1887

Abstract

In order to expedite the discovery of genes coding for either drug targets or antibiotic resistance, we have developed a functional genomic strategy termed Plas-Seq. This technique involves coupling a multicopy suppressor library to next-generation sequencing. We generated an Escherichia coli plasmid genomic library that was transformed into E. coli. These transformants were selected step by step using 0.25× to 2× minimum inhibitory concentrations for ceftriaxone, gentamicin, levofloxacin, tetracycline or trimethoprim. Plasmids were isolated at each selection step and subjected to Illumina sequencing. By searching for genomic loci whose sequencing coverage increased with antibiotic pressure we were able to detect 48 different genomic loci that were enriched by at least one antibiotic. Fifteen of these loci were studied functionally, and we showed that 13 can decrease the susceptibility of E. coli to antibiotics when overexpressed. These genes coded for drug targets, transcription factors, membrane proteins and resistance factors. The technique of Plas-Seq is expediting the discovery of genes associated with the mode of action or resistance to antibiotics and led to the isolation of a novel gene influencing drug susceptibility. It has the potential for being applied to novel molecules and to other microbial species.

Loading

Article metrics loading...

/content/journal/mgen/10.1099/mgen.0.000148
2018-01-10
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/mgen/4/2/mgen000148.html?itemId=/content/journal/mgen/10.1099/mgen.0.000148&mimeType=html&fmt=ahah

References

  1. Laxminarayan R, Duse A, Wattal C, Zaidi AK, Wertheim HF et al. Antibiotic resistance-the need for global solutions. Lancet Infect Dis 2013;13:1057–1098 [CrossRef][PubMed]
    [Google Scholar]
  2. Brown ED, Wright GD. Antibacterial drug discovery in the resistance era. Nature 2016;529:336–343 [CrossRef][PubMed]
    [Google Scholar]
  3. Czaplewski L, Bax R, Clokie M, Dawson M, Fairhead H et al. Alternatives to antibiotics-a pipeline portfolio review. Lancet Infect Dis 2016;16:239–251 [CrossRef][PubMed]
    [Google Scholar]
  4. Li X, Zolli-Juran M, Cechetto JD, Daigle DM, Wright GD et al. Multicopy suppressors for novel antibacterial compounds reveal targets and drug efflux susceptibility. Chem Biol 2004;11:1423–1430 [CrossRef][PubMed]
    [Google Scholar]
  5. Stirrett KL, Ferreras JA, Rossi SM, Moy RL, Fonseca FV et al. A multicopy suppressor screening approach as a means to identify antibiotic resistance determinant candidates in Yersinia pestis. BMC Microbiol 2008;8:122 [CrossRef][PubMed]
    [Google Scholar]
  6. Kündig C, Haimeur A, Légaré D, Papadopoulou B, Ouellette M. Increased transport of pteridines compensates for mutations in the high affinity folate transporter and contributes to methotrexate resistance in the protozoan parasite Leishmania tarentolae. EMBO J 1999;18:2342–2351 [CrossRef][PubMed]
    [Google Scholar]
  7. Cotrim PC, Garrity LK, Beverley SM. Isolation of genes mediating resistance to inhibitors of nucleoside and ergosterol metabolism in Leishmania by overexpression/selection. J Biol Chem 1999;274:37723–37730 [CrossRef][PubMed]
    [Google Scholar]
  8. Gazanion É, Fernández-Prada C, Papadopoulou B, Leprohon P, Ouellette M. Cos-Seq for high-throughput identification of drug target and resistance mechanisms in the protozoan parasite Leishmania. Proc Natl Acad Sci USA 2016;113:E3012E3021 [CrossRef][PubMed]
    [Google Scholar]
  9. Goldstone RJ, Smith DGE. A population genomics approach to exploiting the accessory 'resistome' of Escherichia coli. Microb Genom 2017;3:15 [CrossRef][PubMed]
    [Google Scholar]
  10. World Health Organization 2017; Global priority list of antibiotic-resistant bacteria to guide research, discovery, and development of new antibiotics. www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf
  11. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25:1754–1760 [CrossRef][PubMed]
    [Google Scholar]
  12. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD et al. De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis. Nat Protoc 2013;8:1494–1512 [CrossRef][PubMed]
    [Google Scholar]
  13. Bray NL, Pimentel H, Melsted P, Pachter L. Near-optimal probabilistic RNA-seq quantification. Nat Biotechnol 2016;34:525–527 [CrossRef][PubMed]
    [Google Scholar]
  14. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 2010;26:139–140 [CrossRef][PubMed]
    [Google Scholar]
  15. Larson MH, Gilbert LA, Wang X, Lim WA, Weissman JS et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 2013;8:2180–2196 [CrossRef][PubMed]
    [Google Scholar]
  16. Lupien A, Gingras H, Bergeron MG, Leprohon P, Ouellette M. Multiple mutations and increased RNA expression in tetracycline-resistant Streptococcus pneumoniae as determined by genome-wide DNA and mRNA sequencing. J Antimicrob Chemother 2015;70:1946–1959 [CrossRef][PubMed]
    [Google Scholar]
  17. Datsenko KA, Wanner BL. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA 2000;97:6640–6645 [CrossRef][PubMed]
    [Google Scholar]
  18. Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA et al. A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 2001;73:56–65 [CrossRef][PubMed]
    [Google Scholar]
  19. Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152:1173–1183 [CrossRef][PubMed]
    [Google Scholar]
  20. Hasegawa K, Chiba N, Kobayashi R, Murayama SY, Iwata S et al. Rapidly increasing prevalence of beta-lactamase-nonproducing, ampicillin-resistant Haemophilus influenzae type b in patients with meningitis. Antimicrob Agents Chemother 2004;48:1509–1514 [CrossRef][PubMed]
    [Google Scholar]
  21. Wienholtz NH, Barut A, Nørskov-Lauritsen N. Substitutions in PBP3 confer resistance to both ampicillin and extended-spectrum cephalosporins in Haemophilus parainfluenzae as revealed by site-directed mutagenesis and gene recombinants. J Antimicrob Chemother 2017;72:2544–2547 [CrossRef][PubMed]
    [Google Scholar]
  22. Rood JI, Laird AJ, Williams JW. Cloning of the Escherichia coli K-12 dihydrofolate reductase gene following mu-mediated transposition. Gene 1980;8:255–265 [CrossRef][PubMed]
    [Google Scholar]
  23. Nishino K, Yamasaki S, Hayashi-Nishino M, Yamaguchi A. Effect of NlpE overproduction on multidrug resistance in Escherichia coli. Antimicrob Agents Chemother 2010;54:2239–2243 [CrossRef][PubMed]
    [Google Scholar]
  24. Rahmati S, Yang S, Davidson AL, Zechiedrich EL. Control of the AcrAB multidrug efflux pump by quorum-sensing regulator SdiA. Mol Microbiol 2002;43:677–685 [CrossRef][PubMed]
    [Google Scholar]
  25. Tavío MM, Aquili VD, Poveda JB, Antunes NT, Sánchez-Céspedes J et al. Quorum-sensing regulator sdiA and marA overexpression is involved in in vitro-selected multidrug resistance of Escherichia coli. J Antimicrob Chemother 2010;65:1178–1186 [CrossRef][PubMed]
    [Google Scholar]
  26. Burman LG, Park JT, Lindström EB, Boman HG. Resistance of Escherichia coli to penicillins: identification of the structural gene for the chromosomal penicillinase. J Bacteriol 1973;116:123–130[PubMed]
    [Google Scholar]
  27. Olsson O, Bergström S, Lindberg FP, Normark S. ampC beta-lactamase hyperproduction in Escherichia coli: natural ampicillin resistance generated by horizontal chromosomal DNA transfer from Shigella. Proc Natl Acad Sci USA 1983;80:7556–7560 [CrossRef][PubMed]
    [Google Scholar]
  28. Jair KW, Yu X, Skarstad K, Thöny B, Fujita N et al. Transcriptional activation of promoters of the superoxide and multiple antibiotic resistance regulons by Rob, a binding protein of the Escherichia coli origin of chromosomal replication. J Bacteriol 1996;178:2507–2513 [CrossRef][PubMed]
    [Google Scholar]
  29. Miller PF, Gambino LF, Sulavik MC, Gracheck SJ. Genetic relationship between soxRS and mar loci in promoting multiple antibiotic resistance in Escherichia coli. Antimicrob Agents Chemother 1994;38:1773–1779 [CrossRef][PubMed]
    [Google Scholar]
  30. Chubiz LM, Glekas GD, Rao CV. Transcriptional cross talk within the mar-sox-rob regulon in Escherichia coli is limited to the rob and marRAB operons. J Bacteriol 2012;194:4867–4875 [CrossRef][PubMed]
    [Google Scholar]
  31. George AM, Levy SB. Gene in the major cotransduction gap of the Escherichia coli K-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics. J Bacteriol 1983;155:541–548[PubMed]
    [Google Scholar]
  32. Goldman JD, White DG, Levy SB. Multiple antibiotic resistance (mar) locus protects Escherichia coli from rapid cell killing by fluoroquinolones. Antimicrob Agents Chemother 1996;40:1266–1269[PubMed]
    [Google Scholar]
  33. McDermott PF, McMurry LM, Podglajen I, Dzink-Fox JL, Schneiders T et al. The marC gene of Escherichia coli is not involved in multiple antibiotic resistance. Antimicrob Agents Chemother 2008;52:382–383 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/mgen/10.1099/mgen.0.000148
Loading
/content/journal/mgen/10.1099/mgen.0.000148
Loading

Data & Media loading...

Supplementary File 1

PDF

Most Cited This Month

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error