1887

Microbiology Society logo

Volume 169, Issue 5

Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool Open Access

Abstract

Antimicrobial resistance (AMR) genes are widely disseminated on plasmids. Therefore, interventions aimed at blocking plasmid uptake and transfer may curb the spread of AMR. Previous studies have used CRISPR-Cas-based technology to remove plasmids encoding AMR genes from target bacteria, using either phage- or plasmid-based delivery vehicles that typically have narrow host ranges. To make this technology feasible for removal of AMR plasmids from multiple members of complex microbial communities, an efficient, broad host-range delivery vehicle is needed. We engineered the broad host-range IncP1-plasmid pKJK5 to encode programmed to target an AMR gene. We demonstrate that the resulting plasmid pKJK5::csg has the ability to block the uptake of AMR plasmids and to remove resident plasmids from . Furthermore, due to its broad host range, pKJK5::csg successfully blocked AMR plasmid uptake in a range of environmental, pig- and human-associated coliform isolates, as well as in isolates of two species of . This study firmly establishes pKJK5::csg as a promising broad host-range CRISPR-Cas9 delivery tool for AMR plasmid removal, which has the potential to be applied in complex microbial communities to remove AMR genes from a broad range of bacterial species.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): AMR gene removal , antibiotic resensitization , antimicrobial resistance , broad host-range plasmids , CRISPR-Cas plasmids and plasmid curing
Funding
This study was supported by the:
  • H2020 European Research Council (Award ERC-STG-2016-714478)
    • Principle Award Recipient: EdzeR Westra
  • Bundesministerium für Bildung und Forschung (Award 01LC1904A)
    • Principle Award Recipient: UliKlümper
  • JPI-AMR HARISSA (Award MISTAR)
    • Principle Award Recipient: Stinekevan Houte
  • Lister Institute of Preventive Medicine
    • Principle Award Recipient: Stinekevan Houte
  • Biotechnology and Biological Sciences Research Council (Award BB/S017674/1)
    • Principle Award Recipient: Stinekevan Houte
  • Biotechnology and Biological Sciences Research Council (Award BB/R010781/1)
    • Principle Award Recipient: Stinekevan Houte
  • Medical Research Council (Award MR/N0137941/1)
    • Principle Award Recipient: DavidWalker-Sünderhauf
© 2023 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001334
2023-05-25
2024-05-10
Loading full text...

Full text loading...

/deliver/fulltext/micro/169/5/mic001334.html?itemId=/content/journal/micro/10.1099/mic.0.001334&mimeType=html&fmt=ahah

References

  1. Murray CJL, Ikuta KS, Sharara F, Swetschinski L, Robles Aguilar G et al. Global burden of bacterial antimicrobial resistance in 2019: a systematic analysis. The Lancet 2022; 399:629–655 [View Article]
     [Google Scholar]
  2. Murray AK, Stanton I, Gaze WH, Snape J. Dawning of a new ERA: Environmental Risk Assessment of antibiotics and their potential to select for antimicrobial resistance. Water Res 2021; 200:117233 [View Article] [PubMed]
     [Google Scholar]
  3. Partridge SR, Kwong SM, Firth N, Jensen SO. Mobile genetic elements associated with antimicrobial resistance. Clin Microbiol Rev 2018; 31:e00088-17 [View Article] [PubMed]
     [Google Scholar]
  4. Buckner MMC, Ciusa ML, Piddock LJV. Strategies to combat antimicrobial resistance: anti-plasmid and plasmid curing. FEMS Microbiol Rev 2018; 42:781–804 [View Article] [PubMed]
     [Google Scholar]
  5. Pursey E, Sünderhauf D, Gaze WH, Westra ER, van Houte S. CRISPR-Cas antimicrobials: challenges and future prospects. PLoS Pathog 2018; 14:e1006990 [View Article] [PubMed]
     [Google Scholar]
  6. Vrancianu CO, Popa LI, Bleotu C, Chifiriuc MC. Targeting plasmids to limit acquisition and transmission of antimicrobial resistance. Front Microbiol 2020; 11:761 [View Article] [PubMed]
     [Google Scholar]
  7. Bikard D, Euler CW, Jiang W, Nussenzweig PM, Goldberg GW et al. Exploiting CRISPR-Cas nucleases to produce sequence-specific antimicrobials. Nat Biotechnol 2014; 32:1146–1150 [View Article] [PubMed]
     [Google Scholar]
  8. Citorik RJ, Mimee M, Lu TK. Sequence-specific antimicrobials using efficiently delivered RNA-guided nucleases. Nat Biotechnol 2014; 32:1141–1145 [View Article] [PubMed]
     [Google Scholar]
  9. Gomaa AA, Klumpe HE, Luo ML, Selle K, Barrangou R et al. Programmable removal of bacterial strains by use of genome-targeting CRISPR-Cas systems. mBio 2014; 5:e00928–13 [View Article] [PubMed]
     [Google Scholar]
  10. Dong H, Xiang H, Mu D, Wang D, Wang T. Exploiting a conjugative CRISPR/Cas9 system to eliminate plasmid harbouring the mcr-1 gene from Escherichia coli. Int J Antimicrob Agents 2019; 53:1–8 [View Article] [PubMed]
     [Google Scholar]
  11. Wongpayak P, Meesungnoen O, Saejang S, Subsoontorn P. A highly effective and self-transmissible CRISPR antimicrobial for elimination of target plasmids without antibiotic selection. PeerJ 2021; 9:e11996 [View Article] [PubMed]
     [Google Scholar]
  12. Carattoli A. Plasmids and the spread of resistance. Int J Med Microbiol 2013; 303:298–304 [View Article] [PubMed]
     [Google Scholar]
  13. Bahl MI, Hansen LH, Goesmann A, Sørensen SJ. The multiple antibiotic resistance IncP-1 plasmid pKJK5 isolated from a soil environment is phylogenetically divergent from members of the previously established alpha, beta and delta sub-groups. Plasmid 2007; 58:31–43 [View Article] [PubMed]
     [Google Scholar]
  14. Klümper U, Riber L, Dechesne A, Sannazzarro A, Hansen LH et al. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community. ISME J 2015; 9:934–945 [View Article] [PubMed]
     [Google Scholar]
  15. Li L, Dechesne A, Madsen JS, Nesme J, Sørensen SJ et al. Plasmids persist in a microbial community by providing fitness benefit to multiple phylotypes. ISME J 2020; 14:1170–1181 [View Article] [PubMed]
     [Google Scholar]
  16. Song J, Klümper U, Riber L, Dechesne A, Smets BF et al. A converging subset of soil bacterial taxa is permissive to the IncP-1 plasmid pKJK5 across a range of soil copper contamination. FEMS Microbiol Ecol 2020; 96:fiaa200 [View Article] [PubMed]
     [Google Scholar]
  17. Qiu D, Damron FH, Mima T, Schweizer HP, Yu HD. PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria. Appl Environ Microbiol 2008; 74:7422–7426 [View Article] [PubMed]
     [Google Scholar]
  18. Lanzer M, Bujard H. Promoters largely determine the efficiency of repressor action. Proc Natl Acad Sci 1988; 85:8973–8977 [View Article] [PubMed]
     [Google Scholar]
  19. Bradley DE. Specification of the conjugative pili and surface mating systems of Pseudomonas plasmids. Microbiology 1983; 129:2545–2556 [View Article]
     [Google Scholar]
  20. Li X, Bao N, Yan Z, Yuan X-Z, Wang S-G et al. Tailoring CRISPR-Cas immunity for the degradation of antibiotic resistance genes. Synth Biol 2022 [View Article]
     [Google Scholar]
  21. Rodrigues M, McBride SW, Hullahalli K, Palmer KL, Duerkop BA. Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci. Antimicrob Agents Chemother 2019; 63:e01454–19 [View Article]
     [Google Scholar]
  22. Ruotsalainen P, Penttinen R, Mattila S, Jalasvuori M. Midbiotics: conjugative plasmids for genetic engineering of natural gut flora. Gut Microbes 2019; 10:643–653 [View Article] [PubMed]
     [Google Scholar]
  23. Wang P, He D, Li B, Guo Y, Wang W et al. Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system. J Antimicrob Chemother 2019; 74:2559–2565 [View Article] [PubMed]
     [Google Scholar]
  24. Li P, Wan P, Zhao R, Chen J, Li X et al. Targeted elimination of blaNDM-5 gene in Escherichia coli by conjugative CRISPR-Cas9 system. IDR 2022; 15:1707–1716 [View Article]
     [Google Scholar]
  25. Hamilton TA, Pellegrino GM, Therrien JA, Ham DT, Bartlett PC et al. Efficient inter-species conjugative transfer of a CRISPR nuclease for targeted bacterial killing. Nat Commun 2019; 10:4544 [View Article] [PubMed]
     [Google Scholar]
  26. Crits-Christoph A, Hallowell HA, Koutouvalis K, Suez J. Good microbes, bad genes? The dissemination of antimicrobial resistance in the human microbiome. Gut Microbes 2022; 14:2055944 [View Article] [PubMed]
     [Google Scholar]
  27. United Nations Frontiers 2017: Emerging Issues of Environmental Concern. UNEP - UN Environment Programme; 2017 http://www.unep.org/resources/frontiers-2017-emerging-issues-environmental-concern accessed 10 September 2021
  28. Dimitriu T, Matthews AC, Buckling A. Increased copy number couples the evolution of plasmid horizontal transmission and plasmid-encoded antibiotic resistance. Proc Natl Acad Sci 2021; 118:e2107818118 [View Article]
     [Google Scholar]
  29. Walker-Sünderhauf D, Klümper U, Gaze WH, Westra ER, van Houte S. Interspecific competition can drive the loss of conjugative plasmids from a focal species in a microbial community. Microbiology 2022 [View Article]
     [Google Scholar]
  30. Cho S, Choe D, Lee E, Kim SC, Palsson B et al. High-level dCas9 expression induces abnormal cell morphology in Escherichia coli. ACS Synth Biol 2018; 7:1085–1094 [View Article] [PubMed]
     [Google Scholar]
  31. Jiang Y, Qian F, Yang J, Liu Y, Dong F et al. CRISPR-Cpf1 assisted genome editing of Corynebacterium glutamicum. Nat Commun 2017; 8: [View Article]
     [Google Scholar]
  32. Zhang J, Zong W, Hong W, Zhang Z-T, Wang Y. Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production. Metabolic Engineering 2018; 47:49–59 [View Article]
     [Google Scholar]
  33. Mamontov V, Martynov A, Morozova N, Bukatin A, Staroverov DB et al. Persistence of plasmids targeted by CRISPR interference in bacterial populations. Proc Natl Acad Sci 2022; 119:e2114905119 [View Article]
     [Google Scholar]
  34. Mahendra C, Christie KA, Osuna BA, Pinilla-Redondo R, Kleinstiver BP et al. Author correction: broad-spectrum anti-CRISPR proteins facilitate horizontal gene transfer. Nat Microbiol 2020; 5:872 [View Article] [PubMed]
     [Google Scholar]
  35. Benchling Benchling · Better tools, faster research; 2015 https://benchling.com accessed 11 September 2017
  36. Puigbò P, Guzmán E, Romeu A, Garcia-Vallvé S. OPTIMIZER: a web server for optimizing the codon usage of DNA sequences. Nucleic Acids Res 2007; 35:W126–31 [View Article] [PubMed]
     [Google Scholar]
  37. Nakamura Y, Gojobori T, Ikemura T. Codon usage tabulated from international DNA sequence databases: status for the year 2000. Nucleic Acids Res 2000; 28:292 [View Article] [PubMed]
     [Google Scholar]
  38. Gautheret D, Lambert A. Direct RNA motif definition and identification from multiple sequence alignments using secondary structure profiles. J Mol Biol 2001; 313:1003–1011 [View Article] [PubMed]
     [Google Scholar]
  39. 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 [View Article]
     [Google Scholar]
  40. Briner AE, Donohoue PD, Gomaa AA, Selle K, Slorach EM et al. Guide RNA functional modules direct Cas9 activity and orthogonality. Mol Cell 2014; 56:333–339 [View Article] [PubMed]
     [Google Scholar]
  41. Lee DJ, Bingle LEH, Heurlier K, Pallen MJ, Penn CW et al. Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains. BMC Microbiol 2009; 9:252 [View Article] [PubMed]
     [Google Scholar]
  42. Ferrières L, Hémery G, Nham T, Guérout AM, Mazel D et al. Silent mischief: bacteriophage Mu insertions contaminate products of Escherichia coli random mutagenesis performed using suicidal transposon delivery plasmids mobilized by broad-host-range RP4 conjugative machinery. J Bacteriol 2010; 192:6418–6427 [View Article] [PubMed]
     [Google Scholar]
  43. Leonard AFC, Zhang L, Balfour AJ, Garside R, Hawkey PM et al. Exposure to and colonisation by antibiotic-resistant E. coli in UK coastal water users: environmental surveillance, exposure assessment, and epidemiological study (Beach Bum Survey). Environ Int 2018; 114:326–333 [View Article] [PubMed]
     [Google Scholar]
  44. Schroth MN, Cho JJ, Green SK, Kominos SD. Epidemiology of Pseudomonas aeruginosa in agricultural areas. J Med Microbiol 2018; 67:1191–1201 [View Article] [PubMed]
     [Google Scholar]
  45. Rainey PB, Bailey MJ. Physical and genetic map of the Pseudomonas fluorescens SBW25 chromosome. Mol Microbiol 1996; 19:521–533 [View Article] [PubMed]
     [Google Scholar]
  46. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012; 337:816–821 [View Article] [PubMed]
     [Google Scholar]
  47. Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green fluorescent protein (GFP). Gene 1996; 173:33–38 [View Article] [PubMed]
     [Google Scholar]
  48. Martínez-García E, Calles B, Arévalo-Rodríguez M, de Lorenzo V. pBAM1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. BMC Microbiol 2011; 11:38 [View Article] [PubMed]
     [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001334
Loading
Removal of AMR plasmids using a mobile, broad host-range CRISPR-Cas9 delivery tool
Microbiology 169, 001334 (2023); https://doi.org/10.1099/mic.0.001334
/content/journal/micro/10.1099/mic.0.001334
/content/journal/micro/10.1099/mic.0.001334
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Supplementary material 2

EXCEL

Most cited Most Cited RSS feed

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