflammatory
signaling cascades. Thus, by entering a lysogenic rather
than a lytic state (through incorporation in the bacterial
2702
Commentary
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
Diabetes Volume 64, August 2015
the new data presented by Messad et al. (8), the ROSAlike 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).
Figure 1—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 singlespecies 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.
diabetes.diabetesjournals.org
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.
Duality of Interest. No potential conflicts of interest relevant to this article
were reported.
References
1. Singh N, Armstrong DG, Lipsky BA. Preventing foot ulcers in patients with
diabetes. JAMA 2005;293:217–228
2. Lavery LA, Armstrong DG, Wunderlich RP, Mohler MJ, Wendel CS, Lipsky
BA. Risk factors for foot infections in individuals with diabetes. Diabetes Care
2006;29:1288–1293
3. Lavery LA, Armstrong DG, Murdoch DP, Peters EJG, Lipsky BA. Validation of
the Infectious Diseases Society of America’s diabetic foot infection classification
system. Clin Infect Dis 2007;44:562–565
4. Barshes NR, Sigireddi M, Wrobel JS, et al. The system of care for the diabetic foot: objectives, outcomes, and opportunities. Diabet Foot Ankle 2013;4:
21847
5. Bowler PG, Duerden BI, Armstrong DG. Wound microbiology and associated
approaches to wound management. Clin Microbiol Rev 2001;14:244–269
6. Lipsky BA, Berendt AR, Cornia PB, et al.; Infectious Diseases Society of
America. 2012 Infectious Diseases Society of America clinical practice guideline
for the diagnosis and treatment of diabetic foot infections. Clin Infect Dis 2012;
54:e132–e173
Armstrong, Hurwitz, and Lipsky
2703
7. Game FL, Hinchliffe RJ, Apelqvist J, et al. A systematic review of interventions to enhance the healing of chronic ulcers of the foot in diabetes. Diabetes
Metab Res Rev 2012;28 (Suppl. 1):119–141
8. Messad N, Prajsnar TK, Lina G, et al. Existence of a colonizing Staphylococcus aureus strain isolated in diabetic foot ulcers. Diabetes 2015;64:2991–
2995
9. Gussin GN, Johnson AD, Pabo CO, Sauer RT. Repressor and Cro Protein:
Structure, Function, and Role in Lysogenization [monograph online], 2009. Available
from https://cshmonographs.org/csh/index.php/monographs/article/viewArticle/4997.
Accessed 13 April 2015
10. Virgin HW. The virome in mammalian physiology and disease. Cell 2014;
157:142–150
11. Breitbart M, Hewson I, Felts B, et al. Metagenomic analyses of an uncultured viral community from human feces. J Bacteriol 2003;185:6220–6223
12. Minot S, Sinha R, Chen J, et al. The human gut virome: inter-individual
variation and dynamic response to diet. Genome Res 2011;21:1616–1625
13. Reyes A, Haynes M, Hanson N, et al. Viruses in the faecal microbiota of
monozygotic twins and their mothers. Nature 2010;466:334–338
14. Brüssow H, Canchaya C, Hardt WD. Phages and the evolution of bacterial
pathogens: from genomic rearrangements to lysogenic conversion. Microbiol Mol
Biol Rev 2004;68:560–602
15. Maiques E, Úbeda C, Campoy S, et al. b-Lactam antibiotics induce the SOS
Response and horizontal transfer of virulence factors in Staphylococcus aureus.
J Bacteriol 2006;188:2726–2729
16. Hurwitz BL, Sullivan MB. The Pacific Ocean virome (POV): a marine viral
metagenomic dataset and associated protein clusters for quantitative viral
ecology. PLoS One 2013;8:e57355
17. Brum JR, Sullivan MB. Rising to the challenge: accelerated pace of discovery transforms marine virology. Nat Rev Microbiol 2015;13:147–159
18. Breitbart M. Marine viruses: truth or dare. Ann Rev Mar Sci 2012;4:
425–448
19. Pirnay J-P, De Vos D, Verbeken G, et al. The phage therapy paradigm: prêtà-porter or sur-mesure? Pharm Res 2011;28:934–937
20. Spichler A, Hurwitz BL, Armstrong DG, Lipsky BA. Microbiology of diabetic foot
infections: from Louis Pasteur to “crime scene investigation.” BMC Med 2015;13:2
21. Armstrong DG, Lew EJ, Hurwitz B, Wild T. The quest for tissue repair’s
holy grail: the promise of wound diagnostics or just another fishing expedition? Wound Medicine. 27 March 2015 [Epub ahead of print]. DOI: 10.1016/
j.wndm.2015.03.010