1887

Abstract

Colistin is a last resort antibiotic for treating infections caused by carbapenem-resistant isolates. Mechanisms of resistance to colistin have been widely described in and but have yet to be characterized in and species.

To identify the causative mutations leading to generation of colistin resistance in and spp.

Colistin resistance was generated by culturing in increasing concentrations of colistin or by direct culture in a lethal (above MIC) concentration. Whole-genome sequencing was used to identify mutations. Fitness of resistant strains was determined by changes in growth rate, and virulence in .

We were able to generate colistin resistance upon exposure to sub-MIC levels of colistin, in several but not all strains of and resulting in a 16-fold increase in colistin MIC values for both species. The same individual strains also developed resistance to colistin after a single exposure at 10× MIC, with a similar increase in MIC. Genetic analysis revealed that this increased resistance was attributed to mutations in PmrB for and PhoP in , although we were not able to identify causative mutations in all strains. Colistin-resistant mutants showed little difference in growth rate, and virulence in , although there were strain-to-strain differences.

Stable colistin resistance may be acquired with no loss of fitness in these species. However, only select strains were able to adapt suggesting that acquisition of colistin resistance is dependent upon individual strain characteristics.

Keyword(s): Citrobacter , colistin , Enterobacter , phoPQ and pmrAB
Funding
This study was supported by the:
  • Public Health England (Award 109506)
    • Principle Award Recipient: Matthew E Wand
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2020-03-03
2024-11-12
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References

  1. Falagas ME, Kasiakou SK. Colistin: the revival of polymyxins for the management of multidrug-resistant gram-negative bacterial infections. Clin Infect Dis 2005; 40:1333–1341 [View Article][PubMed]
    [Google Scholar]
  2. Bialvaei AZ, Samadi Kafil H, Colistin SKH. Colistin, mechanisms and prevalence of resistance. Curr Med Res Opin 2015; 31:707–721 [View Article][PubMed]
    [Google Scholar]
  3. Baron S, Hadjadj L, Rolain J-M, Olaitan AO. Molecular mechanisms of polymyxin resistance: knowns and unknowns. Int J Antimicrob Agents 2016; 48:583–591 [View Article][PubMed]
    [Google Scholar]
  4. Poirel L, Jayol A, Nordmann P. Polymyxins: antibacterial activity, susceptibility testing, and resistance mechanisms encoded by plasmids or chromosomes. Clin Microbiol Rev 2017; 30:557–596 [View Article][PubMed]
    [Google Scholar]
  5. Olaitan AO, Morand S, Rolain J-M. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol 2014; 5:643 [View Article][PubMed]
    [Google Scholar]
  6. Liu Y-Y, Wang Y, Walsh TR, Yi L-X, 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 [View Article][PubMed]
    [Google Scholar]
  7. Winfield MD, Latifi T, Groisman EA. Transcriptional regulation of the 4-amino-4-deoxy-L-arabinose biosynthetic genes in Yersinia pestis . J Biol Chem 2005; 280:14765–14772 [View Article][PubMed]
    [Google Scholar]
  8. Arana DM, Ortega A, González-Barberá E, Lara N, Bautista V et al. Carbapenem-resistant Citrobacter spp. isolated in Spain from 2013 to 2015 produced a variety of carbapenemases including VIM-1, OXA-48, KPC-2, NDM-1 and VIM-2. J Antimicrob Chemother 2017; 72:3283–3287 [View Article][PubMed]
    [Google Scholar]
  9. Chen S, Hu F, Liu Y, Zhu D, Wang H et al. Detection and spread of carbapenem-resistant Citrobacter freundii in a teaching hospital in China. Am J Infect Control 2011; 39:e55–e60 [View Article][PubMed]
    [Google Scholar]
  10. Annavajhala MK, Gomez-Simmonds A, Uhlemann A-C. Multidrug-Resistant Enterobacter cloacae complex emerging as a global diversifying threat. Front Microbiol 2019; 10:44 [View Article][PubMed]
    [Google Scholar]
  11. Gomez-Simmonds A, Hu Y, Sullivan SB, Wang Z, Whittier S et al. Evidence from a New York City hospital of rising incidence of genetically diverse carbapenem-resistant Enterobacter cloacae and dominance of ST171, 2007-14. J Antimicrob Chemother 2016; 71:2351–2353 [View Article][PubMed]
    [Google Scholar]
  12. Kanamori H, Parobek CM, Juliano JJ, van Duin D, Cairns BA et al. A Prolonged Outbreak of KPC-3-Producing Enterobacter cloacae and Klebsiella pneumoniae driven by multiple mechanisms of resistance transmission at a large academic burn center. Antimicrob Agents Chemother 2017; 61:pii: e01516-16
    [Google Scholar]
  13. Nordmann P, Jayol A, Poirel L. Rapid detection of polymyxin resistance in Enterobacteriaceae . Emerg Infect Dis 2016; 22:1038–1043 [View Article][PubMed]
    [Google Scholar]
  14. Diene SM, Merhej V, Henry M, El Filali A, Roux V et al. The rhizome of the multidrug-resistant Enterobacter aerogenes genome reveals how new "killer bugs" are created because of a sympatric lifestyle. Mol Biol Evol 2013; 30:369–383 [View Article][PubMed]
    [Google Scholar]
  15. Hong Y-K, Lee J-Y, Ko KS. Colistin resistance in Enterobacter spp. isolates in Korea. J Microbiol 2018; 56:435–440 [View Article][PubMed]
    [Google Scholar]
  16. Norgan AP, Freese JM, Tuin PM, Cunningham SA, Jeraldo PR et al. Carbapenem- and colistin-resistant Enterobacter cloacae from delta, Colorado, in 2015. Antimicrob Agents Chemother 2016; 60:3141–3144 [View Article]
    [Google Scholar]
  17. Zeng K-J, Doi Y, Patil S, Huang X, Tian G-B. Emergence of the plasmid-mediated mcr-1 gene in colistin-resistant Enterobacter aerogenes and Enterobacter cloacae . Antimicrob Agents Chemother 2016; 60:3862–3863 [View Article][PubMed]
    [Google Scholar]
  18. Sennati S, Di Pilato V, Riccobono E, Di Maggio T, Villagran AL et al. Citrobacter braakii carrying plasmid-borne mcr-1 colistin resistance gene from ready-to-eat food from a market in the Chaco region of Bolivia. J Antimicrob Chemother 2017; 72:2127–2129 [View Article][PubMed]
    [Google Scholar]
  19. Wise MG, Estabrook MA, Sahm DF, Stone GG, Kazmierczak KM. Prevalence of mcr-type genes among colistin-resistant Enterobacteriaceae collected in 2014-2016 as part of the INFORM global surveillance program. PLoS One 2018; 13:e0195281 [View Article][PubMed]
    [Google Scholar]
  20. Bock LJ, Hind CK, Sutton JM, Wand ME. Growth media and assay plate material can impact on the effectiveness of cationic biocides and antibiotics against different bacterial species. Lett Appl Microbiol 2018; 66:368–377 [View Article][PubMed]
    [Google Scholar]
  21. Wand ME, Müller CM, Titball RW, Michell SL. Macrophage and Galleria mellonella infection models reflect the virulence of naturally occurring isolates of B. pseudomallei, B. thailandensis and B. oklahomensis . BMC Microbiol 2011; 11:11 [View Article][PubMed]
    [Google Scholar]
  22. Wand ME, Bock LJ, Bonney LC, Sutton JM. Mechanisms of increased resistance to chlorhexidine and cross-resistance to colistin following exposure of Klebsiella pneumoniae clinical isolates to chlorhexidine. Antimicrob Agents Chemother 2017; 61:e01162–16 [View Article][PubMed]
    [Google Scholar]
  23. Afgan E, Baker D, Batut B, van den Beek M, Bouvier D et al. The galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update. Nucleic Acids Res 2018; 46:W537–W544 [View Article][PubMed]
    [Google Scholar]
  24. Jolley KA, Bliss CM, Bennett JS, Bratcher HB, Brehony C et al. Ribosomal multilocus sequence typing: universal characterization of bacteria from domain to strain. Microbiology 2012; 158:1005–1015 [View Article][PubMed]
    [Google Scholar]
  25. Hoffmann H, Roggenkamp A. Population genetics of the nomenspecies Enterobacter cloacae . Appl Environ Microbiol 2003; 69:5306–5318 [View Article][PubMed]
    [Google Scholar]
  26. Guérin F, Isnard C, Sinel C, Morand P, Dhalluin A et al. Cluster-dependent colistin hetero-resistance in Enterobacter cloacae complex. J Antimicrob Chemother 2016; 71:3058–3061 [View Article][PubMed]
    [Google Scholar]
  27. Huang L, Feng Y, Zong Z. Heterogeneous resistance to colistin in Enterobacter cloacae complex due to a new small transmembrane protein. J Antimicrob Chemother 2019; 74:2551–2558 [View Article][PubMed]
    [Google Scholar]
  28. Telke AA, Olaitan AO, Morand S, Rolain J-M. soxRS induces colistin hetero-resistance in Enterobacter asburiae and Enterobacter cloacae by regulating the acrAB-tolC efflux pump. J Antimicrob Chemother 2017; 72:2715–2721 [View Article][PubMed]
    [Google Scholar]
  29. Napier BA, Band V, Burd EM, Weiss DS. Colistin heteroresistance in Enterobacter cloacae is associated with cross-resistance to the host antimicrobial lysozyme. Antimicrob Agents Chemother 2014; 58:5594–5597 [View Article][PubMed]
    [Google Scholar]
  30. Kang KN, Klein DR, Kazi MI, Guérin F, Cattoir V et al. Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ-dependent 4-amino-4-deoxy-L-arabinose addition to lipid A. Mol Microbiol 2019; 111:1604–1616 [View Article][PubMed]
    [Google Scholar]
  31. Wand ME, Bock LJ, Bonney LC, Sutton JM. Retention of virulence following adaptation to colistin in Acinetobacter baumannii reflects the mechanism of resistance. J Antimicrob Chemother 2015; 70:2209–2216 [View Article][PubMed]
    [Google Scholar]
  32. Wand ME, Bock LJ, Sutton JM. Retention of virulence following colistin adaptation in Klebsiella pneumoniae is strain-dependent rather than associated with specific mutations. J Med Microbiol 2017; 66:959–964 [View Article][PubMed]
    [Google Scholar]
  33. Moreira CG, Russell R, Mishra AA, Narayanan S, Ritchie JM et al. Bacterial adrenergic sensors regulate virulence of enteric pathogens in the gut. mBio 2016; 7:e00826–16 [View Article][PubMed]
    [Google Scholar]
  34. Carroll LM, Gaballa A, Guldimann C, Sullivan G, Henderson LO et al. Identification of novel mobilized colistin resistance gene mcr-9 in a multidrug-resistant, colistin-susceptible Salmonella enterica serotype Typhimurium isolate. mBio 2019; 10:e00853–19 [View Article][PubMed]
    [Google Scholar]
  35. Belogurov AA, Delver EP, Rodzevich OV. Plasmid pKM101 encodes two nonhomologous antirestriction proteins (ArdA and ArdB) whose expression is controlled by homologous regulatory sequences. J Bacteriol 1993; 175:4843–4850 [View Article][PubMed]
    [Google Scholar]
  36. Price NL, Raivio TL. Characterization of the Cpx regulon in Escherichia coli strain MC4100. J Bacteriol 2009; 191:1798–1815 [View Article][PubMed]
    [Google Scholar]
  37. Hirakawa H, Inazumi Y, Masaki T, Hirata T, Yamaguchi A. Indole induces the expression of multidrug exporter genes in Escherichia coli . Mol Microbiol 2005; 55:1113–1126 [View Article][PubMed]
    [Google Scholar]
  38. De Wulf P, McGuire AM, Liu X, Lin ECC. Genome-wide profiling of promoter recognition by the two-component response regulator CpxR-P in Escherichia coli . J Biol Chem 2002; 277:26652–26661 [View Article][PubMed]
    [Google Scholar]
  39. Kohanski MA, Dwyer DJ, Wierzbowski J, Cottarel G, Collins JJ. Mistranslation of membrane proteins and two-component system activation trigger antibiotic-mediated cell death. Cell 2008; 135:679–690 [View Article][PubMed]
    [Google Scholar]
  40. Kurabayashi K, Hirakawa Y, Tanimoto K, Tomita H, Hirakawa H. Role of the CpxAR two-component signal transduction system in control of fosfomycin resistance and carbon substrate uptake. J Bacteriol 2014; 196:248–256 [View Article][PubMed]
    [Google Scholar]
  41. Srinivasan VB, Vaidyanathan V, Mondal A, Rajamohan G. Role of the two component signal transduction system CpxAR in conferring cefepime and chloramphenicol resistance in Klebsiella pneumoniae NTUH-K2044. PLoS One 2012; 7:e33777 [View Article][PubMed]
    [Google Scholar]
  42. Weatherspoon-Griffin N, Zhao G, Kong W, Kong Y et al. The CpxR/CpxA two-component system up-regulates two Tat-dependent peptidoglycan amidases to confer bacterial resistance to antimicrobial peptide. J Biol Chem 2011; 286:5529–5539 [View Article][PubMed]
    [Google Scholar]
  43. Zhai Y-J, Huang H, Liu J, Sun H-R, He D et al. CpxR overexpression increases the susceptibility of acrB and cpxR double-deleted Salmonella enterica serovar Typhimurium to colistin. J Antimicrob Chemother 2018; 73:3016–3024 [View Article][PubMed]
    [Google Scholar]
  44. Choi M-J, Ko KS. Mutant prevention concentrations of colistin for Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae clinical isolates. J Antimicrob Chemother 2014; 69:275–277 [View Article][PubMed]
    [Google Scholar]
  45. Choi M-J, Ko KS. Loss of hypermucoviscosity and increased fitness cost in colistin-resistant Klebsiella pneumoniae sequence type 23 strains. Antimicrob Agents Chemother 2015; 59:6763–6773 [View Article][PubMed]
    [Google Scholar]
  46. Nang SC, Morris FC, McDonald MJ, Han M-L, Wang J et al. Fitness cost of mcr-1-mediated polymyxin resistance in Klebsiella pneumoniae . J Antimicrob Chemother 2018; 73:1604–1610 [View Article][PubMed]
    [Google Scholar]
  47. Wang R, Liu Y, Zhang Q, Jin L, Wang Q et al. The prevalence of colistin resistance in Escherichia coli and Klebsiella pneumoniae isolated from food animals in China: coexistence of mcr-1 and bla NDM with low fitness cost. Int J Antimicrob Agents 2018; 51:739–744 [View Article][PubMed]
    [Google Scholar]
  48. MacNair CR, Stokes JM, Carfrae LA, Fiebig-Comyn AA, Coombes BK et al. Overcoming mcr-1 mediated colistin resistance with colistin in combination with other antibiotics. Nat Commun 2018; 9:458 [View Article][PubMed]
    [Google Scholar]
  49. Guérin F, Isnard C, Cattoir V, Giard JC. Complex regulation pathways of AmpC-mediated β-Lactam resistance in Enterobacter cloacae complex. Antimicrob Agents Chemother 2015; 59:7753–7761 [View Article][PubMed]
    [Google Scholar]
  50. Esposito EP, Cervoni M, Bernardo M, Crivaro V, Cuccurullo S et al. Molecular epidemiology and virulence profiles of colistin-resistant Klebsiella pneumoniae blood isolates from the hospital agency “Ospendale dei Colli,” Naples, Italy. Front Microbiol 2018; 9:1463 [View Article][PubMed]
    [Google Scholar]
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