Skip to main content
Log in

Identifying Escherichia coli genes involved in intrinsic multidrug resistance

  • Applied Microbial and Cell Physiology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Multidrug resistance is a major cause of clinical failure in treating bacterial infections. Increasing evidence suggests that bacteria can resist multiple antibiotics through intrinsic mechanisms that rely on gene products such as efflux pumps that expel antibiotics and special membrane proteins that block the penetration of drug molecules. In this study, Escherichia coli was used as a model system to explore the genetic basis of intrinsic multidrug resistance. A random mutant library was constructed in E. coli EC100 using transposon mutagenesis. The library was screened by growth measurement to identify the mutants with enhanced or reduced resistance to chloramphenicol (Cm). Out of the 4,000 mutants screened, six mutants were found to be more sensitive to Cm and seven were more resistant compared to the wild-type EC100. Mutations in 12 out of the 13 mutants were identified by inverse polymerase chain reaction. Mutants of the genes rob, garP, bipA, insK, and yhhX were more sensitive to Cm compared to the wild-type EC100, while the mutation of rhaB, yejM, dsdX, nagA, yccE, atpF, or htrB led to higher resistance. Overexpression of rob was found to increase the resistance of E. coli biofilms to tobramycin (Tob) by 2.7-fold, while overexpression of nagA, rhaB, and yccE significantly enhanced the susceptibility of biofilms by 2.2-, 2.5-, and 2.1-fold respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Ariza RR, Li Z, Ringstad N, Demple B (1995) Activation of multiple antibiotic resistance and binding of stress-inducible promoters by Escherichia coli Rob protein. J Bacteriol 177:1655–1661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H (2006) Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol 2:1–11

    Article  CAS  Google Scholar 

  • Bacic M, Parker AC, Stagg J, Whitley HP, Wells WG, Jacob LA, Smith CJ (2005) Genetic and structural analysis of the Bacteroides conjugative transposon CTn341. J Bacteriol 187:2858–2869

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barber M (1947) Staphylococcal infection due to penicillin-resistanct strains. Br Med J 2:863–865

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barker HC, Kinsella N, Jaspe A, Friedrich T, O’Connor CD (2000) Formate protects stationary-phase Escherichia coli and Salmonella cells from killing by a cationic antimicrobial peptide. Mol Microbiol 35:1518–1529

    Article  CAS  PubMed  Google Scholar 

  • Blattner FR, Plunkett G 3rd, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF et al. (1997) The complete genome sequence of Escherichia coli K-12. Science 277:1453–1474

    CAS  PubMed  Google Scholar 

  • Bush K, Jacoby GA, Medeiros AA (1995) A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 39:1211–1233

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bussiere DE, Muchmore SW, Dealwis CG, Schluckebier G, Nienaber VL, Edalji RP, Walter KA, Ladror US, Holzman TF, Abad-Zapatero C (1998) Crystal structure of ErmC’, an rRNA methyltransferase which mediates antibiotic resistance in bacteria. Biochemistry 37:7103–7112

    Article  CAS  PubMed  Google Scholar 

  • Characklis WG (1981) Bioengineering report: fouling biofilm development: a process analysis. Biotechnol Bioeng 23:1923–1960

    Article  CAS  Google Scholar 

  • Costerton JW, Stewart PS, Greenberg EP (1999) Bacterial biofilms: a common cause of persistent infections. Science 284:1318–1322

    Article  CAS  PubMed  Google Scholar 

  • Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 97:6640–6645

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Farris M, Grant A, Richardson TB, O’Connor CD (1998) BipA: a tyrosine-phosphorylated GTPase that mediates interactions between enteropathogenic Escherichia coli (EPEC) and epithelial cells. Mol Microbiol 28:265–279

    Article  CAS  PubMed  Google Scholar 

  • Fernandes P, Ferreira BS, Cabral JM (2003) Solvent tolerance in bacteria: role of efflux pumps and cross-resistance with antibiotics. Int J Antimicrob Agents 22:211–216

    Article  CAS  PubMed  Google Scholar 

  • Filiatrault MJ, Picardo KF, Ngai H, Passador L, Iglewski BH (2006) Identification of Pseudomonas aeruginosa genes involved in virulence and anaerobic growth. Infect Immun 74:4237–4245

    CAS  PubMed  PubMed Central  Google Scholar 

  • Fratamico PM, Bhunia AK, Smith JL (2005) Foodborne pathogens: microbiology and molecular biology. Caister Academic, Wymondham, Norfolk, UK, p 384

    Google Scholar 

  • Gallegos MT, Schleif R, Bairoch A, Hofmann K, Ramos JL (1997) AraC/XylS family of transcriptional regulators. Microbiol Mol Biol Rev 61:393–410

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gilbert P, Das J, Foley I (1997) Biofilm susceptibility to antimicrobials. Adv Dent Res 11:160–167

    Article  CAS  PubMed  Google Scholar 

  • Gillis RJ, White KG, Choi KH, Wagner VE, Schweizer HP, Iglewski BH (2005) Molecular basis of azithromycin-resistant Pseudomonas aeruginosa biofilms. Antimicrob Agents Chemother 49:3858–3867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Gomez MJ, Neyfakh AA (2006) Genes involved in intrinsic antibiotic resistance of Acinetobacter baylyi. Antimicrob Agents Chemother 50:3562–3567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Goryshin IY, Reznikoff WS (1998) Tn5 in vitro transposition. J Biol Chem 273:7367–7374

    Article  CAS  PubMed  Google Scholar 

  • Hall-Stoodley L, Costerton JW, Stoodley P (2004) Bacterial biofilms: from the natural environment to infectious diseases. Nat Rev Microbiol 2:95–108

    Article  CAS  PubMed  Google Scholar 

  • Hancock RE (1998) Resistance mechanisms in Pseudomonas aeruginosa and other nonfermentative gram-negative bacteria. Clin Infect Dis 27(Suppl 1):S93–S99

    Article  CAS  PubMed  Google Scholar 

  • Hantke K (2001) Iron and metal regulation in bacteria. Curr Opin Microbiol 4:172–177

    Article  CAS  PubMed  Google Scholar 

  • Harrison PF, Lederberg J (1998) Antimicrobial resistance: issues and options. National Academy, Washington, D.C, pp 9–10

    Google Scholar 

  • Heydorn A, Nielsen AT, Hentzer M, Sternberg C, Givskov M, Ersbøll BK, Molin S (2000) Quantification of biofilm structures by the novel computer program COMSTAT. Microbiology 146:2395–2407

    Article  CAS  PubMed  Google Scholar 

  • Hunt DE, Sandham HJ (1969) Improved agar gradient-plate technique. Appl Microbiol 17:329–330

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kitagawa M, Ara T, Arifuzzaman M, Ioka-Nakamichi T, Inamoto E, Toyonaga H, Mori H (2005) Complete set of ORF clones of Escherichia coli ASKA library (a complete set of E. coli K-12 ORF archive): unique resources for biological research. DNA Res 12:291–299

    Article  CAS  PubMed  Google Scholar 

  • Klevens RM, Edwards JR, Richards CL, Horan TC, Gaynes RP, Pollock DA, Cardo DM (2007) Estimating health care-associated infections and deaths in U.S. hospitals, 2002 Atlanta: Centers for Disease Control and Prevention. 160–166 p

  • Lewenza S, Falsafi RK, Winsor G, Gooderham WJ, McPhee JB, Brinkman FS, Hancock RE (2005) Construction of a mini-Tn5-luxCDABE mutant library in Pseudomonas aeruginosa PAO1: a tool for identifying differentially regulated genes. Genome Res 15:583–589

    CAS  PubMed  PubMed Central  Google Scholar 

  • Lewis K (2005) Persister cells and the riddle of biofilm survival. Biochemistry (Mosc) 70:267–274

    Article  CAS  Google Scholar 

  • Ma D, Cook DN, Alberti M, Pon NG, Nikaido H, Hearst JE (1995) Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol 16:45–55

    Article  CAS  PubMed  Google Scholar 

  • Mah TF, Pitts B, Pellock B, Walker GC, Stewart PS, O’Toole GA (2003) A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance. Nature 426:306–310

    Article  CAS  PubMed  Google Scholar 

  • Martin VJJ, Mohn WW (2002) The isolation of DNA sequences flanking Tn5 transposon insertions by inverse PCR. Methods Mol Biol 192:315–323

    CAS  PubMed  Google Scholar 

  • McMurry L, Petrucci RE Jr, Levy SB (1980) Active efflux of tetracycline encoded by four genetically different tetracycline resistance determinants in Escherichia coli. Proc Natl Acad Sci USA 77:3974–3977

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mizuno T, Chou MY, Inouye M (1984) A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci USA 81:1966–1970

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nishino K, Yamaguchi A (2001) Analysis of a complete library of putative drug transporter genes in Escherichia coli. J Bacteriol 183:5803–5812

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ochman H, Gerber AS, Hartl DL (1988) Genetic applications of an inverse polymerase chain reaction. Genetics 120:621–623

    CAS  PubMed  PubMed Central  Google Scholar 

  • Owens RM, Pritchard G, Skipp P, Hodey M, Connell SR, Nierhaus KH, O’Connor CD (2004) A dedicated translation factor controls the synthesis of the global regulator Fis. Embo J 23:3375–3385

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfennig PL, Flower AM (2001) BipA is required for growth of Escherichia coi K12 at low temperature. Mol Genet Genomics 266:313–317

    Article  CAS  PubMed  Google Scholar 

  • Potera C (1999) Microbiology: forging a link between biofilms and disease. Science 283:1837–1839

    Article  CAS  PubMed  Google Scholar 

  • Putman M, van Veen HW, Konings WN (2000) Molecular properties of bacterial multidrug transporters. Microbiol Mol Biol Rev 64:672–693

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qi SY, Li Y, Szyroki A, Giles IG, Moir A, O’Connor CD (1995) Salmonella typhimurium responses to a bactericidal protein from human neutrophils. Mol Microbiol 17:523–531

    Article  CAS  PubMed  Google Scholar 

  • Ren D, Bedzyk L, Setlow P, Thomas S, Ye RW, Wood TK (2004a) Gene expression in Bacillus subtilis surface biofilms with and without sporulation and the importance of yveR for biofilm maintenance. Biotechnol Bioeng 86:344–364

    Article  CAS  PubMed  Google Scholar 

  • Ren D, Bedzyk LA, Thomas SM, Ye RW, Wood TK (2004b) Gene expression in Escherichia coli biofilms. Appl Microbiol Biotechnol 64:515–524

    Article  CAS  PubMed  Google Scholar 

  • Rodriguez RL, Tait RC (1983) Recombinant DNA Techniques: An Introduction. Benjamin/Cummings Publishing, Menlo Park, CA

    Google Scholar 

  • Rosenberg EY, Bertenthal D, Nilles ML, Bertrand KP, Nikaido H (2003) Bile salts and fatty acids induce the expression of Escherichia coli AcrAB multidrug efflux pump through their interaction with Rob regulatory protein. Mol Microbiol 48:1609–1619

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Scharf SJ, Horn GT, Erlich HA (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233:1076–1078

    Article  CAS  PubMed  Google Scholar 

  • Stover CK, Pham XQ, Erwin AL, Mizoguchi SD, Warrener P, Hickey MJ, Brinkman FS, Hufnagle WO, Kowalik DJ, Lagrou M et al. (2000) Complete genome sequence of Pseudomonas aeruginosa PA01, an opportunistic pathogen. Nature 406:959–964

    Article  CAS  PubMed  Google Scholar 

  • Struble JM, Gill RT (2006) Reverse engineering antibiotic sensitivity in a multidrug-resistant Pseudomonas aeruginosa isolate. Antimicrob Agents Chemother 50:2506–2515

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sulavik MC, Houseweart C, Cramer C, Jiwani N, Murgolo N, Greene J, DiDomenico B, Shaw KJ, Miller GH, Hare R et al. (2001) Antibiotic susceptibility profiles of Escherichia coli strains lacking multidrug efflux pump genes. Antimicrob Agents Chemother 45:1126–1136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Thomson KS, Smith Moland E (2000) Version 2000: the new beta-lactamases of Gram-negative bacteria at the dawn of the new millennium. Microbes Infect 2:1225–1235

    Article  CAS  PubMed  Google Scholar 

  • Touze T, Eswaran J, Bokma E, Koronakis E, Hughes C, Koronakis V (2004) Interactions underlying assembly of the Escherichia coli AcrAB-TolC multidrug efflux system. Mol Microbiol 53:697–706

    Article  CAS  PubMed  Google Scholar 

  • Walsh C (2000) Molecular mechanisms that confer antibacterial drug resistance. Nature 406:775–781

    Article  CAS  PubMed  Google Scholar 

  • White-Ziegler CA, Malhowski AJ, Young S (2007) Human body temperature (37 °C) increases the expression of iron, carbohydrate, and amino acid utilization genes in Escherichia coli K-12. J Bacteriol 189:5429–5440

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zgurskaya HI, Nikaido H (1999) AcrA is a highly asymmetric protein capable of spanning the periplasm. J Mol Biol 285:409–420

    Article  CAS  PubMed  Google Scholar 

  • Zottola EA, Sasahara KC (1994) Microbial biofilms in the food processing industry—should they be a concern? Int J Food Microbiol 23:125–148

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Syracuse University for financial support. The authors are also grateful to the National Institute of Genetics of Japan for providing E. coli BW25113, AG1, the mutants of efflux pumps, and strains for gene overexpression.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dacheng Ren.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Duo, M., Hou, S. & Ren, D. Identifying Escherichia coli genes involved in intrinsic multidrug resistance. Appl Microbiol Biotechnol 81, 731–741 (2008). https://doi.org/10.1007/s00253-008-1709-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-008-1709-6

Keywords

Navigation