1887

Abstract

Extensively drug-resistant Klebsiella pneumoniae (XDR-KP) infections cause high mortality and are disseminating globally. Identifying the genetic basis underpinning resistance allows for rapid diagnosis and treatment. XDR isolates sourced from Greece and Brazil, including 19 polymyxin-resistant and five polymyxin-susceptible strains, were subjected to whole genome sequencing. Seventeen of the 19 polymyxin-resistant isolates harboured variations upstream or within mgrB. The most common mutation identified was an insertion at nucleotide position 75 in mgrB via an ISKpn26-like element in the ST258 lineage and ISKpn13 in one ST11 isolate. Three strains acquired an IS1 element upstream of mgrB and another strain had an ISKpn25 insertion at 133 bp. Other isolates had truncations (C28STOP, Q30STOP) or a missense mutation (D29E) affecting mgrB. Complementation assays revealed all mgrB perturbations contributed to resistance. Missense mutations in phoQ (T281M, G385C) were also found to facilitate resistance. Several variants in phoPQ co-segregating with the ISKpn26-like insertion were identified as potential partial suppressor mutations. Three ST258 samples were found to contain subpopulations with different resistance-conferring mutations, including the ISKpn26-like insertion colonizing with a novel mutation in pmrB (P158R), both confirmed via complementation assays. These findings highlight the broad spectrum of chromosomal modifications which can facilitate and regulate resistance against polymyxins in K. pneumoniae.

Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000158
2018-02-12
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/mgen/4/3/mgen000158.html?itemId=/content/journal/mgen/10.1099/mgen.0.000158&mimeType=html&fmt=ahah

References

  1. Ramirez MS, Traglia GM, Lin DL, Tran T, Tolmasky ME. Plasmid-mediated antibiotic resistance and virulence in Gram-negatives: the Klebsiella pneumoniae paradigm. Microbiol Spectr 2014;2:1–15 [CrossRef][PubMed]
    [Google Scholar]
  2. Patel G, Huprikar S, Factor SH, Jenkins SG, Calfee DP. Outcomes of carbapenem-resistant Klebsiella pneumoniae infection and the impact of antimicrobial and adjunctive therapies. Infect Control Hosp Epidemiol 2008;29:1099–1106 [CrossRef][PubMed]
    [Google Scholar]
  3. Biswas S, Brunel JM, Dubus JC, Reynaud-Gaubert M, Rolain JM. Colistin: an update on the antibiotic of the 21st century. Expert Rev Anti Infect Ther 2012;10:917–934 [CrossRef][PubMed]
    [Google Scholar]
  4. Suh JY, Son JS, Chung DR, Peck KR, Ko KS et al. Nonclonal emergence of colistin-resistant Klebsiella pneumoniae isolates from blood samples in South Korea. Antimicrob Agents Chemother 2010;54:560–562 [CrossRef][PubMed]
    [Google Scholar]
  5. Kim SY, Shin J, Shin SY, Ko KS. Characteristics of carbapenem-resistant Enterobacteriaceae isolates from Korea. Diagn Microbiol Infect Dis 2013;76:486–490 [CrossRef][PubMed]
    [Google Scholar]
  6. Mohamudha PR, Harish BN, Parija SC. Emerging carbapenem resistance among nosocomial isolates of Klebsiella pneumoniae in South India. Int J Pharma Bio Sci 2010;1:1–11
    [Google Scholar]
  7. Goel G, Hmar L, Sarkar de M, Bhattacharya S, Chandy M. Colistin-resistant Klebsiella pneumoniae: report of a cluster of 24 cases from a new oncology center in eastern India. Infect Control Hosp Epidemiol 2014;35:1076–1077 [CrossRef][PubMed]
    [Google Scholar]
  8. Cannatelli A, Giani T, D'Andrea MM, Di Pilato V, Arena F et al. MgrB inactivation is a common mechanism of colistin resistance in KPC-producing Klebsiella pneumoniae of clinical origin. Antimicrob Agents Chemother 2014;58:5696–5703 [CrossRef][PubMed]
    [Google Scholar]
  9. Mavroidi A, Katsiari M, Likousi S, Palla E, Roussou Z et al. Characterization of ST258 colistin-resistant, blaKPC-producing Klebsiella pneumoniae in a Greek Hospital. Microb Drug Resist 2016;22:392–398 [CrossRef][PubMed]
    [Google Scholar]
  10. Giamarellou H. Epidemiology of infections caused by polymyxin-resistant pathogens. Int J Antimicrob Agents 2016;48:614–621 [CrossRef][PubMed]
    [Google Scholar]
  11. Monaco M, Giani T, Raffone M, Arena F, Garcia-Fernandez A et al. Colistin resistance superimposed to endemic carbapenem-resistant Klebsiella pneumoniae: a rapidly evolving problem in Italy, November 2013 to April 2014. Euro Surveill 2014;19:pii=20939 [CrossRef][PubMed]
    [Google Scholar]
  12. Pereira GH, Garcia DO, Mostardeiro M, Fanti KS, Levin AS. Outbreak of carbapenem-resistant Klebsiella pneumoniae: two-year epidemiologic follow-up in a tertiary hospital. Mem Inst Oswaldo Cruz 2013;108:113–115 [CrossRef][PubMed]
    [Google Scholar]
  13. Gaspar GG, Bellissimo-Rodrigues F, Andrade LN, Darini AL, Martinez R. Induction and nosocomial dissemination of carbapenem and polymyxin-resistant Klebsiella pneumoniae. Rev Soc Bras Med Trop 2015;48:483–487 [CrossRef][PubMed]
    [Google Scholar]
  14. Ah YM, Kim AJ, Lee JY. Colistin resistance in Klebsiella pneumoniae. Int J Antimicrob Agents 2014;44:8–15 [CrossRef][PubMed]
    [Google Scholar]
  15. Koike M, Iida K, Matsuo T. Electron microscopic studies on mode of action of polymyxin. J Bacteriol 1969;97:448–452[PubMed]
    [Google Scholar]
  16. Dixon RA, Chopra I. Leakage of periplasmic proteins from Escherichia coli mediated by polymyxin B nonapeptide. Antimicrob Agents Chemother 1986;29:781–788 [CrossRef][PubMed]
    [Google Scholar]
  17. Deris ZZ, Akter J, Sivanesan S, Roberts KD, Thompson PE et al. A secondary mode of action of polymyxins against Gram-negative bacteria involves the inhibition of NADH-quinone oxidoreductase activity. J Antibiot 2014;67:147–151 [CrossRef][PubMed]
    [Google Scholar]
  18. Groisman EA, Kayser J, Soncini FC. Regulation of polymyxin resistance and adaptation to low-Mg2+ environments. J Bacteriol 1997;179:7040–7045 [CrossRef][PubMed]
    [Google Scholar]
  19. Velkov T, Thompson PE, Nation RL, Li J. Structure- activity relationships of polymyxin antibiotics. J Med Chem 2010;53:1898–1916 [CrossRef][PubMed]
    [Google Scholar]
  20. Cheng HY, Chen YF, Peng HL. Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43. J Biomed Sci 2010;17:60 [CrossRef][PubMed]
    [Google Scholar]
  21. Helander IM, Kato Y, Kilpeläinen I, Kostiainen R, Lindner B et al. Characterization of lipopolysaccharides of polymyxin-resistant and polymyxin-sensitive Klebsiella pneumoniae O3. Eur J Biochem 1996;237:272–278 [CrossRef][PubMed]
    [Google Scholar]
  22. Velkov T, Deris ZZ, Huang JX, Azad MA, Butler M et al. Surface changes and polymyxin interactions with a resistant strain of Klebsiella pneumoniae. Innate Immun 2014;20:350–363 [CrossRef][PubMed]
    [Google Scholar]
  23. Wright MS, Suzuki Y, Jones MB, Marshall SH, Rudin SD et al. Genomic and transcriptomic analyses of colistin-resistant clinical isolates of Klebsiella pneumoniae reveal multiple pathways of resistance. Antimicrob Agents Chemother 2015;59:536–543 [CrossRef][PubMed]
    [Google Scholar]
  24. Olaitan AO, Diene SM, Kempf M, Berrazeg M, Bakour S et al. Worldwide emergence of colistin resistance in Klebsiella pneumoniae from healthy humans and patients in Lao PDR, Thailand, Israel, Nigeria and France owing to inactivation of the PhoP/PhoQ regulator mgrB: an epidemiological and molecular study. Int J Antimicrob Agents 2014;44:500–507 [CrossRef][PubMed]
    [Google Scholar]
  25. Arena F, Henrici de Angelis L, Cannatelli A, di Pilato V, Amorese M et al. Colistin resistance caused by inactivation of the MgrB regulator is not associated with decreased virulence of sequence type 258 KPC carbapenemase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother 2016;60:2509–2512 [CrossRef][PubMed]
    [Google Scholar]
  26. Lee JY, Choi MJ, Choi HJ, Ko KS. Preservation of acquired colistin resistance in Gram-negative bacteria. Antimicrob Agents Chemother 2015;60:609–612 [CrossRef][PubMed]
    [Google Scholar]
  27. Meletis G, Tzampaz E, Sianou E, Tzavaras I, Sofianou D. Colistin heteroresistance in carbapenemase-producing Klebsiella pneumoniae. J Antimicrob Chemother 2011;66:946–947 [CrossRef][PubMed]
    [Google Scholar]
  28. Zowawi HM, Forde BM, Alfaresi M, Alzarouni A, Farahat Y et al. Stepwise evolution of pandrug-resistance in Klebsiella pneumoniae. Sci Rep 2015;5:15082 [CrossRef][PubMed]
    [Google Scholar]
  29. Liu YY, Wang Y, Walsh TR, Yi LX, Zhang R et al. Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study. Lancet Infect Dis 2016;16:161–168 [CrossRef][PubMed]
    [Google Scholar]
  30. Stoesser N, Mathers AJ, Moore CE, Day NP, Crook DW. Colistin resistance gene mcr-1 and pHNSHP45 plasmid in human isolates of Escherichia coli and Klebsiella pneumoniae. Lancet Infect Dis 2016;16:285–286 [CrossRef][PubMed]
    [Google Scholar]
  31. Wang Y, Tian GB, Zhang R, Shen Y, Tyrrell JM et al. Prevalence, risk factors, outcomes, and molecular epidemiology of mcr-1-positive Enterobacteriaceae in patients and healthy adults from China: an epidemiological and clinical study. Lancet Infect Dis 2017;17:390–399 [CrossRef][PubMed]
    [Google Scholar]
  32. di Pilato V, Arena F, Tascini C, Cannatelli A, Henrici de Angelis L et al. mcr-1.2, a new mcr variant carried on a transferable plasmid from a colistin-resistant KPC carbapenemase-producing Klebsiella pneumoniae strain of sequence type 512. Antimicrob Agents Chemother 2016;60:5612–5615 [CrossRef][PubMed]
    [Google Scholar]
  33. Yin W, Li H, Shen Y, Liu Z, Wang S et al. Novel plasmid-mediated colistin resistance gene mcr-3 in Escherichia coli. MBio 2017;8:e00543-17 [CrossRef][PubMed]
    [Google Scholar]
  34. Tietgen M, Semmler T, Riedel-Christ S, Kempf VAJ, Molinaro A et al. Impact of the colistin resistance gene mcr-1 on bacterial fitness. Int J Antimicrob Agents 2017;pii: S0924-8579 [CrossRef][PubMed]
    [Google Scholar]
  35. Clinical and Laboratory Standards Institute Performance Standards for Antimicrobial Susceptibility Testing– Twenty-sixth Edition: Approved standard M100S Wayne, PA: CLSI; 2016
    [Google Scholar]
  36. Magiorakos AP, Srinivasan A, Carey RB, Carmeli Y, Falagas ME et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012;18:268–281 [CrossRef][PubMed]
    [Google Scholar]
  37. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2014;30:2114–2120 [CrossRef][PubMed]
    [Google Scholar]
  38. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012;19:455–477 [CrossRef][PubMed]
    [Google Scholar]
  39. Overbeek R, Olson R, Pusch GD, Olsen GJ, Davis JJ et al. The SEED and the Rapid Annotation of microbial genomes using Subsystems Technology (RAST). Nucleic Acids Res 2014;42:D206–D214 [CrossRef][PubMed]
    [Google Scholar]
  40. Larsen MV, Cosentino S, Rasmussen S, Friis C, Hasman H et al. Multilocus sequence typing of total-genome-sequenced bacteria. J Clin Microbiol 2012;50:1355–1361 [CrossRef][PubMed]
    [Google Scholar]
  41. Zankari E, Hasman H, Cosentino S, Vestergaard M, Rasmussen S et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother 2012;67:2640–2644 [CrossRef][PubMed]
    [Google Scholar]
  42. Jünemann S, Sedlazeck FJ, Prior K, Albersmeier A, John U et al. Updating benchtop sequencing performance comparison. Nat Biotechnol 2013;31:294–296 [CrossRef][PubMed]
    [Google Scholar]
  43. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25:1754–1760 [CrossRef][PubMed]
    [Google Scholar]
  44. Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv preprint arXiv 2012;12073907
    [Google Scholar]
  45. Cingolani P, Platts A, Wang Lel, Coon M, Nguyen T et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 2012;6:80–92 [CrossRef][PubMed]
    [Google Scholar]
  46. Choi Y, Chan AP. PROVEAN web server: a tool to predict the functional effect of amino acid substitutions and indels. Bioinformatics 2015;31:2745–2747 [CrossRef][PubMed]
    [Google Scholar]
  47. Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: the reference centre for bacterial insertion sequences. Nucleic Acids Res 2006;34:D32–D36 [CrossRef][PubMed]
    [Google Scholar]
  48. Jayol A, Poirel L, Brink A, Villegas MV, Yilmaz M et al. Resistance to colistin associated with a single amino acid change in protein PmrB among Klebsiella pneumoniae isolates of worldwide origin. Antimicrob Agents Chemother 2014;58:4762–4766 [CrossRef][PubMed]
    [Google Scholar]
  49. Neonakis IK, Samonis G, Messaritakis H, Baritaki S, Georgiladakis A et al. Resistance status and evolution trends of Klebsiella pneumoniae isolates in a university hospital in Greece: ineffectiveness of carbapenems and increasing resistance to colistin. Chemotherapy 2010;56:448–452 [CrossRef][PubMed]
    [Google Scholar]
  50. Poirel L, Jayol A, Bontron S, Villegas MV, Ozdamar M et al. The mgrB gene as a key target for acquired resistance to colistin in Klebsiella pneumoniae. J Antimicrob Chemother 2015;70:75–80 [CrossRef][PubMed]
    [Google Scholar]
  51. Cannatelli A, D'Andrea MM, Giani T, di Pilato V, Arena F et al. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrob Agents Chemother 2013;57:5521–5526 [CrossRef][PubMed]
    [Google Scholar]
  52. Aires CA, Pereira PS, Asensi MD, Carvalho-Assef AP. mgrB mutations mediating polymyxin B resistance in Klebsiella pneumoniae isolates from rectal surveillance swabs in Brazil. Antimicrob Agents Chemother 2016;60:6969–6972 [CrossRef][PubMed]
    [Google Scholar]
  53. Kidd TJ, Mills G, Sá-Pessoa J, Dumigan A, Frank CG et al. A Klebsiella pneumoniae antibiotic resistance mechanism that subdues host defences and promotes virulence. EMBO Mol Med 2017;9:430–447 [CrossRef][PubMed]
    [Google Scholar]
  54. Halaby T, Kucukkose E, Janssen AB, Rogers MR, Doorduijn DJ et al. Genomic characterization of colistin heteroresistance in Klebsiella pneumoniae during a nosocomial outbreak. Antimicrob Agents Chemother 2016;60:6837–6843 [CrossRef][PubMed]
    [Google Scholar]
  55. Deleo FR, Chen L, Porcella SF, Martens CA, Kobayashi SD et al. Molecular dissection of the evolution of carbapenem-resistant multilocus sequence type 258 Klebsiella pneumoniae. Proc Natl Acad Sci USA 2014;111:4988–4993 [CrossRef][PubMed]
    [Google Scholar]
  56. Miller AK, Brannon MK, Stevens L, Johansen HK, Selgrade SE et al. PhoQ mutations promote lipid A modification and polymyxin resistance of Pseudomonas aeruginosa found in colistin-treated cystic fibrosis patients. Antimicrob Agents Chemother 2011;55:5761–5769 [CrossRef][PubMed]
    [Google Scholar]
  57. Lee JY, Ko KS. Mutations and expression of PmrAB and PhoPQ related with colistin resistance in Pseudomonas aeruginosa clinical isolates. Diagn Microbiol Infect Dis 2014;78:271–276 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000158
Loading
/content/journal/mgen/10.1099/mgen.0.000158
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error