Browse > Article
http://dx.doi.org/10.4014/jmb.1307.07059

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)
Publication Information
Journal of Microbiology and Biotechnology / v.23, no.9, 2013 , pp. 1293-1303 More about this Journal
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
Bacteria; mutation; antibiotics; norfloxacin; persister; RNA-Seq;
Citations & Related Records
연도 인용수 순위
  • Reference
1 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.   DOI   ScienceOn
2 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.   DOI   ScienceOn
3 Jung J, Baek JH, Park W. 2010. Complete genome sequence of the diesel-degrading Acinetobacter sp. strain DR1. J. Bacteriol. 192: 4794-4795.   DOI   ScienceOn
4 Kang YS, Park W. 2010. Trade-off between antibiotic resistance and biological fitness in Acinetobacter sp. strain DR1. Environ. Microbiol. 12: 1304-1318.   DOI   ScienceOn
5 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.   DOI   ScienceOn
6 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.   DOI   ScienceOn
7 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.   DOI   ScienceOn
8 Keren I, Kaldalu N, Spoering A, Wang Y, Lewis K. 2004. Persister cells and tolerance to antimicrobials. FEMS Microbiol. Lett. 230: 13-18.   DOI   ScienceOn
9 Keren I, Minami S, Rubin E, Lewis K. 2011. Characterization and transcriptome analysis of Mycobacterium tuberculosis persisters. MBio 2: e00100-e00111.
10 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.   DOI
11 LeClerc JE, Li B, Payne WL, Cebula TA. 1996. High mutation frequencies among Escherichia coli and Salmonella pathogens. Science 274: 1208-1211.   DOI   ScienceOn
12 Lewis K. 2010. Persister cells. Annu. Rev. Microbiol. 64: 357-372.   DOI   ScienceOn
13 Martinez JL, Baquero F. 2000. Mutation frequencies and antibiotic resistance. Antimicrob. Agents Chemother. 44: 1771-1777.   DOI   ScienceOn
14 Liu Y, Imlay JA. 2013. Cell death from antibiotics without the involvement of reactive oxygen species. Science 339: 1210-1213.   DOI   ScienceOn
15 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.   DOI   ScienceOn
16 Malik M, Zhao X, Drlica K. 2006. Lethal fragmentation of bacterial chromosomes mediated by DNA gyrase and quinolones. Mol. Microbiol. 61: 810-25.   DOI   ScienceOn
17 Mortazavi A, Williams BA, McCue K, Schaeffer L, Wold B. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat. Methods 5: 621-628.   DOI   ScienceOn
18 Norton MD, Spilkia AJ, Godoy VG. 2013. Antibiotic resistance acquired through a DNA damage-inducible response in Acinetobacter baumannii. J. Bacteriol. 195: 1335-1345.   DOI   ScienceOn
19 Phillips I, Culebras E, Moreno F, Baquero F. 1987. Induction of the SOS response by new 4-quinolones. J. Antimicrob. Chemother. 20: 631-638.   DOI
20 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.   DOI   ScienceOn
21 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.   DOI   ScienceOn
22 Suttle CA, Chen F. 1992. Mechanisms and rates of decay of marine viruses in seawater. Appl. Environ. Microbiol. 58: 3721-3729.
23 Shendure J. 2008. The beginning of the end for microarrays? Nat. Methods 5: 585-587   DOI   ScienceOn
24 Spoering AL, Lewis K. 2001. Biofilms and planktonic cells of Pseudomonas aeruginosa have similar resistance to killing by antimicrobials. J. Bacteriol. 183: 6746-6751.   DOI   ScienceOn
25 Stanier RY, Palleroni NJ, Doudoroff M. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43: 159-271.   DOI   ScienceOn
26 Turton JF, Perry C, Hannah MJ. 2012. Isolation of bacteriophage against currently circulating strains of Acinetobacter baumannii. J. Med. Microb. Diagn. 1: 109.
27 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.   DOI   ScienceOn
28 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.   DOI   ScienceOn
29 Wang Z, Gerstein M, Snyder M. 2009. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10: 57-63.   DOI   ScienceOn
30 Yeom J, Imlay JA, Park W. 2010. Iron homeostasis affects antibiotic-mediated cell death in Pseudomonas species. J. Biol. Chem. 285: 22689-22695.   DOI   ScienceOn
31 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.   DOI   ScienceOn
32 Bigger J. 1944. Treatment of staphylococcal infections with penicillin by intermittent sterilisation. Lancet 244: 497-500.   DOI   ScienceOn
33 Andriole VT. 2005. The quinolones: past, present, and future. Clin. Infect. Dis. 41: 113-119.   DOI   ScienceOn
34 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.   DOI   ScienceOn
35 Berenstein D. 1986. Prophage induction by ultraviolet light in Acinetobacter calcoaceticus. J. Gen. Microbiol. 132: 2633-2636.
36 Bergogne-Berezin E, Towner KJ. 1996. Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin. Microbiol. Rev. 9: 148-165.
37 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.
38 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.   DOI
39 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.   DOI   ScienceOn
40 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.   DOI   ScienceOn
41 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.   DOI   ScienceOn
42 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.   DOI   ScienceOn
43 Dorr T, Lewis K, Vuli M. 2009. SOS response induces persistence to fluoroquinolones in Escherichia coli. PLoS Genet. 5: e1000760.   DOI   ScienceOn
44 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.   DOI
45 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.   DOI   ScienceOn
46 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.   DOI   ScienceOn