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Volume 170, Issue 3

Adaptive response of under serial ciprofloxacin exposure Open Access

Graphical Abstract

 Graphical Abstract 

Sub-MICs of ciprofloxacin result in DNA breakage, triggering DNA damage stress and the SOS stress response. This, in turn, induces changes in regulatory factors and the translation machinery. Modifications to regulatory components, including MvaT, RpoS and RpoB, lead to the upregulation (depicted in green) of efflux pumps such as MexEF-OprN and the downregulation (depicted in red) of porins. Additionally, there are alterations in the expression of virulence factors. Concurrently, ciprofloxacin affects metabolic processes, shortens cell lengths and induces the formation of multinucleated filaments due to disruptions in cell replication.

 

 

Abstract

Understanding the evolution of antibiotic resistance is important for combating drug-resistant bacteria. In this work, we investigated the adaptive response of to ciprofloxacin. Ciprofloxacin-susceptible ATCC 9027, CIP-E1 ( ATCC 9027 exposed to ciprofloxacin for 14 days) and CIP-E2 (CIP-E1 cultured in antibiotic-free broth for 10 days) were compared. Phenotypic responses including cell morphology, antibiotic susceptibility, and production of pyoverdine, pyocyanin and rhamnolipid were assessed. Proteomic responses were evaluated using comparative iTRAQ labelling LC-MS/MS to identify differentially expressed proteins (DEPs). Expression of associated genes coding for notable DEPs and their related regulatory genes were checked using quantitative reverse transcriptase PCR. CIP-E1 displayed a heterogeneous morphology, featuring both filamentous cells and cells with reduced length and width. By contrast, although filaments were not present, CIP-E2 still exhibited size reduction. Considering the MIC values, ciprofloxacin-exposed strains developed resistance to fluoroquinolone antibiotics but maintained susceptibility to other antibiotic classes, except for carbapenems. Pyoverdine and pyocyanin production showed insignificant decreases, whereas there was a significant decrease in rhamnolipid production. A total of 1039 proteins were identified, of which approximately 25 % were DEPs. In general, there were more downregulated proteins than upregulated proteins. Noted changes included decreased OprD and PilP, and increased MexEF-OprN, MvaT and Vfr, as well as proteins of ribosome machinery and metabolism clusters. Gene expression analysis confirmed the proteomic data and indicated the downregulation of and . In summary, the response to CIP involved approximately a quarter of the proteome, primarily associated with ribosome machinery and metabolic processes. Potential targets for bacterial interference encompassed outer membrane proteins and global regulators, such as MvaT.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): antibiotic resistance , ciprofloxacin , iTraq labelling , proteomics and Pseudomonas aeruginosa
Funding
This study was supported by the:
  • Nafosted (Award 108.04-2018.08)
    • Principle Award Recipient: ThiThu Hoai Nguyen
© 2024 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License.
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2024-04-03
2024-04-29
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References

  1. Huynh TQ, Tran NBV, Pham TTV, Le VBT. Truong TP et al adaptive response of Pseudomonas aeruginosa under serial ciprofloxacin exposure Figshare 2024 https://doi.org/10.6084/m9.figshare.25140002
     [Google Scholar]
  2. Mathee K, Narasimhan G, Valdes C, Qiu X, Matewish JM et al. Dynamics of Pseudomonas aeruginosa genome evolution. Proc Natl Acad Sci U S A 2008; 105:3100–3105 [View Article] [PubMed]
     [Google Scholar]
  3. Kung VL, Ozer EA, Hauser AR. The accessory genome of Pseudomonas aeruginosa. Microbiol Mol Biol Rev 2010; 74:621–641 [View Article] [PubMed]
     [Google Scholar]
  4. Gellatly SL, Hancock REW. Pseudomonas aeruginosa: new insights into pathogenesis and host defenses. Pathog Dis 2013; 67:159–173 [View Article] [PubMed]
     [Google Scholar]
  5. Poole K. Pseudomonas aeruginosa: resistance to the max. Front Microbiol 2011; 2:65 [View Article] [PubMed]
     [Google Scholar]
  6. Su H-C, Ramkissoon K, Doolittle J, Clark M, Khatun J et al. The development of ciprofloxacin resistance in Pseudomonas aeruginosa involves multiple response stages and multiple proteins. Antimicrob Agents Chemother 2010; 54:4626–4635 [View Article] [PubMed]
     [Google Scholar]
  7. Higgins PG, Fluit AC, Milatovic D, Verhoef J, Schmitz FJ. Mutations in GyrA, ParC, MexR and NfxB in clinical isolates of Pseudomonas aeruginosa. Int J Antimicrob Agents 2003; 21:409–413 [View Article] [PubMed]
     [Google Scholar]
  8. Baum EZ, Crespo-Carbone SM, Morrow BJ, Davies TA, Foleno BD et al. Effect of MexXY overexpression on ceftobiprole susceptibility in Pseudomonas aeruginosa. Antimicrob Agents Chemother 2009; 53:2785–2790 [View Article] [PubMed]
     [Google Scholar]
  9. Hughes D. Selection and evolution of resistance to antimicrobial drugs. IUBMB Life 2014; 66:521–529 [View Article] [PubMed]
     [Google Scholar]
  10. Lister PD, Wolter DJ, Hanson ND. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms. Clin Microbiol Rev 2009; 22:582–610 [View Article] [PubMed]
     [Google Scholar]
  11. Thuc QH, Thi THN. The effects of sub-MIC ciprofloxacin exposure on antibiotic susceptibility and virulence factors in Pseudomonas aeruginosa ATCC 9027. Int J Life Sci Res Arch 2022; 3:070–077 [View Article]
     [Google Scholar]
  12. Nguyen NHB, Pham TTV, Huynh TQ, Nguyen TH, Nguyen TTH. Sample preparative procedure for Pseudomonas aeruginosa observation under scanning electron microscope. Vietnam J Biotechnol 2022; 20:717–726 [View Article]
     [Google Scholar]
  13. Ahamed F, Devnath P. Extraction, purification and characterization of pyocyanin produced by Pseudomonas aeruginosa and evaluation for its antimicrobial activity. Int Res J Biol Sci 2017; 6:1–7
     [Google Scholar]
  14. Pinzon NM, Ju LK. Improved detection of rhamnolipid production using agar plates containing methylene blue and cetyl trimethylammonium bromide. Biotechnol Lett 2009; 31:1583–1588 [View Article] [PubMed]
     [Google Scholar]
  15. Dao KHT, Hamer KE, Clark CL, Harshman LG. Pyoverdine production by Pseudomonas aeruginosa exposed to metals or an oxidative stress agent. Ecol Appl 1999; 9:441 [View Article]
     [Google Scholar]
  16. Tran NBV, Truong QM, Nguyen LQA, Nguyen NMH, Tran QH et al. Prevalence and virulence of commensal Pseudomonas aeruginosa isolates from healthy individuals in Southern Vietnam (2018-2020). Biomedicines 2022; 11:54 [View Article] [PubMed]
     [Google Scholar]
  17. Thai VC, Lim TK, Le KPU, Lin Q, Nguyen TTH. iTRAQ-based proteome analysis of fluoroquinolone-resistant Staphylococcus aureus. J Glob Antimicrob Resist 2017; 8:82–89 [View Article] [PubMed]
     [Google Scholar]
  18. Nolan T, Hands RE, Bustin SA. Quantification of mRNA using real-time RT-PCR. Nat Protoc 2006; 1:1559–1582 [View Article] [PubMed]
     [Google Scholar]
  19. Riedel G, Rüdrich U, Fekete-Drimusz N, Manns MP, Vondran FWR et al. An extended ΔCT-method facilitating normalisation with multiple reference genes suited for quantitative RT-PCR analyses of human hepatocyte-like cells. PLoS One 2014; 9:e93031 [View Article] [PubMed]
     [Google Scholar]
  20. Kumar A, Lorand D. Robust ΔΔct estimate. Genomics 2021; 113:420–427 [View Article] [PubMed]
     [Google Scholar]
  21. Bos J, Zhang Q, Vyawahare S, Rogers E, Rosenberg SM et al. Emergence of antibiotic resistance from multinucleated bacterial filaments. Proc Natl Acad Sci U S A 2015; 112:178–183 [View Article] [PubMed]
     [Google Scholar]
  22. Buijs J, Dofferhoff ASM, Mouton JW, Wagenvoort JHT, van der Meer JWM. Concentration-dependency of beta-lactam-induced filament formation in gram-negative bacteria. Clin Microbiol Infect 2008; 14:344–349 [View Article] [PubMed]
     [Google Scholar]
  23. Marques CNH, Nelson SM. Pharmacodynamics of ciprofloxacin against Pseudomonas aeruginosa planktonic and biofilm-derived cells. Lett Appl Microbiol 2019; 68:350–359 [View Article] [PubMed]
     [Google Scholar]
  24. Monahan LG, Turnbull L, Osvath SR, Birch D, Charles IG et al. Rapid conversion of Pseudomonas aeruginosa to a spherical cell morphotype facilitates tolerance to carbapenems and penicillins but increases susceptibility to antimicrobial peptides. Antimicrob Agents Chemother 2014; 58:1956–1962 [View Article]
     [Google Scholar]
  25. Vaillancourt M, Limsuwannarot SP, Bresee C, Poopalarajah R, Jorth P. Pseudomonas aeruginosa mexR and mexEF antibiotic efflux pump variants exhibit increased virulence. Antibiot Basel Switz 2021; 10:1164 [View Article]
     [Google Scholar]
  26. Linares JF, López JA, Camafeita E, Albar JP, Rojo F et al. Overexpression of the multidrug efflux pumps MexCD-OprJ and MexEF-OprN is associated with a reduction of type III secretion in Pseudomonas aeruginosa. J Bacteriol 2005; 187:1384–1391 [View Article] [PubMed]
     [Google Scholar]
  27. Ngo HL, Huynh TQ, Tran NBV, Nguyen NHB, Tong TH et al. Proteomic analysis of ceftazidime and meropenem-exposed Pseudomonas aeruginosa ATCC 9027. Proteome Sci 2023; 21:15 [View Article] [PubMed]
     [Google Scholar]
  28. Rehman A, Patrick WM, Lamont IL. Mechanisms of ciprofloxacin resistance in Pseudomonas aeruginosa: new approaches to an old problem. J Med Microbiol 2019; 68:1–10 [View Article] [PubMed]
     [Google Scholar]
  29. Lambert PA. Mechanisms of antibiotic resistance in Pseudomonas aeruginosa. J R Soc Med 2002; 95:22–26 [PubMed]
     [Google Scholar]
  30. Purssell A, Poole K. Functional characterization of the NfxB repressor of the mexCD-oprJ multidrug efflux operon of Pseudomonas aeruginosa. Microbiol Read Engl 2013; 159:2058–2073 [View Article] [PubMed]
     [Google Scholar]
  31. Jeannot K, Elsen S, Köhler T, Attree I, van Delden C et al. Resistance and virulence of Pseudomonas aeruginosa clinical strains overproducing the MexCD-OprJ efflux pump. Antimicrob Agents Chemother 2008; 52:2455–2462 [View Article] [PubMed]
     [Google Scholar]
  32. Tomás M, Doumith M, Warner M, Turton JF, Beceiro A et al. Efflux pumps, OprD porin, AmpC beta-lactamase, and multiresistance in Pseudomonas aeruginosa isolates from cystic fibrosis patients. Antimicrob Agents Chemother 2010; 54:2219–2224 [View Article] [PubMed]
     [Google Scholar]
  33. Feng Y, Hodiamont CJ, van Hest RM, Brul S, Schultsz C et al. Development of antibiotic resistance during simulated treatment of Pseudomonas aeruginosa in chemostats. PLoS One 2016; 11:e0149310 [View Article] [PubMed]
     [Google Scholar]
  34. Ochs MM, McCusker MP, Bains M, Hancock REW. Negative regulation of the Pseudomonas aeruginosa outer membrane porin OprD selective for imipenem and basic amino acids. Antimicrob Agents Chemother 1999; 43:1085–1090 [View Article] [PubMed]
     [Google Scholar]
  35. Schuster M, Hawkins AC, Harwood CS, Greenberg EP. The Pseudomonas aeruginosa RpoS regulon and its relationship to quorum sensing. Mol Microbiol 2004; 51:973–985 [View Article] [PubMed]
     [Google Scholar]
  36. Suh SJ, Silo-Suh L, Woods DE, Hassett DJ, West SE et al. Effect of rpoS mutation on the stress response and expression of virulence factors in Pseudomonas aeruginosa. J Bacteriol 1999; 181:3890–3897 [View Article] [PubMed]
     [Google Scholar]
  37. Kayama S, Murakami K, Ono T, Ushimaru M, Yamamoto A et al. The role of rpoS gene and quorum-sensing system in ofloxacin tolerance in Pseudomonas aeruginosa. FEMS Microbiol Lett 2009; 298:184–192 [View Article] [PubMed]
     [Google Scholar]
  38. Murakami K, Ono T, Viducic D, Kayama S, Mori M et al. Role for rpoS gene of Pseudomonas aeruginosa in antibiotic tolerance. FEMS Microbiol Lett 2005; 242:161–167 [View Article] [PubMed]
     [Google Scholar]
  39. Hall CW, Hinz AJ, Gagnon LB-P, Zhang L, Nadeau J-P et al. Pseudomonas aeruginosa biofilm antibiotic resistance gene ndvB expression requires the RpoS stationary-phase sigma factor. Appl Environ Microbiol 2018; 84:e02762-17 [View Article] [PubMed]
     [Google Scholar]
  40. Hall AR, Iles JC, MacLean RC. The fitness cost of rifampicin resistance in Pseudomonas aeruginosa depends on demand for RNA polymerase. Genetics 2011; 187:817–822 [View Article] [PubMed]
     [Google Scholar]
  41. Pietsch F, Bergman JM, Brandis G, Marcusson LL, Zorzet A et al. Ciprofloxacin selects for RNA polymerase mutations with pleiotropic antibiotic resistance effects. J Antimicrob Chemother 2017; 72:75–84 [View Article] [PubMed]
     [Google Scholar]
  42. López-Causapé C, Cabot G, Del Barrio-Tofiño E, Oliver A. The versatile mutational resistome of Pseudomonas aeruginosa. Front Microbiol 2018; 9:685 [View Article] [PubMed]
     [Google Scholar]
  43. Vallet I, Diggle SP, Stacey RE, Cámara M, Ventre I et al. Biofilm formation in Pseudomonas aeruginosa: fimbrial cup gene clusters are controlled by the transcriptional regulator MvaT. J Bacteriol 2004; 186:2880–2890 [View Article] [PubMed]
     [Google Scholar]
  44. Leighton TL, Buensuceso RNC, Howell PL, Burrows LL. Biogenesis of Pseudomonas aeruginosa type IV pili and regulation of their function. Environ Microbiol 2015; 17:4148–4163 [View Article] [PubMed]
     [Google Scholar]
  45. Roncarati D, Scarlato V, Vannini A. Targeting of regulators as a promising approach in the search for novel antimicrobial agents. Microorganisms 2022; 10:185 [View Article] [PubMed]
     [Google Scholar]
  46. Hernando-Amado S, Laborda P, Valverde JR, Martínez JL. Mutational background influences P. aeruginosa ciprofloxacin resistance evolution but preserves collateral sensitivity robustness. Proc Natl Acad Sci U S A 2022; 119:e2109370119 [View Article] [PubMed]
     [Google Scholar]
  47. Yu XH, Hao ZH, Liu PL, Liu MM, Zhao LL et al. Increased expression of efflux pump norA drives the rapid evolutionary trajectory from tolerance to resistance against ciprofloxacin in Staphylococcus aureus. Antimicrob Agents Chemother 2022; 66:e0059422 [View Article] [PubMed]
     [Google Scholar]
  48. Lloyd MG, Lundgren BR, Hall CW, Gagnon LB-P, Mah T-F et al. Targeting the alternative sigma factor RpoN to combat virulence in Pseudomonas aeruginosa. Sci Rep 2017; 7:12615 [View Article] [PubMed]
     [Google Scholar]
  49. Fraser-Pitt DJ, Dolan SK, Toledo-Aparicio D, Hunt JG, Smith DW et al. Cysteamine inhibits glycine utilisation and disrupts virulence in Pseudomonas aeruginosa. Front Cell Infect Microbiol 2021; 11:718213 [View Article] [PubMed]
     [Google Scholar]
  50. Horna G, López M, Guerra H, Saénz Y, Ruiz J. Interplay between MexAB-OprM and MexEF-OprN in clinical isolates of Pseudomonas aeruginosa. Sci Rep 2018; 8:16463 [View Article] [PubMed]
     [Google Scholar]
  51. Williams McMackin EA, Marsden AE, Yahr TL. H-NS family members MvaT and MvaU regulate the Pseudomonas aeruginosa type III secretion system. J Bacteriol 2019; 201:e00054-19 [View Article] [PubMed]
     [Google Scholar]
  52. Savli H, Karadenizli A, Kolayli F, Gundes S, Ozbek U et al. Expression stability of six housekeeping genes: a proposal for resistance gene quantification studies of Pseudomonas aeruginosa by real-time quantitative RT-PCR. J Med Microbiol 2003; 52:403–408 [View Article] [PubMed]
     [Google Scholar]
  53. Remans T, Keunen E, Bex GJ, Smeets K, Vangronsveld J et al. Reliable gene expression analysis by reverse transcription-quantitative PCR: reporting and minimizing the uncertainty in data accuracy. Plant Cell 2014; 26:3829–3837 [View Article] [PubMed]
     [Google Scholar]
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Adaptive response of Pseudomonas aeruginosa under serial ciprofloxacin exposure
Microbiology 170, 001443 (2024); https://doi.org/10.1099/mic.0.001443
/content/journal/micro/10.1099/mic.0.001443
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