Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지
DOI QR코드

DOI QR Code

Insight into Norfloxacin Resistance of Acinetobacter oleivorans DR1: Target Gene Mutation, Persister, and RNA-Seq Analyses

  • Kim, Jisun (Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Noh, Jaemin (Department of Environmental Science and Ecological Engineering, Korea University) ;
  • Park, Woojun (Department of Environmental Science and Ecological Engineering, Korea University)
  • Received : 2013.07.19
  • Accepted : 2013.07.30
  • Published : 2013.09.28
Download PDF
⟨ Previous Next ⟩

Abstract

Antibiotic resistance of soilborne Acinetobacter species has been poorly explored. In this study, norfloxacin resistance of a soil bacterium, Acinetobacter oleivorans DR1, was investigated. The frequencies of mutant appearance of all tested non-pathogenic Acinetobacter strains were lower than those of pathogenic strains under minimum inhibitory concentration (MIC). When the quinolone-resistance-determining region of the gyrA gene was examined, only one mutant (His78Asn) out of 10 resistant variants had a mutation. Whole transcriptome analysis using a RNA-Seq demonstrated that genes involved in SOS response and DNA repair were significantly up-regulated by norfloxacin. Determining the MICs of survival cells after norfloxacin treatment confirmed some of those cells were indeed persister cells. Ten colonies, randomly selected from among those that survived in the presence of norfloxacin, did not exhibit increased MIC. Thus, both the low mutation frequency of the target gene and SOS response under norfloxacin suggested that persister formation might contribute to the resistance of DR1 against norfloxacin. The persister frequency increased without a change in MIC when stationary phase cells, low growth rates conditions, and growth-deficient dnaJ mutant were used. Taken together, our comprehensive approach, which included mutational analysis of the target gene, persister formation assays, and RNA sequencing, indicated that DR1 survival when exposed to norfloxacin is related not only to target gene mutation but also to persister formation, possibly through up-regulation of the SOS response and DNA repair genes.

Keywords

References

  1. Andriole VT. 2005. The quinolones: past, present, and future. Clin. Infect. Dis. 41: 113-119. https://doi.org/10.1086/428051
  2. Aranda J, Bardina C, Beceiro A, Rumbo S, Cabral MP, Barbe J, et al. 2011. Acinetobacter baumannii RecA protein in repair of DNA damage, antimicrobial resistance, general stress response, and virulence. J. Bacteriol. 193: 3740-3747. https://doi.org/10.1128/JB.00389-11
  3. Berenstein D. 1986. Prophage induction by ultraviolet light in Acinetobacter calcoaceticus. J. Gen. Microbiol. 132: 2633-2636.
  4. Bergogne-Berezin E, Towner KJ. 1996. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin. Microbiol. Rev. 9: 148-165.
  5. Bernier SP, Lebeaux D, DeFrancesco AS, Valomon A, Soubigou G, Coppee JY, et al. 2013. Starvation, together with the SOS response, mediates high biofilm-specific tolerance to the fluoroquinolone ofloxacin. PLoS Genet. 9: e1003144. https://doi.org/10.1371/journal.pgen.1003144
  6. Bigger J. 1944. Treatment of staphylococcal infections with penicillin by intermittent sterilisation. Lancet 244: 497-500. https://doi.org/10.1016/S0140-6736(00)74210-3
  7. Camarena L, Bruno V, Euskirchen G, Poggio S, Snyder M. 2010. Molecular mechanisms of ethanol-induced pathogenesis revealed by RNA-sequencing. PLoS Pathog. 6: e1000834. https://doi.org/10.1371/journal.ppat.1000834
  8. Carver T, Bohme U, Otto TD, Parkhill J, Berriman M. 2010. BamView: viewing mapped read alignment data in the context of the reference sequence. Bioinformatics 26: 676-677. https://doi.org/10.1093/bioinformatics/btq010
  9. Courcelle J, Khodursky A, Peter B, Brown P, Hanawalt P. 2001. Comparative gene expression profiles following UV exposure in wild type and SOS-deficient Escherichia coli. Genetics 158: 41-64.
  10. Coyne S, Rosenfeld N, Lambert T, Courvalin P, Perichon B. 2010. Overexpression of resistance-nodulation-cell division pump AdeFGH confers multidrug resistance in Acinetobacter baumannii. Antimicrob. Agents Chemother. 54: 4389-4393. https://doi.org/10.1128/AAC.00155-10
  11. Dorr T, Lewis K, Vuli M. 2009. SOS response induces persistence to fluoroquinolones in Escherichia coli. PLoS Genet. 5: e1000760. https://doi.org/10.1371/journal.pgen.1000760
  12. Gallert C, Fund K, Winter J. 2005. Antibiotic resistance of bacteria in raw and biologically treated sewage and in groundwater below leaking sewers. Appl. Microbiol. Biotechnol. 69: 106-112. https://doi.org/10.1007/s00253-005-0033-7
  13. Gilbert P, Collier PJ, Brown MR. 1990. Influence of growth rate on susceptibility to antimicrobial agents: biofilms, cell cycle, dormancy, and stringent response. Antimicrob. Agents Chemother. 34: 1865-1868. https://doi.org/10.1128/AAC.34.10.1865
  14. Hare JM, Perkins SN, Gregg-Jolly LA. 2006. A constitutively expressed, truncated umuDC operon regulates the recAdependent DNA damage induction of a gene in Acinetobacter baylyi strain ADP1. Appl. Environ. Microbiol. 72: 4036-4043. https://doi.org/10.1128/AEM.02774-05
  15. Herold S, Siebert J, Huber A, Schmidt H. 2005. Global expression of prophage genes in Escherichia coli O157:H7 strain EDL933 in response to norfloxacin. Antimicrob. Agents Chemother. 49: 931-944. https://doi.org/10.1128/AAC.49.3.931-944.2005
  16. Hojgaard A, Szerlong H, Tabor C, Kuempel P. 1999. Norfloxacin-induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV. Mol. Microbiol. 33: 1027-1036. https://doi.org/10.1046/j.1365-2958.1999.01545.x
  17. Irith W, Kai H, Robert EWH. 2008. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc. 3: 163-175. https://doi.org/10.1038/nprot.2007.521
  18. Jung J, Baek JH, Park W. 2010. Complete genome sequence of the diesel-degrading Acinetobacter sp. strain DR1. J. Bacteriol. 192: 4794-4795. https://doi.org/10.1128/JB.00722-10
  19. Jung J, Madsen EL, Jeon CO, Park W. 2011. Comparative genomic analysis of Acinetobacter oleivorans DR1 to determine strain-specific genomic regions and gentisate biodegradation. Appl. Environ. Microbiol. 77: 7418-7424. https://doi.org/10.1128/AEM.05231-11
  20. Kaneko A, Sasaki J, Shimadzu M, Kanayama A, Saika T, Kobayashi I. 2000. Comparison of gyrA and parC mutations and resistance levels among fluoroquinolone-resistant isolates and laboratory-derived mutants of oral streptococci. J. Antimicrob. Chemother. 45: 771-775. https://doi.org/10.1093/jac/45.6.771
  21. Kang YS, Park W. 2010. Protection against diesel oil toxicity by sodium chloride-induced exopolysaccharides in Acinetobacter sp. strain DR1. J. Biosci. Bioeng. 109: 118-123. https://doi.org/10.1016/j.jbiosc.2009.08.001
  22. Kang YS, Park W. 2010. Trade-off between antibiotic resistance and biological fitness in Acinetobacter sp. strain DR1. Environ. Microbiol. 12: 1304-1318. https://doi.org/10.1111/j.1462-2920.2010.02175.x
  23. Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. 2004. Persister cells and tolerance to antimicrobials. FEMS Microbiol. Lett. 230: 13-18. https://doi.org/10.1016/S0378-1097(03)00856-5
  24. Keren I, Minami S, Rubin E, Lewis K. 2011. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2: e00100-e00111.
  25. Kim JM, Jeon CO. 2009. Isolation and characterization of a new benzene, toluene, and ethylbenzene degrading bacterium, Acinetobacter sp. B113. Curr. Microbiol. 58: 70-75. https://doi.org/10.1007/s00284-008-9268-8
  26. LeClerc JE, Li B, Payne WL, Cebula TA. 1996. High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274: 1208-1211. https://doi.org/10.1126/science.274.5290.1208
  27. Lewis K. 2010. Persister cells. Annu. Rev. Microbiol. 64: 357-372. https://doi.org/10.1146/annurev.micro.112408.134306
  28. Liu Y, Imlay JA. 2013. Cell death from antibiotics without the involvement of reactive oxygen species. Science 339: 1210-1213. https://doi.org/10.1126/science.1232751
  29. Luidalepp H, Joers A, Kaldalu N, Tenson T. 2011. Age of inoculum strongly influences persister frequency and can mask effects of mutations implicated in altered persistence. J. Bacteriol. 193: 3598-3605. https://doi.org/10.1128/JB.00085-11
  30. Malik M, Zhao X, Drlica K. 2006. Lethal fragmentation of bacterial chromosomes mediated by DNA gyrase and quinolones. Mol. Microbiol. 61: 810-25. https://doi.org/10.1111/j.1365-2958.2006.05275.x
  31. Martinez JL, Baquero F. 2000. Mutation frequencies and antibiotic resistance. Antimicrob. Agents Chemother. 44: 1771-1777. https://doi.org/10.1128/AAC.44.7.1771-1777.2000
  32. Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5: 621-628. https://doi.org/10.1038/nmeth.1226
  33. Norton MD, Spilkia AJ, Godoy VG. 2013. Antibiotic resistance acquired through a DNA damage-inducible response in Acinetobacter baumannii. J. Bacteriol. 195: 1335-1345. https://doi.org/10.1128/JB.02176-12
  34. Phillips I, Culebras E, Moreno F, Baquero F. 1987. Induction of the SOS response by new 4-quinolones. J. Antimicrob. Chemother. 20: 631-638. https://doi.org/10.1093/jac/20.5.631
  35. Rauch PJ, Palmen R, Burds AA, Gregg-Jolly LA, van der Zee JR, Hellingwerf KJ. 1996. The expression of the Acinetobacter calcoaceticus recA gene increases in response to DNA damage independently of RecA and of development of competence for natural transformation. Microbiology 142: 1025-1032. https://doi.org/10.1099/00221287-142-4-1025
  36. Robinson A, Brzoska AJ, Turner KM, Withers R, Harry EJ, Lewis PJ, et al. 2010. Essential biological processes of an emerging pathogen: DNA replication, transcription, and cell division in Acinetobacter spp. Microbiol. Mol. Biol. Rev. 74: 273-297. https://doi.org/10.1128/MMBR.00048-09
  37. Shendure J. 2008. The beginning of the end for microarrays? Nat. Methods 5: 585-587 https://doi.org/10.1038/nmeth0708-585
  38. Spoering AL, Lewis K. 2001. Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J. Bacteriol. 183: 6746-6751. https://doi.org/10.1128/JB.183.23.6746-6751.2001
  39. Stanier RY, Palleroni NJ, Doudoroff M. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43: 159-271. https://doi.org/10.1099/00221287-43-2-159
  40. Suttle CA, Chen F. 1992. Mechanisms and rates of decay of marine viruses in seawater. Appl. Environ. Microbiol. 58: 3721-3729.
  41. Turton JF, Perry C, Hannah MJ. 2012. Isolation of bacteriophage against currently circulating strains of Acinetobacter baumannii. J. Med. Microb. Diagn. 1: 109.
  42. Valdezate S, Navarro A, Medina-Pascual MJ, Carrasco G, Saez-Nieto JA. 2010. Molecular screening for rifampicin and fluoroquinolone resistance in a clinical population of Brucella melitensis. J. Antimicrob. Chemother. 65: 51-53. https://doi.org/10.1093/jac/dkp389
  43. Van Dessel H, Dijkshoorn L, Van Der Reijden T, Bakker N, Paauw A, Van Den Broek P, et al. 2004. Identification of a new geographically widespread multiresistant Acinetobacter baumannii clone from European hospitals. Res. Microbiol. 155: 105-112. https://doi.org/10.1016/j.resmic.2003.10.003
  44. Wang Z, Gerstein M, Snyder M. 2009. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10: 57-63. https://doi.org/10.1038/nrg2484
  45. Yeom J, Imlay JA, Park W. 2010. Iron homeostasis affects antibiotic-mediated cell death in Pseudomonas species. J. Biol. Chem. 285: 22689-22695. https://doi.org/10.1074/jbc.M110.127456
  46. Yi H, Cho YJ, Won S, Lee JE, Jin YH, Kim S, et al. 2011. Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-Seq. Nucleic Acids Res. 39: e140. https://doi.org/10.1093/nar/gkr617

Cited by

  1. Horizontal gene transfer in the human gastrointestinal tract: potential spread of antibiotic resistance genes vol.7, pp.None, 2013, https://doi.org/10.2147/idr.s48820
  2. Introduction to RNA‐Seq and its Applications to Drug Discovery and Development vol.75, pp.5, 2014, https://doi.org/10.1002/ddr.21215
  3. Global Transcriptome and Physiological Responses of Acinetobacter oleivorans DR1 Exposed to Distinct Classes of Antibiotics vol.9, pp.10, 2014, https://doi.org/10.1371/journal.pone.0110215
  4. Molecular mechanism involved in the response to hydrogen peroxide stress in Acinetobacter oleivorans DR1 vol.99, pp.24, 2013, https://doi.org/10.1007/s00253-015-6914-5
  5. Acinetobacter species as model microorganisms in environmental microbiology: current state and perspectives vol.99, pp.6, 2015, https://doi.org/10.1007/s00253-015-6439-y
  6. luxS/AI-2 Quorum Sensing Is Involved in Antimicrobial Susceptibility in Streptococcus agalactiae vol.50, pp.1, 2015, https://doi.org/10.3147/jsfp.50.8
  7. A simple and sensitive method for determination of Norfloxacin in pharmaceutical preparations vol.51, pp.2, 2013, https://doi.org/10.1590/s1984-82502015000200020
  8. Antibiotic resistance of pathogenic Acinetobacter species and emerging combination therapy vol.55, pp.11, 2017, https://doi.org/10.1007/s12275-017-7288-4
  9. Lineage-specific SoxR-mediated Regulation of an Endoribonuclease Protects Non-enteric Bacteria from Redox-active Compounds vol.292, pp.1, 2013, https://doi.org/10.1074/jbc.m116.757500