1887

Abstract

Rifampicin is a broad-spectrum antibiotic that binds to the bacterial RNA polymerase (RNAP), compromising DNA transcription. Rifampicin resistance is common in several microorganisms and it is typically caused by point mutations in the gene encoding the β subunit of RNA polymerase, . Different mutations are responsible for various levels of rifampicin resistance and for a range of secondary effects. mutations conferring rifampicin resistance have been shown to be responsible for severe effects on transcription, cell fitness, bacterial stress response and virulence. Such effects have never been investigated in the marine pathogen , even though rifampicin-resistant strains of have been isolated previously. Moreover, spontaneous rifampicin-resistant strains of have an important role in conjugation and mutagenesis protocols, with poor consideration of the effects of mutations. In this work, effects on growth, stress response and virulence of were investigated using a set of nine spontaneous rifampicin-resistant derivatives of CMCP6. Three different mutations (Q513K, S522L and H526Y) were identified with varying incidence rates. These three mutant types each showed high resistance to rifampicin [minimal inhibitory concentration (MIC) >800 µg ml], but different secondary effects. The strains carrying the mutation H526Y had a growth advantage in rich medium but had severely reduced salt stress tolerance in the presence of high NaCl concentrations as well as a significant reduction in ethanol stress resistance. Strains possessing the S522L mutation had reduced growth rate and overall biomass accumulation in rich medium. Furthermore, investigation of virulence characteristics demonstrated that all the rifampicin-resistant strains showed compromised motility when compared with the wild-type, but no major effects on exoenzyme production were observed. These findings reveal a wide range of secondary effects of mutations and indicate that rifampicin resistance is not an appropriate selectable marker for studies that aim to investigate phenotypic behaviour in this organism.

Funding
This study was supported by the:
  • H2020 Marie Skłodowska-Curie Actions, http://dx.doi.org/10.13039/100010665 (Award 721456)
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000991
2020-11-13
2021-08-02
Loading full text...

Full text loading...

/deliver/fulltext/micro/166/12/1160.html?itemId=/content/journal/micro/10.1099/mic.0.000991&mimeType=html&fmt=ahah

References

  1. WHO Antimicrobial resistance global report on surveillance; 2014
  2. Alifano P, Palumbo C, Pasanisi D, Talà A. Rifampicin-resistance, rpoB polymorphism and RNA polymerase genetic engineering. J Biotechnol 2015; 202:60–77 [View Article]
    [Google Scholar]
  3. di Mauro E, Synder L, Marino P, Lamberti A, Coppo A et al. Rifampicin sensitivity of the components of DNA-dependent RNA polymerase. Nature 1969; 222:533–537 [View Article]
    [Google Scholar]
  4. Manten A, Van Wijngaarden LJ. Development of drug resistance to rifampicin. Chemotherapy 1969; 14:93–100 [View Article]
    [Google Scholar]
  5. Ezekiel DH, Hutchins JE. Mutations affecting RNA polymerase associated with rifampicin resistance in Escherichia coli . Nature 1968; 220:276–277 [View Article]
    [Google Scholar]
  6. Jin DJ, Gross CA. Mapping and sequencing of mutations in the Escherichia coli rpoB gene that lead to rifampicin resistance. J Mol Biol 1988; 202:45–58 [View Article]
    [Google Scholar]
  7. Lisitsyn NA, Sverdlov ED, Moiseyeva EP, Danilevskaya ON, Nikiforov VG. Mutation to rifampicin resistance at the beginning of the RNA polymerase β subunit gene in Escherichia coli . Mol Gen Genet 1984; 196:173–174 [View Article]
    [Google Scholar]
  8. Ovchinnikov YA, Monastyrskaya GS, Gubanov VV, Lipkin VM, Sverdlov ED et al. Primary structure of Escherichia coli RNA polymerase nucleotide substitution in the β subunit gene of the rifampicin resistant 255 mutant. Mol Gen Genet 1981; 184:536–538 [View Article]
    [Google Scholar]
  9. Ovchinnikov YA, Monastyrskaya GS, Guriev SO, Kalinina NF, Sverdlov ED et al. RNA polymerase rifampicin resistance mutations in Escherichia coli: sequence changes and dominance. Mol Gen Genet 1983; 190:344–348 [View Article]
    [Google Scholar]
  10. Severinov K, Soushko M, Goldfarb A, Nikiforov V. Rifampicin region revisited. New rifampicin-resistant and streptolydigin-resistant mutants in the β subunit of Escherichia coli RNA polymerase. J Biol Chem 1993; 268:14820–14825
    [Google Scholar]
  11. Vattanaviboon P, Sukchawalit R, Jearanaikoon P, Chuchottaworn C, Ponglikit-mongkol M. Analysis of RNA polymerase gene mutation in three isolatesof rifampicin resistant Mycobacterium tuberculosis . Southeast Asian J Trop MedPublic Health 1995; 26:333–336
    [Google Scholar]
  12. Jin D, Walter W, Gross CA. Characterization of the termination phenotypes of rifampicin resistant rpoB mutants in Escherichia coli . J Mol Biol 1988b; 202:45–63
    [Google Scholar]
  13. Jin DJ, Cashel M, Friedman DI, Nakamura Y, Walter WA et al. Effects of rifampicin resistant rpoB mutations on antitermination and interaction with nusA in Escherichia coli . J Mol Biol 1988a; 204:247–261 [View Article]
    [Google Scholar]
  14. Jin D, Gross CA. RpoB8, a rifampicin-resistant termination-proficient RNA polymerase, has an increased Km for purine nucleotides during transcription elongation. J Biol Chem 1991; 266:14478–14485
    [Google Scholar]
  15. Jin DJ, Walter WA, Gross CA. Characterization of the termination phenotypes of rifampicin-resistant mutants. J Mol Biol 1988; 202:245–253 [View Article]
    [Google Scholar]
  16. Ingham CJ, Furneaux PA. Mutations in the SS subunit of the Bacillus subtilis RNA polymerase that confer both rifampicin resistance and hypersensitivity to NusG. Microbiology 2000; 146:3041–3049 [View Article][PubMed]
    [Google Scholar]
  17. Heisler LM, Feng G, Jin DJ, Gross CA, Landick R. Amino acid substitutions in the two largest subunits of Escherichia coli RNA polymerase that suppress a defective Rho termination factor affect different parts of the transcription complex. J Biol Chem 1996; 271:14572–14583 [View Article][PubMed]
    [Google Scholar]
  18. Reynolds MG. Compensatory evolution in rifampin-resistant Escherichia coli . Genetics 2000; 156:1471–1481
    [Google Scholar]
  19. Billington OJ, McHugh TD, Gillespie SH. Physiological cost of rifampin resistance induced in vitro in Mycobacterium tuberculosis . Antimicrob Agents Chemother 1999; 43:1866–1869 [View Article]
    [Google Scholar]
  20. Wichelhaus TA, Böddinghaus B, Besier S, Schäfer V, Brade V et al. Biological cost of rifampin resistance from the perspective of Staphylococcus aureus . Antimicrob Agents Chemother 2002; 46:3381–3385 [View Article][PubMed]
    [Google Scholar]
  21. Maharjan R, Ferenci T. The fitness costs and benefits of antibiotic resistance in drug-free microenvironments encountered in the human body. Environ Microbiol Rep 2017; 9:635–641 [View Article]
    [Google Scholar]
  22. Gao W, Cameron DR, Davies JK, Kostoulias X, Stepnell J, Howden BP, Stinear TP, Peleg AY et al. The RpoB H₄₈₁Y rifampicin resistance mutation and an active stringent response reduce virulence and increase resistance to innate immune responses in Staphylococcus aureus . J Infect Dis 2013; 207:929–939 [View Article][PubMed]
    [Google Scholar]
  23. Björkman J, Hughes D, Andersson DI. Virulence of antibiotic-resistant Salmonella typhimurium . Proc Natl Acad Sci U S A 1998; 95:3949–3953 [View Article]
    [Google Scholar]
  24. Colicchio R, Pagliuca C, Pastore G, Cicatiello AG, Pagliarulo C et al. Fitness cost of rifampin resistance in Neisseria meningitidis: in vitro study of mechanisms associated with rpoB H553Y mutation. Antimicrob Agents Chemother 2015; 59:7637–7649 [View Article]
    [Google Scholar]
  25. Neri A, Mignogna G, Fazio C, Giorgi A, Schininà ME et al. Neisseria meningitidis rifampicin resistant strains: analysis of protein differentially expressed. BMC Microbiol 2010; 10:246 [View Article]
    [Google Scholar]
  26. Jones MK, Oliver JD. Vibrio vulnificus: disease and pathogenesis. Infect Immun 2009; 77:1723–1733 [View Article]
    [Google Scholar]
  27. Oliver JD. Wound infections caused by Vibrio vulnificus and other marine bacteria. Epidemiol Infect 2005; 133:383–391 [View Article]
    [Google Scholar]
  28. Oliver JD. The biology of Vibrio vulnificus . Microbiol Spectr 2015; 3: [View Article]
    [Google Scholar]
  29. Kim YR, Lee SE, Kim CM, Kim SY, Shin EK et al. Characterization and pathogenic significance of Vibrio vulnificus antigens preferentially expressed in septicemic patients. Infect Immun 2003; 71:5461–5471 [View Article]
    [Google Scholar]
  30. Oliver JD. Vibrio vulnificus . The Biology of Vibrios American Society of Microbiology; 2006
    [Google Scholar]
  31. Heng SP, Letchumanan V, Deng CY, Ab Mutalib NS, Khan TM et al. Vibrio vulnificus: An environmental and clinical burden. Front Microbiol 2017; 8:997 [View Article]
    [Google Scholar]
  32. EG O, Son KT, Yu H, Lee TS, Lee HJ et al. Antimicrobial resistance of Vibrio parahaemolyticus and Vibrio alginolyticus strains isolated from farmed fish in Korea from 2005 through 2007. J Food Prot 2011; 74:380–386
    [Google Scholar]
  33. Ottaviani D, Bacchiocchi I, Masini L, Leoni F, Carraturo A et al. Antimicrobial susceptibility of potentially pathogenic halophilic vibrios isolated from seafood. Int J Antimicrob Agents 2001; 18:135–140 [View Article]
    [Google Scholar]
  34. Zeaiter Z, Mapelli F, Crotti E, Borin S. Methods for the genetic manipulation of marine bacteria. Electronic Journal of Biotechnology 2018; 33:17–28 [View Article]
    [Google Scholar]
  35. Kim E-J, Oh EK, Lee JK. Role of HemF and HemN in the heme biosynthesis of Vibrio vulnificus under S-adenosylmethionine-limiting conditions. Mol Microbiol 2015; 96:497–512 [View Article]
    [Google Scholar]
  36. Kwak JS, Jeong H-G, Satchell KJF. Vibrio vulnificus rtxA1 gene recombination generates toxin variants with altered potency during intestinal infection. Proc Natl Acad Sci U S A 2011; 108:1645–1650 [View Article]
    [Google Scholar]
  37. Deatherage DE, Barrick JE. Identification of mutations in laboratory-evolved microbes from next-generation sequencing data using breseq. Methods Mol Biol 2014; 1151:165–188 [View Article][PubMed]
    [Google Scholar]
  38. Baker-Austin C, Oliver JD. Vibrio vulnificus : new insights into a deadly opportunistic pathogen. Environ Microbiol 2018; 20:423–430 [View Article]
    [Google Scholar]
  39. Koch A, Mizrahi V, Warner DF. The impact of drug resistance on Mycobacterium tuberculosis physiology: what can we learn from rifampicin?. Emerg Microbes Infect 2014; 3:e171–11 [View Article]
    [Google Scholar]
  40. Linkous DA, Oliver JD. Pathogenesis of Vibrio vulnificus . FEMS Microbiol Lett 1999; 174:207–214 [View Article]
    [Google Scholar]
  41. Kim SY, Thanh XT, Jeong K, Kim SB, Pan SO et al. Contribution of six flagellin genes to the flagellum biogenesis of Vibrio vulnificus and in vivo invasion. Infect Immun 2014; 82:29–42 [View Article]
    [Google Scholar]
  42. Ran Kim Y, Haeng Rhee J. Flagellar basal body flg operon as a virulence determinant of Vibrio vulnificus . Biochem Biophys Res Commun 2003; 304:405–410 [View Article]
    [Google Scholar]
  43. Jin DJ, Gross CA. Characterization of the pleiotropic phenotypes of rifampin-resistant rpoB mutants of Escherichia coli . J Bacteriol 1989; 171:5229–5231 [View Article]
    [Google Scholar]
  44. Wrande M, Roth JR, Hughes D. Accumulation of mutants in "aging" bacterial colonies is due to growth under selection, not stress-induced mutagenesis. Proc Natl Acad Sci U S A 2008; 105:11863–11868 [View Article]
    [Google Scholar]
  45. Murphy H, Cashel M. Isolation of RNA polymerase suppressors of a (p)ppGpp deficiency. Methods Enzymol 2003; 371:596–601
    [Google Scholar]
  46. Xiao M, Zhu X, Xu H, Tang J, Liu R et al. A novel point mutation in RpoB improves osmotolerance and succinic acid production in Escherichia coli . BMC Biotechnol 2017; 17:10 [View Article]
    [Google Scholar]
  47. Rodriguez-Verdugo A, Gaut BS, Tenaillon O. Evolution of Escherichia coli rifampicin resistance in an antibiotic-free environment during thermal stress. BMC Evol Biol 2013; 13:50 [View Article]
    [Google Scholar]
  48. Demarre G, Guerout AM, Matsumoto-Mashimo C, Rowe-Magnus DA, Marliere P et al. A new family of mobilizable suicide plasmids based on broad host range R388 plasmid (IncW) and RP4 plasmid (IncPα) conjugative machineries and their cognate Escherichia coli host strains. Res Microbiol 2005; 156:245–255 [View Article]
    [Google Scholar]
  49. Naughton LM, Blumerman SL, Carlberg M, Boyd EF. Osmoadaptation among Vibrio species and unique genomic features and physiological responses of Vibrio parahaemolyticus . Appl Environ Microbiol 2009; 75:2802–2810 [View Article]
    [Google Scholar]
  50. Roberts MF. Organic compatible solutes of halotolerant and halophilic microorganisms. Saline Systems 2005; 1:5 [View Article]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000991
Loading
/content/journal/micro/10.1099/mic.0.000991
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
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