Journal of Biotechnology 187 (2014) 106–107
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of the Gram-negative probiotic Escherichia coli strain Nissle 1917 Marten Reister a,1 , Klaus Hoffmeier a,1 , Nicolas Krezdorn a , Bjoern Rotter a , Chunguang Liang b , Stefan Rund c , Thomas Dandekar b , Ulrich Sonnenborn d , Tobias A. Oelschlaeger c,∗ a
GenXPro GmbH, Altenhoeferallee 3, 60438 Frankfurt/Main, Germany Bioinformatik, Biozentrum, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany c Institut für Molekulare Infektionsbiologie, University of Wuerzburg, Josef-Schneider-Str. 2/D15, 97080 Wuerzburg, Germany d Department of Biological Research, Ardeypharm GmbH, Loerfeldstr. 20, 58313 Herdecke, Germany b
a r t i c l e
i n f o
Article history: Received 10 July 2014 Accepted 24 July 2014 Available online 2 August 2014 Keywords: Escherichia coli Nissle 1917 Genome DNA sequence Probiotic drug
a b s t r a c t Escherichia coli strain Nissle 1917 (EcN) is the active principle of a probiotic preparation (trade name Mutaflor® ) used for the treatment of patients with intestinal diseases such as ulcerative colitis and diarrhea. It has GRAS (generally recognized as save) status and has been shown to be a therapeutically effective drug (Sonnenborn and Schulze, 2009). The complete genomic DNA sequence will help in identifying genes and their products which are essential for the strains probiotic nature. Genbank/EMBL/DDBJ accession number: CP007799 (chromosome). © 2014 Elsevier B.V. All rights reserved.
Escherichia coli strain Nissle 1917 (deposited at DSMZ, Braunschweig, Germany as DSM6601) is of serotype O6:K5:H1 and is one of the few probiotics licensed as a medicine for the treatment of human patients and also one of the best studied probiotic strains (recently reviewed by Jacobi and Malfertheiner, 2011, and by Behnsen et al., 2013). Originally, EcN was isolated in 1917 by Alfred Nissle from the feces of a soldier who was the only one of his unit not suffering from dysentery. EcN is serumsensitive, in contrast to probiotic Lactobacillus strains (Sonnenborn and Schulze, 2009). It harbors two cryptic plasmids, encodes several fitness factors (e.g. seven iron uptake systems, several adhesins) and secretes microcins M and H47 which can kill other E. coli and certain Salmonella strains (Patzer et al., 2003). It also interferes with adhesion and invasion of pathogenic E. coli, Salmonella, Shigella, Listeria, and Legionella (Altenhoefer et al., 2004; Rund et al., 2013; Schierack et al., 2011). Its flagellum is a multipurpose tool not only mediating motility but also inducing expression of human beta-defensin
∗ Corresponding author. Tel.: +49 0931 3182150; fax: +49 0931 3182578. E-mail addresses: [email protected] (M. Reister), [email protected] (K. Hoffmeier), [email protected] (N. Krezdorn), [email protected] (B. Rotter), [email protected] (C. Liang), [email protected] (S. Rund), [email protected] (T. Dandekar), [email protected] (U. Sonnenborn), [email protected] (T.A. Oelschlaeger). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2014.07.442 0168-1656/© 2014 Elsevier B.V. All rights reserved.
2 and functioning as an adhesin for binding to human and porcine mucus (Troge et al., 2012). In vivo EcN is able to modulate immune responses. The therapeutic efficacy of EcN (Mutaflor® ) is well documented in many controlled clinical trials. Several studies showed EcN to be as effective as the “gold standard” mesalazine in maintaining remission of ulcerative colitis, and no serious adverse effects were observed (e.g. Rembacken et al., 1999; Kruis et al., 2004). Duration of diarrhea in young children was significantly reduced in the EcN group compared to the placebo group, and EcN was found to be safe and well tolerated (Henker et al., 2007). In spite of EcN’s long-term use in medicine, its comprehensive investigations by many research groups, and its proven clinical efficacy, the genomic DNA sequence of this probiotic bacterium has only been published as a draft (Cress et al., 2013). Here we report EcN’s complete and annotated genome sequence. For Illumina sequencing, genomic DNA was randomly sheared into pieces of 200–300 bp length and Paired-End libraries were prepared and sequenced on the HiSeq2000. In total, 211,047,644 reads were generated. For PacBio sequencing, genomic DNA was randomly sheared into 10 kb-long pieces and libraries were prepared and sequenced on a PacBio machine. In total 75,152 reads were generated. The reads were processed using an internal protocol of GenXPro for duplicate removal, adapter trimming and elimination of low quality reads. The PacBio reads were corrected using PacBioToCA (Koren et al., 2013) and assembled with Celera Assembler together with the Illumina reads in a hybrid assembly
M. Reister et al. / Journal of Biotechnology 187 (2014) 106–107 Table 1 Escherichia coli Nissle 1917 genome features. Attribute
Value
Genome size (bp) Number of contigs GC content (%) Plasmids rRNAs tRNAs Predicted genes
5,441,200 3 50.6 2 53 117 5324
approach. The resulting contigs were further assembled with CAP3 (Huang and Madan, 1999) to reach 112 contigs. In addition, the Illumina reads were mapped to the reference genome strain E. coli CFT073 (Welch et al., 2002) with Novoalign (www.novocraft.com). The consensus sequences were extracted with samtools (Li et al., 2009). The final DNA sequence was generated and corrected using an internal program that employed MAUVE (Darling et al., 2004) and Segemehl (Hoffmann et al., 2009). The determined genome sequence contains 5,441,200 bp, has a GC-content of 50.6% and encodes 5324 ORFs, 117 tRNAs and 53 rRNAs, as annotated by RAST and Interproscan (Table 1). The closest related E. coli strains are uropathogenic strain CFT073 (genome size 5,231,428 bp; Welch et al., 2002) and asymptomatic bacteriuria causing strain ABU83972 (genome size 5,313,397 bp; Zdziarski et al., 2010). Just 190 ORFs are only present in EcN, but absent in CFT073 and ABU83972. These genes represent about 3,6% of EcN’s genome. However, EcN is more similar to CFT073 with respect to genotype and phenotype than ABU83972 (Vejborg et al., 2010). The now available genome sequence of EcN will be helpful in identifying genes and gene products essential for this strain’s probiotic properties. Acknowledgement This work was supported by the company Ardeypharm GmbH, Herdecke, Germany Fonds 824126. References Altenhoefer, A., Oswald, S., Sonnenborn, U., Enders, C., Schulze, J., Hacker, J., Oelschlaeger, T.A., 2004. The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol. Med. Microbiol. 40, 223–229. Behnsen, J., Deriu, E., Sassone-Corsi, M., Raffatellu, M., 2013. Probiotics: properties, examples, and specific applications. Cold Spring Harb. Perspect. Med. 3, a010074, 1–15.
107
Cress, B.F., Linhardt, R.J., Koffas, M.A.G., 2013. Draft genome sequence of Escherichia coli strain Nissle 1917 (serovar O6:K5:H1). Genome Announc. 1 (2), e00047–13. Darling, A.C., Mau, B., Blattner, F.R., Perna, N.T., 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14, 1394–1403. Henker, J., Laass, M., Blokhin, B.M., Bolbot, Y.K., Maydannik, V.G., Elze, M., Wolff, C., Schulze, J., 2007. The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhea in infants and toddlers. Eur. J. Pediatr. 166, 3111–3318. Hoffmann, S., Otto, C., Kurtz, S., Sharma, C.M., Khaitovich, P., Vogel, J., Stadler, P.F., Hackermueller, J., 2009. Fast mapping of short sequences with mismatches, insertions and deletions using index structures. PLoS Comput. Biol. 5 (9), e1000502. Huang, X., Madan, A., 1999. CAP3: a DNA sequence assembly program. Genome Res. 9, 868–877. Jacobi, C.A., Malfertheiner, P., 2011. Escherichia coli Nissle 1917 (Mutaflor): new insights into an old probiotic bacterium. Dig. Dis. 29, 600–607. Koren, S., Harhay, G.P., Smith, T.P., Bono, J.L., Harhay, D.M., McVey, S.D., Radune, D., Bergman, N.H., Phillippy, A.M., 2013. Reducing assembly complexity of microbial genomes with single-molecule sequencing. Genome Biol. 14 (9), R101. Kruis, W., Fric, P., Pokrotnieks, J., Lukas, M., Fixa, B., Kascak, M., Kamm, M.A., Weismueller, J., Beglinger, C., Stolte, M., Wolff, C., Schulze, J., 2004. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 53, 1617–1623. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079. Patzer, S.I., Baquero, M.R., Bravo, D., Moreno, F., Hantke, K., 2003. The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN. Microbiology 149, 2557–2570. Rembacken, B.J., Snelling, A.M., Hawkey, P.M., Chalmers, D.M., Axon, A.T.R., 1999. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 354, 635–639. Rund, S.A., Rohde, H., Sonnenborn, U., Oelschlaeger, T.A., 2013. Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7. Int. J. Med. Microbiol. 303, 1–8. Schierack, P., Kleta, S., Tedin, K., Babila, J.T., Oswald, S., Oelschlaeger, T.A., Hiemann, R., Paetzold, S., Wieler, L.H., 2011. E. coli Nissle 1917 affects Salmonella adhesion to porcine intestinal epithelial cells. PLoS ONE 6 (2), e14712. Sonnenborn, U., Schulze, J., 2009. The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic. Microb. Ecol. Health Dis. 21, 122–158. Troge, A., Scheppach, W., Schroeder, B.O., Rund, S.A., Heuner, K., Wehkamp, J., Stange, E.F., Oelschlaeger, T.A., 2012. More than a marine propeller – the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus. Int. J. Med. Microbiol. 302, 304–314. Vejborg, R.M., Friis, C., Hancock, V., Schembri, M.A., Klemm, P., 2010. A virulent parent with probiotic progeny: comparative genomics of Escherichia coli strains CFT073, Nissle 1917 and ABU 83972. Mol. Genet. Genomics 283, 469–484. Welch, R.A., Burland, V., Plunkett III, G., Redford, P., Roesch, P., Rasko, D., Buckles, E.L., Liou, S.R., Boutin, A., Hackett, J., Stroud, D., Mayhew, G.F., Rose, D.J., Zhou, S., Schwartz, D.C., Perna, N.T., Mobley, H.L., Donnenberg, M.S., Blattner, F.R., 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 99, 17020– 17024. Zdziarski, J., Brzuszkiewicz, E., Wullt, B., Liesegang, H., Biran, D., Voigt, B., GrönbergHernandez, J., Ragnarsdottir, B., Hecker, M., Ton, Z.E., Daniel, R., Gottschalk, G., Hacker, J., Svanborg, C., Dobrindt, U., 2010. Host imprints on bacterial genomes – rapid, divergent evolution in individual patients. PLoS Pathog. 6 (8), e1001078.
Contents lists available at ScienceDirect
Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec
Genome Announcement
Complete genome sequence of the Gram-negative probiotic Escherichia coli strain Nissle 1917 Marten Reister a,1 , Klaus Hoffmeier a,1 , Nicolas Krezdorn a , Bjoern Rotter a , Chunguang Liang b , Stefan Rund c , Thomas Dandekar b , Ulrich Sonnenborn d , Tobias A. Oelschlaeger c,∗ a
GenXPro GmbH, Altenhoeferallee 3, 60438 Frankfurt/Main, Germany Bioinformatik, Biozentrum, University of Wuerzburg, Am Hubland, 97074 Wuerzburg, Germany c Institut für Molekulare Infektionsbiologie, University of Wuerzburg, Josef-Schneider-Str. 2/D15, 97080 Wuerzburg, Germany d Department of Biological Research, Ardeypharm GmbH, Loerfeldstr. 20, 58313 Herdecke, Germany b
a r t i c l e
i n f o
Article history: Received 10 July 2014 Accepted 24 July 2014 Available online 2 August 2014 Keywords: Escherichia coli Nissle 1917 Genome DNA sequence Probiotic drug
a b s t r a c t Escherichia coli strain Nissle 1917 (EcN) is the active principle of a probiotic preparation (trade name Mutaflor® ) used for the treatment of patients with intestinal diseases such as ulcerative colitis and diarrhea. It has GRAS (generally recognized as save) status and has been shown to be a therapeutically effective drug (Sonnenborn and Schulze, 2009). The complete genomic DNA sequence will help in identifying genes and their products which are essential for the strains probiotic nature. Genbank/EMBL/DDBJ accession number: CP007799 (chromosome). © 2014 Elsevier B.V. All rights reserved.
Escherichia coli strain Nissle 1917 (deposited at DSMZ, Braunschweig, Germany as DSM6601) is of serotype O6:K5:H1 and is one of the few probiotics licensed as a medicine for the treatment of human patients and also one of the best studied probiotic strains (recently reviewed by Jacobi and Malfertheiner, 2011, and by Behnsen et al., 2013). Originally, EcN was isolated in 1917 by Alfred Nissle from the feces of a soldier who was the only one of his unit not suffering from dysentery. EcN is serumsensitive, in contrast to probiotic Lactobacillus strains (Sonnenborn and Schulze, 2009). It harbors two cryptic plasmids, encodes several fitness factors (e.g. seven iron uptake systems, several adhesins) and secretes microcins M and H47 which can kill other E. coli and certain Salmonella strains (Patzer et al., 2003). It also interferes with adhesion and invasion of pathogenic E. coli, Salmonella, Shigella, Listeria, and Legionella (Altenhoefer et al., 2004; Rund et al., 2013; Schierack et al., 2011). Its flagellum is a multipurpose tool not only mediating motility but also inducing expression of human beta-defensin
∗ Corresponding author. Tel.: +49 0931 3182150; fax: +49 0931 3182578. E-mail addresses: [email protected] (M. Reister), [email protected] (K. Hoffmeier), [email protected] (N. Krezdorn), [email protected] (B. Rotter), [email protected] (C. Liang), [email protected] (S. Rund), [email protected] (T. Dandekar), [email protected] (U. Sonnenborn), [email protected] (T.A. Oelschlaeger). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.jbiotec.2014.07.442 0168-1656/© 2014 Elsevier B.V. All rights reserved.
2 and functioning as an adhesin for binding to human and porcine mucus (Troge et al., 2012). In vivo EcN is able to modulate immune responses. The therapeutic efficacy of EcN (Mutaflor® ) is well documented in many controlled clinical trials. Several studies showed EcN to be as effective as the “gold standard” mesalazine in maintaining remission of ulcerative colitis, and no serious adverse effects were observed (e.g. Rembacken et al., 1999; Kruis et al., 2004). Duration of diarrhea in young children was significantly reduced in the EcN group compared to the placebo group, and EcN was found to be safe and well tolerated (Henker et al., 2007). In spite of EcN’s long-term use in medicine, its comprehensive investigations by many research groups, and its proven clinical efficacy, the genomic DNA sequence of this probiotic bacterium has only been published as a draft (Cress et al., 2013). Here we report EcN’s complete and annotated genome sequence. For Illumina sequencing, genomic DNA was randomly sheared into pieces of 200–300 bp length and Paired-End libraries were prepared and sequenced on the HiSeq2000. In total, 211,047,644 reads were generated. For PacBio sequencing, genomic DNA was randomly sheared into 10 kb-long pieces and libraries were prepared and sequenced on a PacBio machine. In total 75,152 reads were generated. The reads were processed using an internal protocol of GenXPro for duplicate removal, adapter trimming and elimination of low quality reads. The PacBio reads were corrected using PacBioToCA (Koren et al., 2013) and assembled with Celera Assembler together with the Illumina reads in a hybrid assembly
M. Reister et al. / Journal of Biotechnology 187 (2014) 106–107 Table 1 Escherichia coli Nissle 1917 genome features. Attribute
Value
Genome size (bp) Number of contigs GC content (%) Plasmids rRNAs tRNAs Predicted genes
5,441,200 3 50.6 2 53 117 5324
approach. The resulting contigs were further assembled with CAP3 (Huang and Madan, 1999) to reach 112 contigs. In addition, the Illumina reads were mapped to the reference genome strain E. coli CFT073 (Welch et al., 2002) with Novoalign (www.novocraft.com). The consensus sequences were extracted with samtools (Li et al., 2009). The final DNA sequence was generated and corrected using an internal program that employed MAUVE (Darling et al., 2004) and Segemehl (Hoffmann et al., 2009). The determined genome sequence contains 5,441,200 bp, has a GC-content of 50.6% and encodes 5324 ORFs, 117 tRNAs and 53 rRNAs, as annotated by RAST and Interproscan (Table 1). The closest related E. coli strains are uropathogenic strain CFT073 (genome size 5,231,428 bp; Welch et al., 2002) and asymptomatic bacteriuria causing strain ABU83972 (genome size 5,313,397 bp; Zdziarski et al., 2010). Just 190 ORFs are only present in EcN, but absent in CFT073 and ABU83972. These genes represent about 3,6% of EcN’s genome. However, EcN is more similar to CFT073 with respect to genotype and phenotype than ABU83972 (Vejborg et al., 2010). The now available genome sequence of EcN will be helpful in identifying genes and gene products essential for this strain’s probiotic properties. Acknowledgement This work was supported by the company Ardeypharm GmbH, Herdecke, Germany Fonds 824126. References Altenhoefer, A., Oswald, S., Sonnenborn, U., Enders, C., Schulze, J., Hacker, J., Oelschlaeger, T.A., 2004. The probiotic Escherichia coli strain Nissle 1917 interferes with invasion of human intestinal epithelial cells by different enteroinvasive bacterial pathogens. FEMS Immunol. Med. Microbiol. 40, 223–229. Behnsen, J., Deriu, E., Sassone-Corsi, M., Raffatellu, M., 2013. Probiotics: properties, examples, and specific applications. Cold Spring Harb. Perspect. Med. 3, a010074, 1–15.
107
Cress, B.F., Linhardt, R.J., Koffas, M.A.G., 2013. Draft genome sequence of Escherichia coli strain Nissle 1917 (serovar O6:K5:H1). Genome Announc. 1 (2), e00047–13. Darling, A.C., Mau, B., Blattner, F.R., Perna, N.T., 2004. Mauve: multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 14, 1394–1403. Henker, J., Laass, M., Blokhin, B.M., Bolbot, Y.K., Maydannik, V.G., Elze, M., Wolff, C., Schulze, J., 2007. The probiotic Escherichia coli strain Nissle 1917 (EcN) stops acute diarrhea in infants and toddlers. Eur. J. Pediatr. 166, 3111–3318. Hoffmann, S., Otto, C., Kurtz, S., Sharma, C.M., Khaitovich, P., Vogel, J., Stadler, P.F., Hackermueller, J., 2009. Fast mapping of short sequences with mismatches, insertions and deletions using index structures. PLoS Comput. Biol. 5 (9), e1000502. Huang, X., Madan, A., 1999. CAP3: a DNA sequence assembly program. Genome Res. 9, 868–877. Jacobi, C.A., Malfertheiner, P., 2011. Escherichia coli Nissle 1917 (Mutaflor): new insights into an old probiotic bacterium. Dig. Dis. 29, 600–607. Koren, S., Harhay, G.P., Smith, T.P., Bono, J.L., Harhay, D.M., McVey, S.D., Radune, D., Bergman, N.H., Phillippy, A.M., 2013. Reducing assembly complexity of microbial genomes with single-molecule sequencing. Genome Biol. 14 (9), R101. Kruis, W., Fric, P., Pokrotnieks, J., Lukas, M., Fixa, B., Kascak, M., Kamm, M.A., Weismueller, J., Beglinger, C., Stolte, M., Wolff, C., Schulze, J., 2004. Maintaining remission of ulcerative colitis with the probiotic Escherichia coli Nissle 1917 is as effective as with standard mesalazine. Gut 53, 1617–1623. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079. Patzer, S.I., Baquero, M.R., Bravo, D., Moreno, F., Hantke, K., 2003. The colicin G, H and X determinants encode microcins M and H47, which might utilize the catecholate siderophore receptors FepA, Cir, Fiu and IroN. Microbiology 149, 2557–2570. Rembacken, B.J., Snelling, A.M., Hawkey, P.M., Chalmers, D.M., Axon, A.T.R., 1999. Non-pathogenic Escherichia coli versus mesalazine for the treatment of ulcerative colitis: a randomised trial. Lancet 354, 635–639. Rund, S.A., Rohde, H., Sonnenborn, U., Oelschlaeger, T.A., 2013. Antagonistic effects of probiotic Escherichia coli Nissle 1917 on EHEC strains of serotype O104:H4 and O157:H7. Int. J. Med. Microbiol. 303, 1–8. Schierack, P., Kleta, S., Tedin, K., Babila, J.T., Oswald, S., Oelschlaeger, T.A., Hiemann, R., Paetzold, S., Wieler, L.H., 2011. E. coli Nissle 1917 affects Salmonella adhesion to porcine intestinal epithelial cells. PLoS ONE 6 (2), e14712. Sonnenborn, U., Schulze, J., 2009. The non-pathogenic Escherichia coli strain Nissle 1917 – features of a versatile probiotic. Microb. Ecol. Health Dis. 21, 122–158. Troge, A., Scheppach, W., Schroeder, B.O., Rund, S.A., Heuner, K., Wehkamp, J., Stange, E.F., Oelschlaeger, T.A., 2012. More than a marine propeller – the flagellum of the probiotic Escherichia coli strain Nissle 1917 is the major adhesin mediating binding to human mucus. Int. J. Med. Microbiol. 302, 304–314. Vejborg, R.M., Friis, C., Hancock, V., Schembri, M.A., Klemm, P., 2010. A virulent parent with probiotic progeny: comparative genomics of Escherichia coli strains CFT073, Nissle 1917 and ABU 83972. Mol. Genet. Genomics 283, 469–484. Welch, R.A., Burland, V., Plunkett III, G., Redford, P., Roesch, P., Rasko, D., Buckles, E.L., Liou, S.R., Boutin, A., Hackett, J., Stroud, D., Mayhew, G.F., Rose, D.J., Zhou, S., Schwartz, D.C., Perna, N.T., Mobley, H.L., Donnenberg, M.S., Blattner, F.R., 2002. Extensive mosaic structure revealed by the complete genome sequence of uropathogenic Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 99, 17020– 17024. Zdziarski, J., Brzuszkiewicz, E., Wullt, B., Liesegang, H., Biran, D., Voigt, B., GrönbergHernandez, J., Ragnarsdottir, B., Hecker, M., Ton, Z.E., Daniel, R., Gottschalk, G., Hacker, J., Svanborg, C., Dobrindt, U., 2010. Host imprints on bacterial genomes – rapid, divergent evolution in individual patients. PLoS Pathog. 6 (8), e1001078.
