bioactivation pathways of pro-drugs. This approach could be used to limit the frequency of
resistance by systematically destroying subpopulations of resistant bacteria that may emerge
REFERENCES AND NOTES
1. B. Spellberg, D. N. Gilbert, Clin. Infect. Dis. 59 (suppl. 2),
2. World Health Organization (WHO), Antimicrobial Resistance:
Global Report on Surveillance (WHO, 2014).
3. S. Hoffner, Lancet 380, 1367–1369 (2012).
4. WHO, Global Tuberculosis Report (WHO, 2015).
5. C. Barry, Science 348, 633–634 (2015).
6. C. Walsh, Antibiotics: Actions, Origins, Resistance (ASM Press, 2003).
7. C. Reading, M. Cole, Antimicrob. Agents Chemother. 11, 852–857
8. Y. Zhang, B. Heym, B. Allen, D. Young, S. Cole, Nature 358,
9. A. Scorpio, Y. Zhang, Nat. Med. 2, 662–667 (1996).
10. A. E. DeBarber, K. Mdluli, M. Bosman, L. G. Bekker, C. E. Barry 3rd,
Proc. Natl. Acad. Sci. U.S.A. 97, 9677–9682 (2000).
11. A. R. Baulard et al., J. Biol. Chem. 275, 28326–28331 (2000).
12. U. H. Manjunatha et al., Proc. Natl. Acad. Sci. U.S.A. 103,
13. M. Matsumoto et al., PLOS Med. 3, e466 (2006).
14. G. V. Bloemberg et al., N. Engl. J. Med. 373, 1986–1988
15. J. Zheng et al., J. Biol. Chem. 288, 23447–23456 (2013).
16. T. A. Vannelli, A. Dykman, P. R. Ortiz de Montellano, J. Biol.
Chem. 277, 12824–12829 (2002).
17. X. Hanoulle et al., J. Antimicrob. Chemother. 58, 768–772 (2006).
18. F. Wang et al., J. Exp. Med. 204, 73–78 (2007).
19. A. Banerjee et al., Science 263, 227–230 (1994).
20. J. Engohang-Ndong et al., Mol. Microbiol. 51, 175–188
21. N. Willand et al., ACS Chem. Biol. 5, 1007–1013 (2010).
22. M. Flipo et al., J. Med. Chem. 54, 2994–3010 (2011).
23. M. Flipo et al., J. Med. Chem. 55, 68–83 (2012).
24. M. Flipo et al., J. Med. Chem. 55, 6391–6402 (2012).
25. N. Willand et al., Nat. Med. 15, 537–544 (2009).
26. L. Cuthbertson, J. R. Nodwell, Microbiol. Mol. Biol. Rev. 77,
27. M. A. Schumacher et al., Science 294, 2158–2163 (2001).
28. F. Frénois, J. Engohang-Ndong, C. Locht, A. R. Baulard,
V. Villeret, Mol. Cell 16, 301–307 (2004).
29. F. Frénois, A. R. Baulard, V. Villeret, Tuberculosis (Edinb.) 86,
30. W. Weber et al., Proc. Natl. Acad. Sci. U.S.A. 105, 9994–9998
31. C. K. Stover et al., Nature 351, 456–460 (1991).
32. M. Coscolla, S. Gagneux, Semin. Immunol. 26, 431–444 (2014).
33. I. Comas et al., Nat. Genet. 45, 1176–1182 (2013).
34. N. Casali et al., Nat. Genet. 46, 279–286 (2014).
35. F. Ardito, B. Posteraro, M. Sanguinetti, S. Zanetti, G. Fadda,
J. Clin. Microbiol. 39, 4440–4444 (2001).
36. S. D. Ahuja et al., PLOS Med. 9, e1001300 (2012).
37. S. S. Grant et al., Cell Chem. Biol. 23, 666–677 (2016).
All data and code to understand and assess the conclusions of
this research are available in the main text, supplementary materials,
and via the following repositories: The refined coordinates and
the structure factors of Rv0078 were deposited in the Protein Data
Bank under the accession numbers 5N1C (iodinated form), 5N1I
(unliganded form), and 5ICJ (liganded form); raw data of RNA-seq
analysis have been deposited in Datadryad.org under the doi 10.5061/
dryad.mb463. We are indebted to the Soleil [Block Allocation Group
(BAG) proposal 20141408] and the European Synchrotron Radiation
Facility (BAG proposal MX-1677) synchrotrons for beam-time
allocations on this project. Sequencing analyses were performed at
the sciCORE ( http://scicore.unibas.ch) scientific computing core
facility at the University of Basel. The funders had no role in study
design, data collection and analysis, decision to publish, or
preparation of the manuscript. We are indebted to E. Willery for
technical support in molecular biology, F. Leroux for screening
management, L. Agouridas and N. Probst for chemical synthesis, and
C. Piveteau for bioanalysis. This work was supported by l’Agence
Nationale de la Recherche (ANR), France (Tea-4-Two, ANR-14-CE14-
0027-01) (ANR-10-EQPX-04-01), by EU grants ERC-STG
INTRACELLTB no 260901, the Feder (12001407 (D-AL), PRIM
(NewBio4Tb), European Research Council (grant 309540-EVODRTB),
SystemsX.ch, Institut National de la Santé et de la Recherche
Médicale, Université de Lille, Institut Pasteur de Lille, Centre National de
la Recherche Scientifique, the Région Hauts-de-France (convention no.
12000080), and Société d’Accélération du Transfert de Technologie Nord.
R. W. is Research Associate at the National Fund for Scientific Research
(FNRS-FRS) (Belgium). M.M. was supported by PRIM (NewBio4Tb),
V.D. was supported by EU grant 260872, and V. T. was suppported by the
Marie Curie Initial Training Network (ITN-2013-607694-Translocation).
The authors also thank the Unité Mixte de Recherche UMR 8199, Lille
Integrated Genomics Network for Advanced Personalized Medicine
(LIGAN-PM) Genomics platform (Lille, France), which belongs to the
Federation de Recherche 3508 Labex EGID (European Genomics
Institute for Diabetes; ANR-10-LABX-46) and was supported by the
Agence Nationale de la Recherche (ANR) Equipex 2010 session (ANR-
10-EQPX-07-01; LIGAN-PM). The LIGAN-PM Genomics platform is
supported by the Fonds Européen de Développement Régional and the
Region Hauts-de-France. We thank A. Wagner and Roquettes-Frères
(Lestrem, France) for their gift of Cyclodextrine Kleptose HP. We greatly
appreciate the fruitful discussions with all the members of the
GlaxoSmithKline team “Diseases of the Developing World (DDW)” and
their invaluable support in the continuation of this project. C.K. is
employed at Bioversys; S.G. is a consultant for the Foundation of
Innovative Diagnostics (FIND) in Geneva, Switzerland. M.G. is CEO
of Bioversys and a Board Member of the BEAM Alliance, a group of
Biopharmaceutical companies from Europe innovating in antimicrobial
resistance research. The BEAM Alliance is a not-for-profit association,
and there is no financial remuneration of any kind for its Board members.
All companies included are working within the field of antimicrobial
drug discovery. N. W., B.D., A.R.B., P.B., and M.D. are inventors
on patent PCT/EP2013/077706, which covers BDM41420; WO/2014/
096369. B.D., N. W., C.L., and A. R.B. are inventors on patent PCT/
FR2007/001138; WO/2008/003861, which covers BDM41906.
The facilities conformed to Directive 86/609/EEC on the Protection
of Animals Used for Experimental and Other Scientific Purposes
and norms published in the European Council E TS123 Appendix
A. Facilities and procedures complied with the Belgian Law
of 14 August 1986 on animal protection and welfare. Training of
experimental leaders, biotechnicians, and animal caretakers was in
accordance with Royal decree of 13 September 2004, which specifies
the training of persons working with laboratory animals. All animal
experimentation and procedures performed at the National Reference
Center for Tuberculosis and Mycobacteria, Bacterial Diseases Service,
Scientific Institute of Public Health (WIV-ISP), Brussels, Belgium, were
validated and approved by the Ethical Committee of the IPH-VAR
(Scientific Institute of Public Health–Veterinary and Agrochemical
Research Centre, Belgium) under file no. 120323-01. The animal facilities
and procedures were under the supervision of an expert on animal
welfare in accordance with the Belgian Ministry of Health. pET-15b-ethR2
and pET-15b-ethR are available from A.R.B. under a materials transfer
agreement with Institut Pasteur de Lille. pCK289 and pCK287 are
available from M.G. under a materials transfer agreement with Bioversys.
Materials and Methods
Figs. S1 to S5
Tables S1 to S6
14 May 2016; accepted 20 February 2017
Inflammation boosts bacteriophage
transfer between Salmonella spp.
Médéric Diard,1 Erik Bakkeren,1 Jeffrey K. Cornuault,2 Kathrin Moor,1†
Annika Hausmann,1† Mikael E. Sellin,1†‡ Claude Loverdo,3 Abram Aertsen,4
Martin Ackermann,5 Marianne De Paepe,2 Emma Slack,1 Wolf-Dietrich Hardt1*
Bacteriophage transfer (lysogenic conversion) promotes bacterial virulence evolution. There
is limited understanding of the factors that determine lysogenic conversion dynamics within
infected hosts. A murine Salmonella Typhimurium (S. Tm) diarrhea model was used to study the
transfer of SopEF, a prophage from S. Tm SL1344, to S. Tm ATCC14028S. Gut inflammation
and enteric disease triggered >55% lysogenic conversion of ATCC14028S within 3 days.
Without inflammation, SopEF transfer was reduced by up to 105-fold. This was because
inflammation (e.g., reactive oxygen species, reactive nitrogen species, hypochlorite) triggers the
bacterial SOS response, boosts expression of the phage antirepressor Tum, and thereby
promotes free phage production and subsequent transfer. Mucosal vaccination prevented a
dense intestinal S. Tm population from inducing inflammation and consequently abolished
SopEF transfer. Vaccination may be a general strategy for blocking pathogen evolution that
requires disease-driven transfer of temperate bacteriophages.
Bacteriophages (phages) often encode viru- lence factors and are important drivers of bacterial pathogen evolution (1, 2). This also holds true for pathogenic enterobacteriaceae, such as Salmonella enterica Typhimurium
(S. Tm). S. Tm genomes typically harbor several
prophages (1). Phage transfer (lysogenic conver-
sion) is a key driver of genomic diversification
between closely related enterobacteriaceae and
is thought to allow rapid adaptation to new host
species, notably by reassorting the virulence factor
repertoire (1). Phage transfer has been studied
extensively in vitro. This has established its mo-
lecular basis and delivered numerous tools for
molecular biology. In hosts infected by Staphy-
lococcus aureus, Escherichia coli, Pseudomonas
aeruginosa, or Streptococcus pyogenes, the re-
activation or transfer of phages has also been
observed (3–8). However, we have limited infor-
mation about the factors controlling phage-transfer
dynamics in vivo.
To address this knowledge gap, we characterized SopEF transfer between two well-established
S. Tm strains (SL1344 and ATCC14028S) commonly
used in virulence studies in mice (fig. S1, A and B).
SopEF belongs to the P2 family of temperate
phages (9, 10). Its tail-fiber region carries a gene
encoding SopE, a virulence factor enhancing S. Tm’s