Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae

Abstract

Among the most urgent public health threats is the worldwide emergence of carbapenem-resistant Enterobacteriaceae1,2,3,4, which are resistant to the antibiotic class of ‘last resort’. In the United States and Europe, carbapenem-resistant strains of the Klebsiella pneumoniae ST258 (ref. 5) sequence type are dominant, endemic6,7,8 and associated with high mortality6,9,10. We report the global evolution of pathogenicity in carbapenem-resistant K. pneumoniae, resulting in the repeated convergence of virulence and carbapenem resistance in the United States and Europe, dating back to as early as 2009. We demonstrate that K. pneumoniae can enhance its pathogenicity by adopting two opposing infection programs through easily acquired gain- and loss-of-function mutations. Single-nucleotide polymorphisms in the capsule biosynthesis gene wzc lead to hypercapsule production, which confers phagocytosis resistance, enhanced dissemination and increased mortality in animal models. In contrast, mutations disrupting capsule biosynthesis genes impair capsule production, which enhances epithelial cell invasion, in vitro biofilm formation and persistence in urinary tract infections. These two types of capsule mutants have emerged repeatedly and independently in Europe and the United States, with hypercapsule mutants associated with bloodstream infections and capsule-deficient mutants associated with urinary tract infections. In the latter case, drug-tolerant K. pneumoniae can persist to yield potentially untreatable, persistent infection.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Capsule heterogeneity in screened collection of 54 clinical ST258 isolates.
Fig. 2: Gain- and loss-of-function mutations increase the virulence or the persistence of K. pneumoniae ST258.
Fig. 3: Hypercapsule and capsule-deficient mutants are widespread across the ST258 phylogeny.
Fig. 4: A model for how capsule remodeling results in different infection phenotypes of K. pneumoniae.

Data availability

Sequencing data from this study can be found in the SRA of the NCBI database under Bioproject accession no. PRJNA506070. All other datasets are available from the corresponding author on reasonable request.

Change history

  • 25 June 2020

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

References

  1. 1.

    Antibiotic Resistance Threats in the United States (Centers for Disease Control and Prevention, 2013).

  2. 2.

    Rapid Risk Assessment: Carbapenem-resistant Enterobacteriaceae—First update (European Centre for Disease Prevention and Control, 2018).

  3. 3.

    Pitout, J. D., Nordmann, P. & Poirel, L. Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrob. Agents Chemother. 59, 5873–5884 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Antimicrobial Resistance—Global Report on Surveillance (World Health Organization, 2014).

  5. 5.

    Deleo, F. R. et al. Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae. Proc. Natl Acad. Sci. USA 111, 4988–4993 (2014).

    CAS  PubMed  Google Scholar 

  6. 6.

    Munoz-Price, L. S. et al. Clinical epidemiology of the global expansion of Klebsiella pneumoniae carbapenemases. Lancet Infect. Dis. 13, 785–796 (2013).

    PubMed  PubMed Central  Google Scholar 

  7. 7.

    Lee, C. R. et al. Global dissemination of carbapenemase-producing Klebsiella pneumoniae: epidemiology, genetic context, treatment options, and detection methods. Front. Microbiol. 7, 895 (2016).

    PubMed  PubMed Central  Google Scholar 

  8. 8.

    Bowers, J. R. et al. Genomic analysis of the emergence and rapid global dissemination of the clonal group 258 Klebsiella pneumoniae pandemic. PLoS ONE 10, e0133727 (2015).

    PubMed  PubMed Central  Google Scholar 

  9. 9.

    Dautzenberg, M. J., Haverkate, M. R., Bonten, M. J. & Bootsma, M. C. Epidemic potential of Escherichia coli ST131 and Klebsiella pneumoniae ST258: a systematic review and meta-analysis. BMJ Open 6, e009971 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Snitkin, E. S. et al. Tracking a hospital outbreak of carbapenem-resistant Klebsiella pneumoniae with whole-genome sequencing. Sci. Transl. Med. 4, 148ra116 (2012).

    PubMed  PubMed Central  Google Scholar 

  11. 11.

    Gu, D. et al. A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study. Lancet Infect. Dis. 18, 37–46 (2018).

    PubMed  Google Scholar 

  12. 12.

    Lam, M. M. C. et al. Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae. Genome Med. 10, 77 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Cerqueira, G. C. et al. Multi-institute analysis of carbapenem resistance reveals remarkable diversity, unexplained mechanisms, and limited clonal outbreaks. Proc. Natl Acad. Sci. USA 114, 1135–1140 (2017).

    CAS  PubMed  Google Scholar 

  14. 14.

    Siu, L. K., Yeh, K. M., Lin, J. C., Fung, C. P. & Chang, F. Y. Klebsiella pneumoniae liver abscess: a new invasive syndrome. Lancet Infect. Dis. 12, 881–887 (2012).

    PubMed  Google Scholar 

  15. 15.

    Sahly, H. et al. Capsule impedes adhesion to and invasion of epithelial cells by Klebsiella pneumoniae. Infect. Immun. 68, 6744–6749 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Domenico, P., Schwartz, S. & Cunha, B. A. Reduction of capsular polysaccharide production in Klebsiella pneumoniae by sodium salicylate. Infect. Immun. 57, 3778–3782 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Broberg, C. A., Palacios, M. & Miller, V. L. Klebsiella: a long way to go towards understanding this enigmatic jet-setter. F1000Prime Rep. 6, 64 (2014).

    PubMed  PubMed Central  Google Scholar 

  18. 18.

    Whitfield, C. Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu. Rev. Biochem. 75, 39–68 (2006).

    CAS  PubMed  Google Scholar 

  19. 19.

    Hunstad, D. A. & Justice, S. S. Intracellular lifestyles and immune evasion strategies of uropathogenic Escherichia coli. Annu. Rev. Microbiol. 64, 203–221 (2010).

    CAS  PubMed  Google Scholar 

  20. 20.

    Mysorekar, I. U. & Hultgren, S. J. Mechanisms of uropathogenic Escherichia coli persistence and eradication from the urinary tract. Proc. Natl Acad. Sci. USA 103, 14170–14175 (2006).

    CAS  PubMed  Google Scholar 

  21. 21.

    Flores-Mireles, A. L., Walker, J. N., Caparon, M. & Hultgren, S. J. Urinary tract infections: epidemiology, mechanisms of infection and treatment options. Nat. Rev. Microbiol. 13, 269–284 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. 22.

    Blango, M. G. & Mulvey, M. A. Persistence of uropathogenic Escherichia coli in the face of multiple antibiotics. Antimicrob. Agents Chemother. 54, 1855–1863 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Hung, C. S., Dodson, K. W. & Hultgren, S. J. A murine model of urinary tract infection. Nat. Protoc. 4, 1230–1243 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Lam, M. M. C. et al. Population genomics of hypervirulent Klebsiella pneumoniae clonal-group 23 reveals early emergence and rapid global dissemination. Nat. Commun. 9, 2703 (2018).

    PubMed  PubMed Central  Google Scholar 

  25. 25.

    Nassif, X., Fournier, J. M., Arondel, J. & Sansonetti, P. J. Mucoid phenotype of Klebsiella pneumoniae is a plasmid-encoded virulence factor. Infect. Immun. 57, 546–552 (1989).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Walker, K. A. et al. A Klebsiella pneumoniae regulatory mutant has reduced capsule expression but retains hypermucoviscosity. MBio 10, e00089-19 (2019).

  27. 27.

    Gottesman, S. & Stout, V. Regulation of capsular polysaccharide synthesis in Escherichia coli K12. Mol. Microbiol. 5, 1599–1606 (1991).

    CAS  PubMed  Google Scholar 

  28. 28.

    Geisinger, E. & Isberg, R. R. Antibiotic modulation of capsular exopolysaccharide and virulence in Acinetobacter baumannii. PLoS Pathog. 11, e1004691 (2015).

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Fang, C. T., Chuang, Y. P., Shun, C. T., Chang, S. C. & Wang, J. T. A novel virulence gene in Klebsiella pneumoniae strains causing primary liver abscess and septic metastatic complications. J. Exp. Med. 199, 697–705 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. 30.

    Wozniak, J. E. et al. A nationwide screen of carbapenem-resistant Klebsiella pneumoniae reveals an isolate with enhanced virulence and clinically undetected colistin heteroresistance. Antimicrob. Agents Chemother. 63, e00107-19 (2019).

  31. 31.

    Ruppe, E. et al. Clonal or not clonal? Investigating hospital outbreaks of KPC-producing Klebsiella pneumoniae with whole-genome sequencing. Clin. Microbiol. Infect. 23, 470–475 (2017).

    CAS  PubMed  Google Scholar 

  32. 32.

    Spencer, M. D. et al. Whole genome sequencing detects inter-facility transmission of carbapenem-resistant Klebsiella pneumoniae. J. Infect. 78, 187–199 (2019).

  33. 33.

    Zilberberg, M. D., Nathanson, B. H., Sulham, K., Fan, W. & Shorr, A. F. Carbapenem resistance, inappropriate empiric treatment and outcomes among patients hospitalized with Enterobacteriaceae urinary tract infection, pneumonia and sepsis. BMC Infect. Dis. 17, 279 (2017).

    PubMed  PubMed Central  Google Scholar 

  34. 34.

    Skurnik, D. et al. Extended-spectrum antibodies protective against carbapenemase-producing Enterobacteriaceae. J. Antimicrob. Chemother. 71, 927–935 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Russo, T. A. & Marr, C. M. Hypervirulent Klebsiella pneumoniae. Clin. Microbiol. Rev. 32, e00001-19 (2019).

  36. 36.

    Uhlemann, A. C., Otto, M., Lowy, F. D. & DeLeo, F. R. Evolution of community- and healthcare-associated methicillin-resistant Staphylococcus aureus. Infect. Genet. Evol. 21, 563–574 (2014).

    PubMed  Google Scholar 

  37. 37.

    Valentini, M., Gonzalez, D., Mavridou, D. A. & Filloux, A. Lifestyle transitions and adaptive pathogenesis of Pseudomonas aeruginosa. Curr. Opin. Microbiol. 41, 15–20 (2018).

    CAS  PubMed  Google Scholar 

  38. 38.

    Kobayashi, S. D. et al. Phagocytosis and killing of carbapenem-resistant ST258 Klebsiella pneumoniae by human neutrophils. J. Infect. Dis. 213, 1615–1622 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Yoshida, K. et al. Role of bacterial capsule in local and systemic inflammatory responses of mice during pulmonary infection with Klebsiella pneumoniae. J. Med. Microbiol. 49, 1003–1010 (2000).

    PubMed  Google Scholar 

  40. 40.

    Kobayashi, S. D. & DeLeo, F. R. Re-evaluating the potential of immunoprophylaxis and/or immunotherapy for infections caused by multidrug resistant Klebsiella pneumoniae. Future Microbiol. 13, 1343–1346 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Edwards, R. A., Keller, L. H. & Schifferli, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207, 149–157 (1998).

    CAS  PubMed  Google Scholar 

  42. 42.

    Gibson, D. G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).

    CAS  PubMed  Google Scholar 

  43. 43.

    Ferrieres, L. et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J. Bacteriol. 192, 6418–6427 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Guzman, L. M., Belin, D., Carson, M. J. & Beckwith, J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177, 4121–4130 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Chang, A. C. & Cohen, S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J. Bacteriol. 134, 1141–1156 (1978).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Shaner, N. C. et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat. Methods 10, 407–409 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Crepin, S., Harel, J. & Dozois, C. M. Chromosomal complementation using Tn7 transposon vectors in Enterobacteriaceae. Appl. Environ. Microbiol. 78, 6001–6008 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48.

    Davis, J. H., Rubin, A. J. & Sauer, R. T. Design, construction and characterization of a set of insulated bacterial promoters. Nucleic Acids Res. 39, 1131–1141 (2011).

    CAS  PubMed  Google Scholar 

  49. 49.

    Coffey, B. M. & Anderson, G. G. Biofilm formation in the 96-well microtiter plate. Methods Mol. Biol. 1149, 631–641 (2014).

    PubMed  Google Scholar 

  50. 50.

    Blumenkrantz, N. & Asboe-Hansen, G. New method for quantitative determination of uronic acids. Anal. Biochem. 54, 484–489 (1973).

    CAS  PubMed  Google Scholar 

  51. 51.

    Hsu, C. R., Lin, T. L., Chen, Y. C., Chou, H. C. & Wang, J. T. The role of Klebsiella pneumoniae rmpA in capsular polysaccharide synthesis and virulence revisited. Microbiology 157, 3446–3457 (2011).

    CAS  PubMed  Google Scholar 

  52. 52.

    Siguier, P., Perochon, J., Lestrade, L., Mahillon, J. & Chandler, M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res. 34, D32–D36 (2006).

    CAS  PubMed  Google Scholar 

  53. 53.

    Lam, M. M. C. et al. Genetic diversity, mobilisation and spread of the yersiniabactin-encoding mobile element ICEKp in Klebsiella pneumoniae populations. Microb. Genom. 4, e000196 (2018).

    PubMed Central  Google Scholar 

  54. 54.

    Walker, B. J. et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE 9, e112963 (2014).

    PubMed  PubMed Central  Google Scholar 

  55. 55.

    Wyres, K. L. et al. Identification of Klebsiella capsule synthesis loci from whole genome data. Microb. Genom. 2, e000102 (2016).

    PubMed  PubMed Central  Google Scholar 

  56. 56.

    Sperlea, T. et al. γBOriS: identification of origins of replication in gammaproteobacteria using motif-based machine learning. Preprint at bioRxiv https://doi.org/10.1101/597070 (2019).

  57. 57.

    Wilm, A. et al. LoFreq: a sequence-quality aware, ultra-sensitive variant caller for uncovering cell-population heterogeneity from high-throughput sequencing datasets. Nucleic Acids Res. 40, 11189–11201 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Goris, J. et al. DNA–DNA hybridization values and their relationship to whole-genome sequence similarities. Int. J. Syst. Evol. Microbiol. 57, 81–91 (2007).

    CAS  PubMed  Google Scholar 

  61. 61.

    Inouye, M. et al. SRST2: rapid genomic surveillance for public health and hospital microbiology labs. Genome Med. 6, 90 (2014).

    PubMed  PubMed Central  Google Scholar 

  62. 62.

    Arndt, D. et al. PHASTER: a better, faster version of the PHAST phage search tool. Nucleic Acids Res. 44, W16–W21 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. 63.

    Mulvey, M. A., Schilling, J. D. & Hultgren, S. J. Establishment of a persistent Escherichia coli reservoir during the acute phase of a bladder infection. Infect. Immun. 69, 4572–4579 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Eto, D. S., Sundsbak, J. L. & Mulvey, M. A. Actin-gated intracellular growth and resurgence of uropathogenic Escherichia coli. Cell Microbiol. 8, 704–717 (2006).

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank L. Van Dijk for his help with the illustration of the phylogenetic analysis and Z. Ackermann-Bloom for advice on imaging. This work was supported by a generous gift from A. and J. Bekenstein and by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIH) under award no. R01AI117043 to D.T.H and award number U19AI110818 to the Broad Institute. C.M.E. was supported by a Research Fellowship from the German Research Foundation (Deutsche Forschungsgemeinschaft) and by a Fund for Medical Discovery Postdoctoral Fellowship from MGH. C.A.R. was supported by the Mexican National Council for Science and Technology and Fundacion Mexico en Harvard. The Microscopy Core of the Program in Membrane Biology was partially supported by a Center for the Study of Inflammatory Bowel Disease grant (no. DK043351) and a Boston Area Diabetes and Endocrinology Research Center award (no. DK057521). The Zeiss LSM 800 Airyscan confocal microscope was purchased using an NIH Shared Instrumentation grant (no. 1S10OD021577-01).

Author information

Affiliations

Authors

Contributions

C.M.E. and D.T.H. conceptualized the study. C.M.E, J.R.B., C.A.R.-O., A.P.Z., L.L., A.P. and A.L.M. investigated it. C.M.E., J.R.B. and C.A.R.-O. carried out the methodology. M.B., K.C., A.E.C. and A.V.N. provided the resources. C.M.E. and D.T.H. administered the project. C.M.E., D.T.H., A.M.E. and L.A.C. supervised it. C.M.E. and D.T.H. wrote the original draft. C.M.E., D.T.H., A.M.E., L.A.C., A.L.M. and A.P. reviewed and edited the manuscript.

Corresponding author

Correspondence to Deborah T. Hung.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Alison Farrell is the primary editor on this article, and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Mucoidity phenotypes of clinical ST258 isolates.

a, Representative colony phenotypes of clinical isolates plated on blood agar plates. The colony morphologies of UCI_38 (mucoid), UCI_37 (hypomucoid) and BIDMC_13 (hypermucoid) are shown. b, Hypomucoid clinical isolates display a translucent appearance on LB agar plates. All of the tested colony-purified ST258 isolates displayed the same colony mucoidity phenotype on blood or LB agar plates. 1, UCI_38 (control normal mucoid); 2, BIDMC_18A; 3, BIDMC_14; 4, BIDMC_54; 5, BIDMC_34; 6, UCI_37; 7, BWH_41; 8, UCI_43; 9, MGH_51; 10, MGH_71; 11, BIDMC_68; 12, MGH_73. c, Identification of capsule mutants on LB agar plates. In order to illustrate the feasibility of the identification of capsule mutant subpopulations on LB agar plates, UCI_38 was mixed with UCI_38wzcG565A (left) or UCI_38ΔwbaP (right) to achieve a concentration of mutants of approximately 5%. Hypermucoid colonies could be easily determined by the different colony morphology and bigger size (red arrow, left), while hypomucoid colonies could be easily identified by their translucent appearance and smaller size (red arrow, right). a-c were repeated 3 times independently with similar results.

Extended Data Fig. 2 Capsule-deficiency improves biofilm formation and invasion of bladder epithelial cells.

a, wbaP deletion in BWH_36 and BWH_45 abolishes capsule production. Transmission electron microscopy of BWH_36, BWH_45 and corresponding wbaP deletion mutants shown. For every isolate, one representative image from four images obtained from one section is shown. b, Impaired capsule production improves biofilm formation. The isogenic set of ST258 capsule mutants derived from UCI_38 is shown, as well as clinical ST258 capsule mutants and the regular and hypomucoid isolate from the patient specimen UR_5. UCI_37, BIDMC_54 and UR_5_hypo are hypomucoid; BIDMC_13 and BIDMC_16 are hypermucoid. Number of biologically independent experiments indicated. Mean and SEM of at least three independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. c, Capsule production impairs the invasion of bladder epithelial cells. wbaP deletion in UCI_38, BWH_36 and BWH_45 increases the invasion of bladder epithelial cells, while wbaP expression in the isogenic wbaP deletion mutant of UCI_38 impairs bladder epithelial cell invasion. Mean and SEM shown. Significance calculated with an unparalleled two-tailed t-test. n = 3 biologically independent experiments. d, Impaired capsule production increases bladder epithelial cell invasion resulting in a larger intracellular reservoir of K. pneumoniae ST258. UCI_38 and the isogenic wbaP deletion mutant are shown. The net increase in CFUs over 48 h is indicated. Mean and standard deviation of n = 4 biologically independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. e, Antibiotics of last resort are unable to clear intracellular carbapenem-resistant Klebsiella pneumoniae from bladder epithelial cells. Intracellular survival of the clinical capsule-deficient isolate BIDMC_54 shown. Mean and standard deviation of n = 3 biologically independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. f, Intracellular survival of UCI_38_mNeon and UCI_38ΔwbaP_mNeon after 1-minute synchronization of infection (via centrifugation). The centrifugation time was reduced for confocal microscopy in order to more effectively wash off extracellular bacteria from host cells, which decreased invasion efficiency but did not affect the observed net increase in recovered CFUs over 48 h seen in Extended Data Fig. 2d. Mean and standard deviation of n = 4 biologically independent experiments shown. Significance calculated with an unparalleled two-tailed t-test. g, The number of host cells infected with the wild type and mutant is consistent over time, with the mutant displaying a 3fold higher frequency of infection compared to the wildtype. Percentage of infected cells at indicated timepoints, as determined by confocal microscopy shown. n = 2 biologically independent experiments. h, Average number of bacteria per infected cell, as determined by recovered CFUs (Extended Data Fig. 2f) and by the number of infected host cells (Extended Data Fig. 2g) at indicated timepoints. For the 4-hour time point, the number of infected cells determined at 8 hpi was used in order to better distinguish between live and dead bacteria. From 8 to 48 hours post infection, all identified intracellular fluorescent Klebsiella were in LAMP1-positive vacuoles. Mean and SEM shown. n = 2 biologically independent experiments. i, Intracellular Klebsiella grow in Klebsiella-containing LAMP1-positive vacuoles. The fluorescence intensity of mNeon-expressing Klebsiella in LAMP1-positive vacuoles determined via integrated density (IntDen) from confocal microscopy images taken under identical conditions. n = 2 biologically independent experiments. j, Representative image taken with a confocal microscope of bladder epithelial cells infected with mNeon expressing UCI_38ΔwbaP (green, 24 hpi), labeled with anti-LAMP1 antibodies (red) and Hoechst dye (blue). The experiments were repeated 3 times independently with similar results. k, The same image is shown without bacteria to visualize the LAMP1-positive compartments in which Klebsiella pneumoniae persist during infection. l, Heterogeneity of intracellular growth observed in Klebsiella-containing vacuoles. The capsule-deficient mutant displays growth more frequently resulting in 2-fold enhanced net-growth vs the wild type (Extended Data Fig. 2f,h). The integrated density of mNeon-expressing Klebsiella in LAMP1-positive vacuoles is shown (n = 70 UCI_38 vs n = 73 UCI_38ΔwbaP containing compartments, from n = 2 biologically independent experiments shown). Median indicated with a dashed line, quartiles indicated with dashed lines. m, Bacteria recovered from the bladder and the kidney of mice infected transurethrally with UCI_38 or UCI_38ΔwbaP, 3 days post infection. n = 10 mice per group. Significance was calculated with the Mann-Whitney two-tailed U test. P values indicated in the figures. n, The capsule-deficient mutant UCI_38ΔwbaP causes localized infection with no observed immunogenicity at 3 days post infection. Representative histological images of haematoxylin and eosin (H&E) stained liver tissue sections from mouse UTIs, 3 days post infection (n = 3 bladders analyzed). For f-i the mean and SEM is shown. P values are indicated in the figures.

Extended Data Fig. 3 SNPs in wzc confer phagocytosis resistance via hypercapsule production.

a, Engineered isogenic strains containing wzc mutations identified in the clinical ST258 isolates produce a hypercapsule. The wzc genes from normal and hypercapsule producing isolates and from the same patient samples (UR_5, UR_11, UR_35) were cloned into the expression plasmid pBAD33 or pBAD33tet (ptet, a tetracycline resistance conferring variant of pBAD33). The ST258 strain UCI_38 and other phylogenetically more distantly related ST258 strains BWH_36 and BWH_4513 were transformed with the resulting constructs and were tested for the ability to resist centrifugation as a measure of hypercapsule production51. After a brief centrifugation step the optical density of the supernatant was determined. b, Clinical isolates with wzc mutations produce a hypercapsule. Overnight cultures were tested for centrifugation resistance as indicator of excessive capsule production51. After a brief centrifugation step the optical density of the supernatant was determined. c, Hypercapsule producing clinical isolates are phagocytosis resistant. UCI_38 and isogenic hypercapsule producing wzc mutants, as well as other clinical isolates with wzc SNPs shown. UR_5 isolates and BIDMC_16 could not be tested for phagocytosis resistance due to extensive drug resistance. d, Mutated wzc induces a hypercapsule in distantly related ST258 strains (for a phylogenetic analysis of these isolates, see Cerqueira et al, 201713). For a-d the mean and SEM of n = 3 biologically independent experiments is shown. Significance calculated with an unparalleled two-tailed t-test. e, Hypercapsule conferring point mutations affect different regions of Wzc. The C-terminal domain of the Wzc protein from ST258 clade 2 and ST512 strains is shown. Wzc controls high level polymerization of capsule in E. coli18. Its activity depends on a C-terminal autokinase domain, which consists of Walker A and B boxes that are crucial for the phosphorylation of tyrosine residues located in the tyrosine cluster and which results in the negative regulation capsule polymerization28. Hypercapsule production has also been observed in clinical Acinetobacter baumanii isolates with point mutations in wzc and have only in some cases been found to affect autokinase activity28. The precise function of Wzc is unknown but it has been hypothesized that it regulates capsule biosynthesis via distinct interactions with other capsule biosynthesis proteins and may have a complex structural role in capsule assembly18. Outside of its autokinase activity, Wzc proteins are also known to form oligomers, independent of phosphorylation18. A535E is located in a Walker A box, while the G565 mutations are located just outside of the canonical Walker A’ box. However, additional conserved amino acids can be found in Walker A’ boxes of Enterobacteriaceae, potentially extending the Walker A’ box to amino acid 566. L74 is not located in a region of any known activity of Wzc but may affect oligomerization or other complex interactions.

Extended Data Fig. 4 Hypercapsule production increases ST258 virulence.

a, Hypercapsule production does not affect the growth rate of UCI_38 (ST258) or SGH_10 (ST23) in axenic culture. UCI_38 and the isogenic hypercapsule mutant UCI_38wzcG565A, as well as the hypervirulent isolate SGH_10 (ST23) were grown in LB medium or in M9 salts supplemented with 0.2% casamino acids. The experiments were repeated 3 times independently with similar results. b, The hypercapsule producing clinical isolate BIDMC_13 displays increased toxicity in a zebrafish bloodstream infection model compared to normal capsule strain UCI_38. The lethal dose (LD50) required to kill 50% of the zebrafish larvae population is indicated. Meanwhile, a capsule-deficient clinical isolate UCI_37 is more attenuated. Mean and SEM shown. n = 170 UCI_38, n = 111 BIDMC_13, n = 104 UCI_37. P-values indicated relative to the normal capsule UCI_38 strain. c, Survival of zebrafish larvae after bloodstream-infection with UCI_38 (n = 1773 + /- 453 CFUs / fish) and the clinical hypercapsule mutant BIDMC_13 (n = 2213 + /-1113 CFUs / fish). P = 0.0024 (log-rank test). d, Hypercapsule facilitates the dissemination of murine UTIs (compare to Fig. 2h). C3H/HeN mice were infected transurethrally with UCI_38 and the isogenic hypercapsule mutant UCI_38wzcG565A. n = 10 mice were used per strain and timepoint. At day 3 post infection only the hypercapsule mutants were isolated from the bladder and the kidney. Significance was calculated with a two-tailed Mann-Whitney U test. The dashed line indicates the limit of detection. Dpi, days post infection. e, Representative histological images of haematoxylin and eosin (H&E) stained liver tissue sections from mouse UTIs 3 days post infection. The livers of n = 3 infected mice were analyzed per group. f, The hypercapsule mutant disseminates more effectively to the spleen in a mouse model of bloodstream infection. Recovered CFUs from homogenized spleens 3 days post infection shown. n = 5 mice were used per group. Significance was calculated with a two-tailed Mann-Whitney U test. g, Transurethral infection of TLR4-deficient C3H/HeJ mice with the normal capsule strain UCI_38 results in dissemination and persistent infection. Infections were established in 20 mice and CFU counts were determined 7 days post infection. The dashed line indicates the limit of detection. Hypercapsule mutants were isolated. h,i,j, A hypercapsulated ST258 mutant that emerged as a subpopulation in a patient displays enhanced virulence in mouse models of infection. h, Mouse UTI model. Recovered CFUs from the liver of mice infected transurethrally with normal (UC_11) and hyper-capsule producing (UCI_11wzcL74P) isolates from the urine specimen UR_11 (3 days post infection). The hypercapsule mutant killed 4 of 15 mice, while the parent isolate killed 1 of 15 mice, which is a degree of virulence that is usually not observed in mouse UTIs. Dead mice are indicated by the number of crosses. Livers from deceased mice are not included in the analysis. n = 15 mice per group. Significance calculated with a two-tailed Mann-Whitney U test. i, Pyogenic liver abscess caused by the hypercapsule mutant from UR_11 observed in 4 of 11 mouse UTIs. j, Mouse bloodstream model. The hypercapsule mutant from UR_11 (UCI_11wzcL74P) displays more rapid lethality in a mouse model of bloodstream infection compared to the normal capsule producing parent at an inoculum of 4x107. n = 5 C3H/HeN mice were infected per group. Mice infected with the normal capsule producing parent were sacrificed due to poor health scores at 16 hpi. Significance calculated with a log-rank test. k, Comparison of capsule production between the representative hypercapsulated ST23 isolate SGH_10 and the hypercapsulated ST258 strain UCI_38wzcG565A. The capsule was isolated and quantified by determining the amount of uronic acids. Mean and SEM of n = 2 independent experiments shown. l, The representative hypervirulent K. pneumoniae isolate SGH_10 displays rapid lethality in a mouse model of intraperitoneal infection. n = 5 BALB/c mice were infected with an inoculum of 1-2x106 CFUs and mouse survival was followed over time. SGH_10 is a representative Asian ST23 isolate harboring all known hypervirulence- associated genes.

Extended Data Fig. 5 Identification of isolates with wzc mutations suspected to confer a hypercapsule.

a. ST258 clade 2 reference tree (including single locus ST512 variants), comprised of 117 reference genomes that represent the phylogenetic diversity of the 966 ST258 clade 2 strains identified in the Refseq database (see Methods). b. Phylogenetic distribution of wzc mutants mapped to the closest strain in the reference tree. Mutants harboring mutations close to the hypercapsule-conferring mutations positions that were found to confer a hypercapsule in this study are shown (Extended Data Fig. 3e).

Extended Data Fig. 6 Direct isolation of hypercapsule mutants from patients infected with multidrug-resistant K. pneumoniae.

The percentage of hypercapsule and capsule-deficient mutants (Hyper/Deficient) as determined by visual inspection of mucoidity and string-test positivity is shown, for 3 patient urine specimens plated and analyzed directly, as well as the identified wzc mutations (resulting amino acid substitutions shown) and sequence types (ST) for the hypercapsule population isolated from each specimen. The clonality of subpopulations isolated from patients was verified by whole genome sequencing. Point mutations in wzc were identified by comparing the wzc sequence of mutant isolates to regular capsule producing isolates in the same specimen or, in the case of the ST14 isolate, by comparing the sequence to the consensus ST14 sequence in NCBI. The ST307 hypercapsule mutant harbors an additional frameshift mutation (fs) in the tyrosine cluster of wzc (Extended Data Fig. 3e) that affects the last 7 codons (G713-Ter720) and extends the open reading frame by two codons.

Supplementary information

Supplementary Information

Supplementary Fig. 1 and Supplementary Tables 1–13.

Reporting Summary

Supplementary Table 12

Supplementary Table 12

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Ernst, C.M., Braxton, J.R., Rodriguez-Osorio, C.A. et al. Adaptive evolution of virulence and persistence in carbapenem-resistant Klebsiella pneumoniae. Nat Med 26, 705–711 (2020). https://doi.org/10.1038/s41591-020-0825-4

Download citation