Potentiation of curing by a broad-host-range self-transmissible vector for displacing resistance plasmids to tackle AMR

Autoři: Alessandro Lazdins aff001;  Anand Prakash Maurya aff001;  Claire E. Miller aff001;  Muhammad Kamruzzaman aff003;  Shuting Liu aff001;  Elton R. Stephens aff001;  Georgina S. Lloyd aff001;  Mona Haratianfar aff001;  Melissa Chamberlain aff001;  Anthony S. Haines aff001;  Jan-Ulrich Kreft aff001;  Mark. A. Webber aff002;  Jonathan Iredell aff003;  Christopher M. Thomas aff001
Působiště autorů: Institute of Microbiology & Infection and School of Biosciences, University of Birmingham, Edgbaston, Birmingham, England, United Kingdom aff001;  Institute of Microbiology and Infection, College of Medical and Dental Sciences, University of Birmingham, Edgbaston, Birmingham, England, United Kingdom aff002;  University of Sydney, Centre for Infectious Disease & Microbiology, Westmead Institute of Medical Research, Westmead, New South Wales, Australia aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: https://doi.org/10.1371/journal.pone.0225202


Plasmids are potent vehicles for spread of antibiotic resistance genes in bacterial populations and often persist in the absence of selection due to efficient maintenance mechanisms. We previously constructed non-conjugative high copy number plasmid vectors that efficiently displace stable plasmids from enteric bacteria in a laboratory context by blocking their replication and neutralising their addiction systems. Here we assess a low copy number broad-host-range self-transmissible IncP-1 plasmid as a vector for such curing cassettes to displace IncF and IncK plasmids. The wild type plasmid carrying the curing cassette displaces target plasmids poorly but derivatives with deletions near the IncP-1 replication origin that elevate copy number about two-fold are efficient. Verification of this in mini IncP-1 plasmids showed that elevated copy number was not sufficient and that the parB gene, korB, that is central to its partitioning and gene control system, also needs to be included. The resulting vector can displace target plasmids from a laboratory population without selection and demonstrated activity in a mouse model although spread is less efficient and requires additional selection pressure.

Klíčová slova:

Antibiotic resistance – Antibiotics – DNA-binding proteins – Plasmid construction – Plasmids – Polymerase chain reaction – Sucrose – Tetracyclines


1. O’Neill J, Audi H, Knox J, Hall W, McDonnell A, Seshadri A, et al., Review on Antimicrobial Resistance. https://amr-review.org/Publications.html. 2016.

2. Thomas CM, Nielsen K. Mechanisms of and barriers to horizontal gene transfer between bacteria. Nature Reviews Microbiology 2005; 3: 711–721. doi: 10.1038/nrmicro1234 16138099

3. Lopatkin AJ, Meredith HR, Srimani JK, Pfeiffer C, Durrett R, You L. Persistence and reversal of plasmid-mediated antibiotic resistance. Nature Communications 2017; 8: 1689–1698. doi: 10.1038/s41467-017-01532-1 29162798

4. Dicks LMT, Mikkelsen LS, Brandsborg E, Marcotte H. Clostridium difficile, the Difficult “Kloster” Fuelled by Antibiotics. Current Microbiology 2019; 76: 774–782. doi: 10.1007/s00284-018-1543-8 30084095

5. Trevors JT. Plasmid curing in bacteria. FEMS Microbiol Rev 1986; 32: 149–157.

6. Thomas CM. Paradigms of plasmid organization. Molecular Microbiology 2000; 37: 485–491. doi: 10.1046/j.1365-2958.2000.02006.x 10931342

7. Kroll J, Klinter S, Schneider C, Voss I, Steinbuchel A. Plasmid addiction systems: perspectives and applications in biotechnology. Microbial Biotechnology 2010; 3: 634–657. doi: 10.1111/j.1751-7915.2010.00170.x 21255361

8. Petersen J. Phylogeny and compatibility: plasmid classification in the genomics era. Archives of Microbiology 2011; 193: 313–321. doi: 10.1007/s00203-011-0686-9 21374058

9. Kamruzzaman M, Shoma S, Thomas C, Partridge S, Iredell J. Plasmid interference for curing antibiotic resistance plasmids in vivo. PLoS ONE 2017; 12: e0172913. doi: 10.1371/journal.pone.0172913 28245276

10. Villa L, Garcia-Fernandez A, Fortini D, Carattoli A. Replicon sequence typing of IncF plasmids carrying virulence and resistance determinants. Journal of Antimicrobial Chemotherapy 2010; 65: 2518–2529. doi: 10.1093/jac/dkq347 20935300

11. Koraimann G. Spread and persistance of virulence and antibiotic resistance genes: a ride on the F plasmid conjugation module. EcoSal Plus 2018; doi: 10.1128/ecosalplus.ESP-0003-2018 30022749

12. Hale L, Lazos O, Haines AS, Thomas CM. An efficient stress-free strategy to displace stable bacterial plasmids. BioTechniques 2010; 48: 223–228. doi: 10.2144/000113366 20359304

13. Burland V, Shao Y, Perna NT, Plunkett G, Sofia HJ, Blattner FR. The complete DNA sequence and analysis of the large virulence plasmid of Escherichia coli O157: H7. Nucleic Acids Research 1998; 26: 4196–4204. doi: 10.1093/nar/26.18.4196 9722640

14. Makino K, Ishil K, Yasunaga T, Hattori M, Yokoyama K, Yutsudo CH, et al. Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157: H7 derived from Sakai outbreak. DNA Research 1998; 5: 1–9. doi: 10.1093/dnares/5.1.1 9628576

15. Zienkiewicz M, Kern-Zdanowicz I, Gobliewski M, Zylinksa J, Mieczkowski P, Gniadkowska M, et al. Mosaic structure of p1658/97, a 125-kilobase plasmid harboring an active amplicon with the extended-spectrum beta-lactamase gene blaSHV-5. Antimicrobial Agents and Chemotherapy 2007; 51: 1164–1171. doi: 10.1128/AAC.00772-06 17220406

16. Haneda T., Okada N., Nakazawa T., Kawakami T. & Danbara H. Complete DNA sequence and comparative analysis of the 50-kilobase virulence plasmid of Salmonella enterica serovar Choleraesuis. Infection and Immunity 2001; 69: 2612–2620. doi: 10.1128/IAI.69.4.2612-2620.2001 11254626

17. Yanisch-Perron C, Vieira J, Messing J. Improved M13 phage cloning vectors and host strains–nucleotide-sequences of the M13mp18 and pUC19 vectors. Gene 1985; 33: 103–119. doi: 10.1016/0378-1119(85)90120-9 2985470

18. Freire-Martin I, Thomas CM, Laing E, AbuOun M, La Ragione RM, Woodward MJ. Curing vector for IncI1 Plasmids and its use to provide evidence for a metabolic burden of IncI1 CTX-M-1 plasmid pIFM3971 on Klebsiella pneumonia. Journal of Medical Microbiology 2016; 65: 611–618. doi: 10.1099/jmm.0.000271 27166141

19. Pansegrau W, Lanka E, Barth PT, Figurski DH, Guiney DG, Haas D, et al. Complete nucleotide sequence of Birmingham IncPα plasmids. Journal of Molecular Biology 1994; 239: 623–663. doi: 10.1006/jmbi.1994.1404 8014987

20. Lowbury EJL, Kidson A, Lilly HA, Ayliffe GA, Jones RJ. Sensitivity of Pseudomonas aeruginosa to antibiotics: emergence of strains highly resistant to carbenicillin. Lancet 1969; 2: 448–452. doi: 10.1016/s0140-6736(69)90163-9 4183901

21. Ingram LC, Richmond MH, Sykes RB. Molecular characterisation of the R factors implicated in the carbenicillin resistance of a sequence of Pseudomonas aeruginosa strains isolated from burns. Antimicrobial Agents and Chemotherapy 1973; 3: 279–288. doi: 10.1128/aac.3.2.279 4208284

22. Kluemper U, Riber L, Dechesne A, Sannazzarro A, Hansen LH, Sørensen SJ, et al. Broad host range plasmids can invade an unexpectedly diverse fraction of a soil bacterial community. ISME Journal 2015; 9: 934–945. doi: 10.1038/ismej.2014.191 25333461

23. Grinsted J, Bennett PM, Richmond MH. A restriction enzyme map of R-plasmid RP1. Plasmid 1977; 1: 34–37. doi: 10.1016/0147-619x(77)90006-3 618183

24. Haines AS. Studies on the active Partitioning system of the IncP-1 plasmids RK2 and R751. PhD Thesis, University of Birmingham, UK. 2001.

25. Bhattacharyya A, Figurski DH. A small protein-protein interaction domain common to KlcB and global regulators KorA and TrbA of promiscuous IncP plasmids. Journal of Molecular Biology 2001; 310: 51–67. doi: 10.1006/jmbi.2001.4729 11419936

26. Thomas CM, Ibbotson JP, Wang N, Smith CA, Tipping R, Loader N. Gene regulation on broad host range plasmid RK2: identification of three novel operons whose transcription is repressed by both KorA and KorC. Nucleic Acids Research 1988; 16: 5345–5359. doi: 10.1093/nar/16.12.5345 2838814

27. Kittell BL, Helinksi DR. Iteron inhibition of plasmid RK2 replication in vitro–evidence for intermolecular coupling of replication origins as a mechanism for RK2 replication control. Proceedings of the National Academy of Sciences USA 1991; 88: 1389–1393.

28. Thomas CM, Cross MA, Hussain AAK, Smith CA. Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2. EMBO Journal 1984; 3: 57–63. 6323170

29. Larsen MH, Figurski DH. Structure, expression, and regulation of the kilC operon of promiscuous IncP alpha plasmids. Journal of Bacteriology 1994; 176: 5022–5032. doi: 10.1128/jb.176.16.5022-5032.1994 7519596

30. Hedges RW, Datta N. fi-R-factors giving chloramphenicol resistance. Nature, London 1971; 234: 220–221. doi: 10.1038/234220a0

31. Praszkier J, Wei T, Siemering K, Pittard J. Comparative analysis of the replication regions of IncB, IncK and IncZ plasmids. Journal Bacteriology 1991; 173: 2393–2397.

32. Thomas CM, Hussain AAK. The korB gene of broad host range plasmid RK2 is a major copy number control element which may act together with trfB by limiting trfA expression. EMBO Journal 1984; 3: 1513–1519. 6378626

33. Kahn M, Kolter R, Thomas C, Figurski D, Meyer R, Remaut E, et al. Plasmid Cloning vehicles derived from plasmids ColE1, F, R6K and RK2. Methods in Enzymology 1979; 68: 268–280. doi: 10.1016/0076-6879(79)68019-9 232215

34. Williams DR, Macartney DP, Thomas CM. The partitioning activity of the RK2 central control region requires only incC, korB and KorB binding site OB3 but other binding sites for destabilising complexes in the absence of OB3. Microbiology 1998; 144: 3369–3378. doi: 10.1099/00221287-144-12-3369 9884229

35. Fox RE, Zhong X, Krone SM, Top EM. Spatial structure and nutrients promote invasion of IncP-1 plasmids in bacterial populations. ISME Journal 2008; 2: 1024–1039. doi: 10.1038/ismej.2008.53 18528415

36. Woodford N, Carattoli A, Karisik E, Underwood A, Ellington MJ, Livermore DM. Complete nucleotide sequences of plasmids pEK204, pEK499, and pEK516, encoding CTX-M enzymes in three major Escherichia coli lineages from the United Kingdom, all belonging to the international O25:H4-ST131 clone. Antimicrobial Agents and Chemotherapy 2009; 53: 4472–4482. doi: 10.1128/AAC.00688-09 19687243

37. Cottell JL, Webber MA, Coldham NG, Taylor DL, Cerdeno-Tarraga AM, Hauser H, et al. Complete Sequence and Molecular Epidemiology of IncK Epidemic Plasmid Encoding bla(CTX-M-14) Emerging Infectious Diseases 2011; 17: 645–652.

38. Wassenaar TM. Insights from 100 years of research with probiotic E. coli. European Journal of Microbiology and Immunology 2016; 6: 147–161. doi: 10.1556/1886.2016.00029 27766164

39. Czaplewski L, Bax R, Clokie M, Dawson M, Fairhead H, Fischetti VA, et al. Alternatives to antibiotics–a pipeline portfolio review. Lancet Infectious Diseases 2016; 16: 239–251. doi: 10.1016/S1473-3099(15)00466-1 26795692

40. Stalker DM, Thomas CM, Helinski DR. Nucleotide sequence of the region of the origin of replication of the broad host range plasmid RK2. Molecular and General Genetics 1981; 181: 8–12. doi: 10.1007/bf00338997 6261086

41. Norberg P, Bergström M, Jethava V, Dubhashi D, Hermansson M. The IncP-1 plasmid backbone adapts to different host bacterial species and evolves through homologous recombination. Nature Communications 2011; 2: 268. doi: 10.1038/ncomms1267 21468020

42. Bechhofer DH, Kornacki JA, Firschein WB, Figurski DH. Gene control in broad host range plasmid RK2: expression, polypeptide production and multiple regulatory functions of korB. Proceedings of the National Academy of Sciences USA 1986; USA 83: 394–98.

43. Bignell C, Thomas CM. The bacterial ParA-ParB partitioning proteins. Journal of Biotechnology 2001; 91: 1–34. doi: 10.1016/s0168-1656(01)00293-0 11522360

44. Fisher GLM, Pastrana CL, Higman VA, Koh A, Taylor JA, Butterer A, et al. The structural basis for dynamic DNA binding and bridging interactions which condense the bacterial centromere. E-Life 2017; 6: e28086. doi: 10.7554/eLife.28086 29244022

45. Song D, Rodrigues K, Graham TGW, Loparo JJ. A network of cis and trans interactions is required for ParB spreading. Nucleic Acids Research 2017; 45: 7106–7117. doi: 10.1093/nar/gkx271 28407103

46. Kawalek A, Bartosik AA, Glabski K, Jagura-Burdzy G. Pseudomonas aeruginosa partitioning protein ParB acts as a nucleoid-associated protein binding to multiple copies of a parS-related motif. Nucleic Acids Research 2018; 46: 4592–4606. doi: 10.1093/nar/gky257 29648658

47. Carattoli A. Plasmids and the spread of resistance. International Journal of Medical Microbiology 2013; 303: 298–304. doi: 10.1016/j.ijmm.2013.02.001 23499304

48. Bradley DE. Specification of the Conjugative Pili and Surface Mating Systems of Pseudomonas Plasmids. Journal of General Microbiology 1983. 129: 2545–2556. doi: 10.1099/00221287-129-8-2545 6138393

49. Licht TR, Struve C, Christensen BB, Poulsen RL, Molin S, Krogfelt KA. Evidence of increased spread and establishment of plasmid RP4 in the intestine under sub-inhibitory tetracycline concentrations. FEMS Microbiology and Ecology 2003. 44: 217–223.

50. Bahl MI, Hansen LH, Licht TR, Sorensen SJ. Conjugative transfer facilitates stable maintenance of IncP-1 plasmid pKJK5 in Escherichia coli cells colonizing the gastrointestinal tract of the germfree rat. Applied and Environmental Microbiology 2007; 73: 341–343. doi: 10.1128/AEM.01971-06 17085707

51. Lauritsen I, Porse A, Sommer MOA, Norholm MHH. A versatile one-step CRISPR-Cas9 based approach to plasmid-curing. Microbial Cell Factories 2017; 16: 135–144. doi: 10.1186/s12934-017-0748-z 28764701

52. Hanahan D. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology 1983; 166: 557–580. doi: 10.1016/s0022-2836(83)80284-8 6345791

53. Bachmann B. Pedigrees of Some Mutant Strains of Escherichia coli K-12. Bacterioogyl Reviews 1972; 36: 525–557.

54. Hershfield V, Boyer HW, Yanofsky C, Lovett MA, Helinski DR. Plasmid ColEl as a molecular vehicle for cloning and amplification of DNA. Proceedings of the National Academy of Sciences USA 1974; 71: 3455–3459.

55. Boyer HW, Roulland-Dussoix D. A complementation analysis of restriction and modification of DNA in Escherichia coli. Journal of Molercular Biology 1969; 41: 459–472.

56. Reister M, Hoffmeier K, Krezdorn N, Rotter B, Liang C, Rund S, et al. Complete genome sequence of the Gram-negative probiotic Escherichia coli strain Nissle 1917. Journal of Biotechnology 2014; 187: 106–107. doi: 10.1016/j.jbiotec.2014.07.442 25093936

57. Vallejo AN, Pogulis RJ, Pease LR. In vitro synthesis of novel genes: mutagenesis and recombination by PCR. Genome Research 1994; 4: S123–S130.

58. Birnboim HC, Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Research 1979; 7: 1513–1523. doi: 10.1093/nar/7.6.1513 388356

59. Sanger F, Nicklen S, Coulson AR. 1977. DNA sequencing with chain terminating inhibitors. Proceedings of the National Academy of Sciences USA 74: 5463–5467.

60. Horton RM, Cai ZL, Ho SN, Pease LR. Gene splicing by overlap extension: tailor-made genes using the polymerase chain reaction. Biotechniques 1990; 8: 528–535. 2357375

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