1887

Abstract

Several genetic regulators belonging to AraC family are involved in the emergence of MDR isolates of due to alterations in membrane permeability. Compared with the genetic regulator Mar, RamA may be more relevant towards the emergence of antibiotic resistance.

Focusing on the global regulators, Mar and Ram, we compared the amino acid sequences of the Ram repressor in 59 clinical isolates and laboratory strains of Sequence types were associated with their corresponding multi-drug resistance phenotypes and membrane protein expression profiles using MIC and immunoblot assays. Quantitative gene expression analysis of the different regulators and their targets (porins and efflux pump components) were performed.

In the majority of the MDR isolates tested, and a region upstream of were mutated but or were unchanged. Expression and cloning experiments highlighted the involvement of the locus in the modification of membrane permeability. Overexpression of RamA lead to decreased porin production and increased expression of efflux pump components, whereas overexpression of RamR had the opposite effects.

Mutations or deletions in leading to the overexpression of RamA predominated in clinical MDR isolates and were associated with a higher-level of expression of efflux pump components. It was hypothesised that mutations in and the self-regulating region proximal to probably altered the binding properties of the RamR repressor; thereby producing the MDR phenotype. Consequently, mutability of RamR may play a key role in predisposing towards the emergence of a MDR phenotype.

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2018-02-01
2024-03-28
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References

  1. Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ. Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol 2015; 13:42–51 [View Article][PubMed]
    [Google Scholar]
  2. Davin-Regli A, Bolla JM, James CE, Lavigne JP, Chevalier J et al. Membrane permeability and regulation of drug "influx and efflux" in enterobacterial pathogens. Curr Drug Targets 2008; 9:750–759 [View Article][PubMed]
    [Google Scholar]
  3. Davin-Regli A, Pagès JM. Enterobacter aerogenes and Enterobacter cloacae; versatile bacterial pathogens confronting antibiotic treatment. Front Microbiol 2015; 6:392 [View Article][PubMed]
    [Google Scholar]
  4. Vonberg RP, Wolter A, Kola A, Ziesing S, Gastmeier P. The endemic situation of Enterobacter aerogenes and Enterobacter cloacae: you will only discover what you are looking for. J Hosp Infect 2007; 65:372–374 [View Article][PubMed]
    [Google Scholar]
  5. 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]
  6. Bornet C, Davin-Regli A, Bosi C, Pages JM, Bollet C. Imipenem resistance of enterobacter aerogenes mediated by outer membrane permeability. J Clin Microbiol 2000; 38:1048–1052[PubMed]
    [Google Scholar]
  7. Pagès JM, James CE, Winterhalter M. The porin and the permeating antibiotic: a selective diffusion barrier in Gram-negative bacteria. Nat Rev Microbiol 2008; 6:893–903 [View Article][PubMed]
    [Google Scholar]
  8. Lavigne JP, Sotto A, Nicolas-Chanoine MH, Bouziges N, Bourg G et al. Membrane permeability, a pivotal function involved in antibiotic resistance and virulence in Enterobacter aerogenes clinical isolates. Clin Microbiol Infect 2012; 18:539–545 [View Article][PubMed]
    [Google Scholar]
  9. Lavigne JP, Sotto A, Nicolas-Chanoine MH, Bouziges N, Pagès JM et al. An adaptive response of Enterobacter aerogenes to imipenem: regulation of porin balance in clinical isolates. Int J Antimicrob Agents 2013; 41:130–136 [View Article][PubMed]
    [Google Scholar]
  10. Chevalier J, Mulfinger C, Garnotel E, Nicolas P, Davin-Régli A et al. Identification and evolution of drug efflux pump in clinical Enterobacter aerogenes strains isolated in 1995 and 2003. PLoS One 2008; 3:e3203 [View Article][PubMed]
    [Google Scholar]
  11. Chollet R, Chevalier J, Bollet C, Pages JM, Davin-Regli A. RamA is an alternate activator of the multidrug resistance cascade in Enterobacter aerogenes. Antimicrob Agents Chemother 2004; 48:2518–2523 [View Article][PubMed]
    [Google Scholar]
  12. Keeney D, Ruzin A, Bradford PA. RamA, a transcriptional regulator, and AcrAB, an RND-type efflux pump, are associated with decreased susceptibility to tigecycline in Enterobacter cloacae. Microb Drug Resist 2007; 13:1–6 [View Article][PubMed]
    [Google Scholar]
  13. Nikaido E, Yamaguchi A, Nishino K. AcrAB multidrug efflux pump regulation in Salmonella enterica serovar Typhimurium by RamA in response to environmental signals. J Biol Chem 2008; 283:24245–24253 [View Article][PubMed]
    [Google Scholar]
  14. O'Regan E, Quinn T, Pagès JM, Mccusker M, Piddock L et al. Multiple regulatory pathways associated with high-level ciprofloxacin and multidrug resistance in Salmonella enterica serovar enteritidis: involvement of RamA and other global regulators. Antimicrob Agents Chemother 2009; 53:1080–1087 [View Article][PubMed]
    [Google Scholar]
  15. Bailey AM, Ivens A, Kingsley R, Cottell JL, Wain J et al. RamA, a member of the AraC/XylS family, influences both virulence and efflux in Salmonella enterica serovar Typhimurium. J Bacteriol 2010; 192:1607–1616 [View Article][PubMed]
    [Google Scholar]
  16. Lawler AJ, Ricci V, Busby SJ, Piddock LJ. Genetic inactivation of acrAB or inhibition of efflux induces expression of ramA. J Antimicrob Chemother 2013; 68:1551–1557 [View Article][PubMed]
    [Google Scholar]
  17. Chollet R, Bollet C, Chevalier J, Malléa M, Pagès JM et al. mar operon involved in multidrug resistance of Enterobacter aerogenes. Antimicrob Agents Chemother 2002; 46:1093–1097 [View Article][PubMed]
    [Google Scholar]
  18. Bratu S, Landman D, George A, Salvani J, Quale J. Correlation of the expression of acrB and the regulatory genes marA, soxS and ramA with antimicrobial resistance in clinical isolates of Klebsiella pneumoniae endemic to New York City. J Antimicrob Chemother 2009; 64:278–283 [View Article][PubMed]
    [Google Scholar]
  19. Martin RG, Bartlett ES, Rosner JL, Wall ME. Activation of the Escherichia coli marA/soxS/rob regulon in response to transcriptional activator concentration. J Mol Biol 2008; 380:278–284 [View Article][PubMed]
    [Google Scholar]
  20. de Majumdar S, Yu J, Fookes M, Mcateer SP, Llobet E et al. Elucidation of the RamA regulon in Klebsiella pneumoniae reveals a role in LPS regulation. PLoS Pathog 2015; 11:e1004627 [View Article][PubMed]
    [Google Scholar]
  21. Rosenblum R, Khan E, Gonzalez G, Hasan R, Schneiders T. Genetic regulation of the ramA locus and its expression in clinical isolates of Klebsiella pneumoniae. Int J Antimicrob Agents 2011; 38:39–45 [View Article][PubMed]
    [Google Scholar]
  22. Chen Y, Hu D, Zhang Q, Liao XP, Liu YH et al. Efflux pump overexpression contributes to tigecycline heteroresistance in Salmonella enterica serovar Typhimurium. Front Cell Infect Microbiol 2017; 7:37 [View Article][PubMed]
    [Google Scholar]
  23. Fàbrega A, Ballesté-Delpierre C, Vila J. Differential impact of ramRA mutations on both ramA transcription and decreased antimicrobial susceptibility in Salmonella Typhimurium. J Antimicrob Chemother 2016; 71:617–624 [View Article][PubMed]
    [Google Scholar]
  24. Jiménez-Castellanos JC, Wan Ahmad Kamil WN, Cheung CH, Tobin MS, Brown J et al. Comparative effects of overproducing the AraC-type transcriptional regulators MarA, SoxS, RarA and RamA on antimicrobial drug susceptibility in Klebsiella pneumoniae. J Antimicrob Chemother 2016; 71:1820–1825 [View Article][PubMed]
    [Google Scholar]
  25. Yamasaki S, Nikaido E, Nakashima R, Sakurai K, Fujiwara D et al. The crystal structure of multidrug-resistance regulator RamR with multiple drugs. Nat Commun 2013; 4:2078 [View Article][PubMed]
    [Google Scholar]
  26. Maneewannakul K, Levy SB. Identification of Mar mutants among clinical isolates of quinolone resistant Escherichia coli. Antimicrob Agents Chemother 1996; 40:1695–1698
    [Google Scholar]
  27. Yaron S, White DG, Matthews KR. Characterization of an Escherichia coli O157:H7 marR mutant. Int J Food Microbiol 2003; 85:281–291 [View Article][PubMed]
    [Google Scholar]
  28. Abouzeed YM, Baucheron S, Cloeckaert A. ramR mutations involved in efflux-mediated multidrug resistance in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 2008; 52:2428–2434 [View Article][PubMed]
    [Google Scholar]
  29. Philippe N, Maigre L, Santini S, Pinet E, Claverie JM et al. In vivo evolution of bacterial resistance in two cases of Enterobacter aerogenes infections during treatment with imipenem. PLoS One 2015; 10:e0138828 [View Article][PubMed]
    [Google Scholar]
  30. Bialek-Davenet S, Marcon E, Leflon-Guibout V, Lavigne JP, Bert F et al. In vitro selection of ramR and soxR mutants overexpressing efflux systems by fluoroquinolones as well as cefoxitin in Klebsiella pneumoniae. Antimicrob Agents Chemother 2011; 55:2795–2802 [View Article][PubMed]
    [Google Scholar]
  31. Kehrenberg C, Cloeckaert A, Klein G, Schwarz S. Decreased fluoroquinolone susceptibility in mutants of Salmonella serovars other than Typhimurium: detection of novel mutations involved in modulated expression of ramA and soxS. J Antimicrob Chemother 2009; 64:1175–1180 [View Article][PubMed]
    [Google Scholar]
  32. Hentschke M, Wolters M, Sobottka I, Rohde H, Aepfelbacher M. ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to tigecycline. Antimicrob Agents Chemother 2010; 54:2720–2723 [View Article][PubMed]
    [Google Scholar]
  33. Bialek-Davenet S, Leflon-Guibout V, Tran Minh O, Marcon E, Moreau R et al. Complete deletion of the ramR gene in an in vitro-selected mutant of Klebsiella pneumoniae overexpressing the AcrAB efflux pump. Antimicrob Agents Chemother 2013; 57:672–673 [View Article][PubMed]
    [Google Scholar]
  34. Seoane AS, Levy SB. Characterization of MarR, the repressor of the multiple antibiotic resistance (mar) operon in Escherichia coli. J Bacteriol 1995; 177:3414–3419 [View Article][PubMed]
    [Google Scholar]
  35. Alekshun MN, Kim YS, Levy SB. Mutational analysis of MarR, the negative regulator of marRAB expression in Escherichia coli, suggests the presence of two regions required for DNA binding. Mol Microbiol 2000; 35:1394–1404 [View Article][PubMed]
    [Google Scholar]
  36. Linde HJ, Notka F, Metz M, Kochanowski B, Heisig P et al. In vivo increase in resistance to ciprofloxacin in Escherichia coli associated with deletion of the C-terminal part of MarR. Antimicrob Agents Chemother 2000; 44:1865–1868 [View Article][PubMed]
    [Google Scholar]
  37. Baucheron S, Le Hello S, Doublet B, Giraud E, Weill FX et al. ramR mutations affecting fluoroquinolone susceptibility in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198. Front Microbiol 2013; 4:213 [View Article][PubMed]
    [Google Scholar]
  38. Villa L, Feudi C, Fortini D, García-Fernández A, Carattoli A. Genomics of KPC-producing Klebsiella pneumoniae sequence type 512 clone highlights the role of RamR and ribosomal S10 protein mutations in conferring tigecycline resistance. Antimicrob Agents Chemother 2014; 58:1707–1712 [View Article][PubMed]
    [Google Scholar]
  39. Campos CB, Aepfelbacher M, Hentschke M. Molecular analysis of the ramRA locus in clinical Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline. New Microbiol 2017; 40:135–138[PubMed]
    [Google Scholar]
  40. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001; 25:402–408 [View Article][PubMed]
    [Google Scholar]
  41. Veleba M, de Majumdar S, Hornsey M, Woodford N, Schneiders T. Genetic characterization of tigecycline resistance in clinical isolates of Enterobacter cloacae and Enterobacter aerogenes. J Antimicrob Chemother 2013; 68:1011–1018 [View Article][PubMed]
    [Google Scholar]
  42. Wang X, Chen H, Zhang Y, Wang Q, Zhao C et al. Genetic characterisation of clinical Klebsiella pneumoniae isolates with reduced susceptibility to tigecycline: role of the global regulator RamA and its local repressor RamR. Int J Antimicrob Agents 2015; 45:635–640 [View Article][PubMed]
    [Google Scholar]
  43. Deochand DK, Grove A. MarR family transcription factors: dynamic variations on a common scaffold. Crit Rev Biochem Mol Biol 2017; 52:595–613 [View Article][PubMed]
    [Google Scholar]
  44. Ghisalberti D, Masi M, Pagès JM, Chevalier J. Chloramphenicol and expression of multidrug efflux pump in Enterobacter aerogenes. Biochem Biophys Res Commun 2005; 328:1113–1118 [View Article][PubMed]
    [Google Scholar]
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