Thymol tolerance in Escherichia coli induces morphological, metabolic and genetic changes

Fatemah Al-Kandari 1 2, Rabeah Al-Temaimi 3, Arnoud H M van Vliet 4, Martin J Woodward 5

Affiliations

16 December 2019

-

doi: 10.1186/s12866-019-1663-8

Abstract

Background: Thymol is a phenolic compound used for its wide spectrum antimicrobial activity. There is a limited understanding of the antimicrobial mechanisms underlying thymol activity. To investigate this, E. coli strain JM109 was exposed to thymol at sub-lethal concentrations and after 16 rounds of exposure, isolates with a 2-fold increased minimal inhibitory concentration (MIC) were recovered (JM109-Thyr). The phenotype was stable after multiple sub-cultures without thymol.

Results: Cell morphology studies by scanning electron microscopy (SEM) suggest that thymol renders bacterial cell membranes permeable and disrupts cellular integrity. 1H Nuclear magnetic resonance (NMR) data showed an increase in lactate and the lactic acid family amino acids in the wild type and JM109-Thyr in the presence of thymol, indicating a shift from aerobic respiration to fermentation. Sequencing of JM109-Thyr defined multiple mutations including a stop mutation in the acrR gene resulting in a truncation of the repressor of the AcrAB efflux pump. AcrAB is a multiprotein complex traversing the cytoplasmic and outer membrane, and is involved in antibiotic clearance.

Conclusions: Our data suggests that thymol tolerance in E. coli induces morphological, metabolic and genetic changes to adapt to thymol antimicrobial activity.

Keywords: Acriflavine resistance regulator; Efflux pump; Escherichia coli; Resistance; Thymol.

Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1 The effects of increasing concentrations of thymol on the growth of the wildtype JM109 E. coli (A), and JM109-Thyr (B)

Fig. 1 The effects of increasing concentrations of thymol on the growth of the wildtype JM109 E. coli (A), and JM109-Thyr (B)

Fig. 2 Scanning electron image of E. coli JM109 cells. (A) Thymol untreated wildtype JM109 cells; (B) JM109-Thyr thymol untreated cells; (C) wildtype JM109 thymol treated cells; (D) JM109-Thyr thymol treated cells

Fig. 2 Scanning electron image of E. coli JM109 cells. (A) Thymol untreated wildtype JM109 cells; (B) JM109-Thyr thymol untreated cells; (C) wildtype JM109 thymol treated cells; (D) JM109-Thyr thymol treated cells

Fig. 3 PCA-score plot illustrating the effect of different solvents on metabolic footprints derived from E. coli JM109 wildtype and JM109-Thyr untreated and treated with a sub-lethal concentration of thymol (50μg l− 1). N = 6 for each sample (JM109thy: wildtype JM109 with thymol; JM109M: JM109 thymol tolerant derivative; JM109Mthy: JM109 tolerant derivative with thymol)

Fig. 3 PCA-score plot illustrating the effect of different solvents on metabolic footprints derived from E. coli JM109 wildtype and JM109-Thyr untreated and treated with a sub-lethal concentration of thymol (50μg l− 1). N = 6 for each sample (JM109thy: wildtype JM109 with thymol; JM109M: JM109 thymol tolerant derivative; JM109Mthy: JM109 tolerant derivative with thymol)

Fig. 4 NMR spectra of JM109 wild-type and JM109-Thyr strain grown with and without thymol. (A) S-line plot of wild-type JM109 (bottom) and JM109-Thyr (top) grown without thymol, (B) partially assigned 700 MHz 1D spectra of wild-type (black) and JM109-Thyr (red). (C) S-line plot of wild-type JM109 grown without thymol (top) and thymol treated (bottom), (D) partially assigned 700 MHz 1D-spectra of wild-type JM109 without thymol (black) and thymol treated (red). (E) S-line plot of JM109-Thyr grown without thymol (top) and thymol treated (bottom), (F) partially assigned 700 MHz 1D-spectra of JM109-Thyr grown without thymol (black) and thymol treated (red). Heat map indicates product concentration

Fig. 4 NMR spectra of JM109 wild-type and JM109-Thyr strain grown with and without thymol. (A) S-line plot of wild-type JM109 (bottom) and JM109-Thyr (top) grown without thymol, (B) partially assigned 700 MHz 1D spectra of wild-type (black) and JM109-Thyr (red). (C) S-line plot of wild-type JM109 grown without thymol (top) and thymol treated (bottom), (D) partially assigned 700 MHz 1D-spectra of wild-type JM109 without thymol (black) and thymol treated (red). (E) S-line plot of JM109-Thyr grown without thymol (top) and thymol treated (bottom), (F) partially assigned 700 MHz 1D-spectra of JM109-Thyr grown without thymol (black) and thymol treated (red). Heat map indicates product concentration

Similar articles

Genetic characterization of phenicol-resistant Escherichia coli and role of wild-type repressor/regulator gene (acrR) on phenicol resistance.

Yaqoob M, Wang LP, Kashif J, Memon J, Umar S, Iqbal MF, Fiaz M, Lu CP.Folia Microbiol (Praha). 2018 Jul;63(4):443-449. doi: 10.1007/s12223-017-0579-7. Epub 2018 Jan 6.PMID: 29307119

Tigecycline Nonsusceptibility Occurs Exclusively in Fluoroquinolone-Resistant Escherichia coli Clinical Isolates, Including the Major Multidrug-Resistant Lineages O25b:H4-ST131-H30R and O1-ST648.

Sato T, Suzuki Y, Shiraishi T, Honda H, Shinagawa M, Yamamoto S, Ogasawara N, Takahashi H, Takahashi S, Tamura Y, Yokota SI.Antimicrob Agents Chemother. 2017 Jan 24;61(2):e01654-16. doi: 10.1128/AAC.01654-16. Print 2017 Feb.PMID: 27855067 Free PMC article.

Natural extracts stimulate membrane-associated mechanisms of resistance in Gram-negative bacteria.

Fadli M, Chevalier J, Hassani L, Mezrioui NE, Pagès JM.Lett Appl Microbiol. 2014 May;58(5):472-7. doi: 10.1111/lam.12216. Epub 2014 Feb 6.PMID: 24447247

Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF).

Bohnert JA, Schuster S, Fähnrich E, Trittler R, Kern WV.J Antimicrob Chemother. 2007 Jun;59(6):1216-22. doi: 10.1093/jac/dkl426. Epub 2006 Oct 24.PMID: 17062614

Exploring the contribution of efflux on the resistance to fluoroquinolones in clinical isolates of Escherichia coli.

Paltansing S, Tengeler AC, Kraakman ME, Claas EC, Bernards AT.Microb Drug Resist. 2013 Dec;19(6):469-76. doi: 10.1089/mdr.2013.0058. Epub 2013 Aug 2.PMID: 23909485

Cited by

Terpenes as bacterial efflux pump inhibitors: A systematic review.

Dias KJSO, Miranda GM, Bessa JR, Araújo ACJ, Freitas PR, Almeida RS, Paulo CLR, Neto JBA, Coutinho HDM, Ribeiro-Filho J.Front Pharmacol. 2022 Oct 13;13:953982. doi: 10.3389/fphar.2022.953982. eCollection 2022.PMID: 36313340 Free PMC article.

Thymol as an Adjuvant to Restore Antibiotic Efficacy and Reduce Antimicrobial Resistance and Virulence Gene Expression in Enterotoxigenic Escherichia coli Strains.

Bonetti A, Tugnoli B, Piva A, Grilli E.Antibiotics (Basel). 2022 Aug 8;11(8):1073. doi: 10.3390/antibiotics11081073.PMID: 36009942 Free PMC article.

Biogenic Silver Nanoparticles Strategically Combined With Origanum vulgare Derivatives: Antibacterial Mechanism of Action and Effect on Multidrug-Resistant Strains.

Scandorieiro S, Rodrigues BCD, Nishio EK, Panagio LA, de Oliveira AG, Durán N, Nakazato G, Kobayashi RKT.Front Microbiol. 2022 May 6;13:842600. doi: 10.3389/fmicb.2022.842600. eCollection 2022.PMID: 35602016 Free PMC article.

Effect of Essential Oils Supplemented with Caprylic Acid and Sodium Chloride against Faecal ESBL-Producing Escherichia coli Isolated from Pigs.

Gāliņa D, Radenkovs V, Kviesis J, Valdovska A.Antibiotics (Basel). 2022 Mar 29;11(4):461. doi: 10.3390/antibiotics11040461.PMID: 35453213 Free PMC article.

Polyphenolic phytochemicals as natural feed additives to control bacterial pathogens in the chicken gut.

Al-Mnaser A, Dakheel M, Alkandari F, Woodward M.Arch Microbiol. 2022 Apr 12;204(5):253. doi: 10.1007/s00203-022-02862-5.PMID: 35412092 Free PMC article. Review.

KMEL References

https://pubmed.ncbi.nlm.nih.gov

References

  1.  
    1. Burt S. Essential oils: their antibacterial properties and potential applications in foods--a review. Int J Food Microbiol. 2004;94(3):223–253. doi: 10.1016/j.ijfoodmicro.2004.03.022. - DOI - PubMed
  2.  
    1. Nazzaro F, Fratianni F, De Martino L, Coppola R, De Feo V. Effect of essential oils on pathogenic bacteria. Pharmaceuticals (Basel) 2013;6:1451–1474. doi: 10.3390/ph6121451. - DOI - PMC - PubMed
  3.  
    1. Escudero J, Lopez JC, Rabanal RM, Valverde S. Secondary metabolites form Satureja species-new triterpenoid from Satureja-Acinos. J Nat Prod. 1985;48:128–132. doi: 10.1021/np50037a025. - DOI
  4.  
    1. Nagoor Meeran MF, Javed H, Al Taee H, Azimullah S, Ojha SK. Pharmacological properties and molecular mechanisms of Thymol: prospects for its therapeutic potential and pharmaceutical development. Front Pharmacol. 2017;8:380. doi: 10.3389/fphar.2017.00380. - DOI - PMC - PubMed
  5.  
    1. Nerio LS, Olivero-Verbel J, Stashenko E. Repellent activity of essential oils: a review. Bioresour Technol. 2010;101(1):372–378. doi: 10.1016/j.biortech.2009.07.048. - DOI - PubMed
  6.  
    1. Marchese A, Orhan IE, Daglia M, Barbieri R, Di Lorenzo A, Nabavi SF, et al. Antibacterial and antifungal activities of thymol: a brief review of the literature. Food Chem. 2016;210:402–414. doi: 10.1016/j.foodchem.2016.04.111. - DOI - PubMed
  7.  
    1. Cosentino S, Tuberoso CI, Pisano B, Satta M, Mascia V, Arzedi E, et al. In-vitro antimicrobial activity and chemical composition of Sardinian Thymus essential oils. Lett Appl Microbiol. 1999;29(2):130–135. doi: 10.1046/j.1472-765X.1999.00605.x. - DOI - PubMed
  8.  
    1. Yuan W, Seng ZJ, Kohli GS, Yang L, Yuk HG. Stress resistance development and genome-wide transcriptional response of Escherichia coli O157:H7 adapted to sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde. Appl Environ Microbiol. 2018;15:84(22). - PMC - PubMed
  9.  
    1. Hartman Hassan B., Fell David A., Rossell Sergio, Jensen Peter Ruhdal, Woodward Martin J., Thorndahl Lotte, Jelsbak Lotte, Olsen John Elmerdahl, Raghunathan Anu, Daefler Simon, Poolman Mark G. Identification of potential drug targets in Salmonella enterica sv. Typhimurium using metabolic modelling and experimental validation. Microbiology. 2014;160(6):1252–1266. doi: 10.1099/mic.0.076091-0. - DOI - PubMed
  10.  
    1. Tiwari V, Tiwari M. Quantitative proteomics to study carbapenem resistance in Acinetobacter baumannii. Front Microbiol. 2014;5:512. doi: 10.3389/fmicb.2014.00512. - DOI - PMC - PubMed
  11.  
    1. Editors PM. Antimicrobial Resistance: Is the World UNprepared. PLoS medicine. 2016;13(9):e1002130. http://journals.plos.org/plosmedicine/s/staff-editors. Accessed 26 Aug 2019. PLOS is funded partly through manuscript publication charges, but the PLOS Medicine Editors are paid a fixed salary (their salaries are not linked to the number of papers published in the journal).
  12.  
    1. Naylor NR, Atun R, Zhu N, Kulasabanathan K, Silva S, Chatterjee A, et al. Estimating the burden of antimicrobial resistance: a systematic literature review. Antimicrob Resist Infect Control. 2018;7:58. doi: 10.1186/s13756-018-0336-y. - DOI - PMC - PubMed
  13.  
    1. Chantziaras I, Boyen F, Callens B, Dewulf J. Correlation between veterinary antimicrobial use and antimicrobial resistance in food-producing animals: a report on seven countries. J Antimicrob Chemother. 2014;69(3):827–834. doi: 10.1093/jac/dkt443. - DOI - PubMed
  14.  
    1. Muloi D, Ward MJ, Pedersen AB, Fevre EM, Woolhouse MEJ, van Bunnik BAD. Are food animals responsible for transfer of antimicrobial-resistant Escherichia coli or their resistance determinants to human populations? A systematic review. Foodborne Pathog Dis. 2018;15(8):467–474. doi: 10.1089/fpd.2017.2411. - DOI - PMC - PubMed
  15.  
    1. Burt SA, Reinders RD. Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7. Lett Appl Microbiol. 2003;36(3):162–167. doi: 10.1046/j.1472-765X.2003.01285.x. - DOI - PubMed
  16.  
    1. Borges A, Abreu AC, Ferreira C, Saavedra MJ, Simoes LC, Simoes M. Antibacterial activity and mode of action of selected glucosinolate hydrolysis products against bacterial pathogens. J Food Sci Technol. 2015;52(8):4737–4748. doi: 10.1007/s13197-014-1533-1. - DOI - PMC - PubMed
  17.  
    1. Du E, Gan L, Li Z, Wang W, Liu D, Guo Y. In vitro antibacterial activity of thymol and carvacrol and their effects on broiler chickens challenged with Clostridium perfringens. Journal of animal science and biotechnology. 2015;6:58. doi: 10.1186/s40104-015-0055-7. - DOI - PMC - PubMed
  18.  
    1. van Hoek AH, Mevius D, Guerra B, Mullany P, Roberts AP, Aarts HJ. Acquired antibiotic resistance genes: an overview. Front Microbiol. 2011;2:203. - PMC - PubMed
  19.  
    1. Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ. Molecular mechanisms of antibiotic resistance. Nat Rev Microbiol. 2015;13(1):42–51. doi: 10.1038/nrmicro3380. - DOI - PubMed
  20.  
    1. Winska K, Maczka W, Lyczko J, Grabarczyk M, Czubaszek A, Szumny A. Essential Oils as Antimicrobial Agents-Myth or Real Alternative? Molecules (Basel, Switzerland). 2019;5;24(11). - PMC - PubMed
  21.  
    1. Dubois-Brissonnet F, Naitali M, Mafu AA, Briandet R. Induction of fatty acid composition modifications and tolerance to biocides in salmonella enterica serovar Typhimurium by plant-derived terpenes. Appl Environ Microbiol. 2011;77(3):906–910. doi: 10.1128/AEM.01480-10. - DOI - PMC - PubMed
  22.  
    1. Wesche AM, Gurtler JB, Marks BP, Ryser ET. Stress, sublethal injury, resuscitation, and virulence of bacterial foodborne pathogens. J Food Prot. 2009;72(5):1121–1138. doi: 10.4315/0362-028X-72.5.1121. - DOI - PubMed
  23.  
    1. Zengin H, Baysal AH. Antibacterial and antioxidant activity of essential oil terpenes against pathogenic and spoilage-forming bacteria and cell structure-activity relationships evaluated by SEM microscopy. Molecules (Basel, Switzerland) 2014;03;19(11):17773–17798. doi: 10.3390/molecules191117773. - DOI - PMC - PubMed
  24.  
    1. Lambert RJ, Skandamis PN, Coote PJ, Nychas GJ. A study of the minimum inhibitory concentration and mode of action of oregano essential oil, thymol and carvacrol. J Appl Microbiol. 2001;91(3):453–462. doi: 10.1046/j.1365-2672.2001.01428.x. - DOI - PubMed
  25.  
    1. Skandamis P, Koutsoumanis K, Fasseas K, Nychas G-JE. Inhibition of oregano essential oil and EDTA on Escherichia coli O157:H7. Italian Journal of Food Science. 2001;13(1):65–75.
  26.  
    1. Fitzgerald DJ, Stratford M, Gasson MJ, Ueckert J, Bos A, Narbad A. Mode of antimicrobial action of vanillin against Escherichia coli, lactobacillus plantarum and Listeria innocua. J Appl Microbiol. 2004;97(1):104–113. doi: 10.1111/j.1365-2672.2004.02275.x. - DOI - PubMed
  27.  
    1. Picone G, Laghi L, Gardini F, Lanciotti R, Siroli L, Capozzi F. Evaluation of the effect of carvacrol on the Escherichia coli 555 metabolome by using 1H-NMR spectroscopy. Food Chem. 2013;141(4):4367–4374. doi: 10.1016/j.foodchem.2013.07.004. - DOI - PubMed
  28.  
    1. Ma D, Cook DN, Alberti M, Pon NG, Nikaido H, Hearst JE. Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol. 1995;16(1):45–55. doi: 10.1111/j.1365-2958.1995.tb02390.x. - DOI - PubMed
  29.  
    1. Tsukagoshi N, Aono R. Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump. J Bacteriol. 2000;182(17):4803–4810. doi: 10.1128/JB.182.17.4803-4810.2000. - DOI - PMC - PubMed
  30.  
    1. Sun J, Deng Z, Yan A. Bacterial multidrug efflux pumps: mechanisms, physiology and pharmacological exploitations. Biochem Biophys Res Commun. 2014;453(2):254–267. doi: 10.1016/j.bbrc.2014.05.090. - DOI - PubMed
  31.  
    1. Venter H, Mowla R, Ohene-Agyei T, Ma S. RND-type drug e ffl ux pumps from gram-negative bacteria: molecular mechanism and inhibition. Front Microbiol. 2015;6:377. doi: 10.3389/fmicb.2015.00377. - DOI - PMC - PubMed
  32.  
    1. Ma D, Alberti M, Lynch C, Nikaido H, Hearst JE. The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stress signals. Mol Microbiol. 1996;19(1):101–112. doi: 10.1046/j.1365-2958.1996.357881.x. - DOI - PubMed
  33.  
    1. Lee JO, Cho KS, Kim OB. Overproduction of AcrR increases organic solvent tolerance mediated by modulation of SoxS regulon in Escherichia coli. Appl Microbiol Biotechnol. 2014;98(20):8763–8773. doi: 10.1007/s00253-014-6024-9. - DOI - PubMed
  34.  
    1. Ramos JL, Martinez-Bueno M, Molina-Henares AJ, Teran W, Watanabe K, Zhang X, et al. The TetR family of transcriptional repressors. Microbiology and molecular biology reviews : MMBR. 2005;69(2):326–356. doi: 10.1128/MMBR.69.2.326-356.2005. - DOI - PMC - PubMed
  35.  
    1. Su CC, Rutherford DJ, Yu EW. Characterization of the multidrug efflux regulator AcrR from Escherichia coli. Biochem Biophys Res Commun. 2007;361(1):85–90. doi: 10.1016/j.bbrc.2007.06.175. - DOI - PMC - PubMed
  36.  
    1. Watanabe R, Doukyu N. Contributions of mutations in acrR and marR genes to organic solvent tolerance in Escherichia coli. AMB Express. 2012;2(1):58. doi: 10.1186/2191-0855-2-58. - DOI - PMC - PubMed
  37.  
    1. Wang H, Dzink-Fox JL, Chen M, Levy SB. Genetic characterization of highly fluoroquinolone-resistant clinical Escherichia coli strains from China: role of acrR mutations. Antimicrobial agents and chemotherapy. 2001;45(5):1515–1521. doi: 10.1128/AAC.45.5.1515-1521.2001. - DOI - PMC - PubMed
  38.  
    1. Webber MA, Talukder A, Piddock LJ. Contribution of mutation at amino acid 45 of AcrR to acrB expression and ciprofloxacin resistance in clinical and veterinary Escherichia coli isolates. Antimicrob Agents Chemother. 2005;49(10):4390–4392. doi: 10.1128/AAC.49.10.4390-4392.2005. - DOI - PMC - PubMed
  39.  
    1. Corvec S, Lepelletier D, Reynaud A, Dauvergne S, Giraudeau C, Caroff N. In vivo selection of an Escherichia coli isolate highly resistant to ciprofloxacin and ceftazidime: role of a 4-bp duplication in acrR and ampC overexpression. Int J Antimicrob Agents. 2008;32(2):196–198. doi: 10.1016/j.ijantimicag.2008.04.001. - DOI - PubMed
  40.  
    1. Adler M, Anjum M, Andersson DI, Sandegren L. Combinations of mutations in envZ, ftsI, mrdA, acrB and acrR can cause high-level carbapenem resistance in Escherichia coli. J Antimicrob Chemother. 2016;71(5):1188–1198. doi: 10.1093/jac/dkv475. - DOI - PubMed
  41.  
    1. Chueca B, Renzoni A, Berdejo D, Pagan R, Kelley WL, Garcia-Gonzalo D. Whole-genome sequencing and genetic analysis reveal novel stress responses to individual constituents of essential oils in Escherichia coli. Appl Environ Microbiol. 2018;1:84(7). - PMC - PubMed
  42.  
    1. Yaqoob M, Wang LP, Kashif J, Memon J, Umar S, Iqbal MF, et al. Genetic characterization of phenicol-resistant Escherichia coli and role of wild-type repressor/regulator gene (acrR) on phenicol resistance. Folia Microbiol. 2018;63(4):443–449. doi: 10.1007/s12223-017-0579-7. - DOI - PubMed
  43.  
    1. Deutscher MP. Degradation of RNA in bacteria: comparison of mRNA and stable RNA. Nucleic Acids Res. 2006;34(2):659–666. doi: 10.1093/nar/gkj472. - DOI - PMC - PubMed
  44.  
    1. Li Z, Pandit S, Deutscher MP. RNase G (CafA protein) and RNase E are both required for the 5′ maturation of 16S ribosomal RNA. EMBO J. 1999;18(10):2878–2885. doi: 10.1093/emboj/18.10.2878. - DOI - PMC - PubMed
  45.  
    1. Kaga N, Umitsuki G, Nagai K, Wachi M. RNase G-dependent degradation of the eno mRNA encoding a glycolysis enzyme enolase in Escherichia coli. Biosci Biotechnol Biochem. 2002;66(10):2216–2220. doi: 10.1271/bbb.66.2216. - DOI - PubMed
  46.  
    1. Umitsuki G, Wachi M, Takada A, Hikichi T, Nagai K. Involvement of RNase G in in vivo mRNA metabolism in Escherichia coli. Genes to cells : devoted to molecular & cellular mechanisms. 2001;6(5):403–410. doi: 10.1046/j.1365-2443.2001.00430.x. - DOI - PubMed
  47.  
    1. Chung DH, Min Z, Wang BC, Kushner SR. Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants. RNA (New York, NY) 2010;16(7):1371–1385. doi: 10.1261/rna.2104810. - DOI - PMC - PubMed
  48.  
    1. Manow R, Wang J, Wang Y, Zhao J, Garza E, Iverson A, et al. Partial deletion of rng (RNase G)-enhanced homoethanol fermentation of xylose by the non-transgenic Escherichia coli RM10. J Ind Microbiol Biotechnol. 2012;39(7):977–985. doi: 10.1007/s10295-012-1100-6. - DOI - PubMed
  49.  
    1. Borges A, Ferreira C, Saavedra MJ, Simoes M. Antibacterial activity and mode of action of ferulic and gallic acids against pathogenic bacteria. Microb Drug Resist. 2013;19(4):256–265. doi: 10.1089/mdr.2012.0244. - DOI - PubMed
  50.  
    1. Hsueh PR, Ko WC, Wu JJ, Lu JJ, Wang FD, Wu HY, et al. Consensus statement on the adherence to clinical and laboratory standards institute (CLSI) antimicrobial susceptibility testing guidelines (CLSI-2010 and CLSI-2010-update) for Enterobacteriaceae in clinical microbiology laboratories in Taiwan. Journal of microbiology, immunology, and infection = Wei mian yu gan ran za zhi. 2010;43(5):452–455. doi: 10.1016/S1684-1182(10)60070-9. - DOI - PubMed
  51.  
    1. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. Journal of computational biology : a journal of computational molecular cell biology. 2012;19(5):455–477. doi: 10.1089/cmb.2012.0021. - DOI - PMC - PubMed
  52.  
    1. Gurevich A, Saveliev V, Vyahhi N, Tesler G. QUAST: quality assessment tool for genome assemblies. Bioinformatics. 2013;29(8):1072–1075. doi: 10.1093/bioinformatics/btt086. - DOI - PMC - PubMed
  53.  
    1. Darling AE, Tritt A, Eisen JA, Facciotti MT. Mauve assembly metrics. Bioinformatics. 2011;27(19):2756–2757. doi: 10.1093/bioinformatics/btr451. - DOI - PMC - PubMed
  54.  
    1. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics. 2014;30(14):2068–2069. doi: 10.1093/bioinformatics/btu153. - DOI - PubMed
  55.  
    1. Edwards JS, Palsson BO. The Escherichia coli MG1655 in silico metabolic genotype: its definition, characteristics, and capabilities. Proc Natl Acad Sci U S A. 2000;97(10):5528–5533. doi: 10.1073/pnas.97.10.5528. - DOI - PMC - PubMed
  56.  
    1. Lee JW, Choi S, Park JH, Vickers CE, Nielsen LK, Lee SY. Development of sucrose-utilizing Escherichia coli K-12 strain by cloning beta-fructofuranosidases and its application for L-threonine production. Appl Microbiol Biotechnol. 2010;88(4):905–913. doi: 10.1007/s00253-010-2825-7. - DOI - PubMed
  57.  
    1. Kumar A, Ernst RR, Wuthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980;95(1):1–6. doi: 10.1016/0006-291X(80)90695-6. - DOI - PubMed
  58.  
    1. Nicholson JK, Foxall PJ, Spraul M, Farrant RD, Lindon JC. 750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma. Anal Chem. 1995;67(5):793–811. doi: 10.1021/ac00101a004. - DOI - PubMed

Page Navigation