Plasmids persist in a microbial community by providing fitness benefit to multiple phylotypes

Abstract

The current epidemic of antibiotic resistance has been facilitated by the wide and rapid horizontal dissemination of antibiotic resistance genes (ARGs) in microbial communities. Indeed, ARGs are often located on plasmids, which can efficiently shuttle genes across diverse taxa. While the existence conditions of plasmids have been extensively studied in a few model bacterial populations, their fate in complex bacterial communities is poorly understood. Here, we coupled plasmid transfer assays with serial growth experiments to investigate the persistence of the broad-host-range IncP-1 plasmid pKJK5 in microbial communities derived from a sewage treatment plant. The cultivation conditions combined different nutrient and oxygen levels, and were non-selective and non-conducive for liquid-phase conjugal transfer. Following initial transfer, the plasmid persisted in almost all conditions during a 10-day serial growth experiment (equivalent to 60 generations), with a transient transconjugant incidence up to 30%. By combining cell enumeration and sorting with amplicon sequencing, we mapped plasmid fitness effects across taxa of the microbial community. Unexpected plasmid fitness benefits were observed in multiple phylotypes of Aeromonas, Enterobacteriaceae, and Pseudomonas, which resulted in community-level plasmid persistence. We demonstrate, for the first time, that plasmid fitness effects across community members can be estimated in high-throughput without prior isolation. By gaining a fitness benefit when carrying plasmids, members within complex microbial communities might have a hitherto unrecognised potential to maintain plasmids for long-term community-wide access.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Transconjugant density (%) detected by FACS in the initial filter mating samples (0 day) and in the daily samples through 10-day serial growth experiments (1–10 days).
Fig. 2: Diversity and absolute abundance of the dominant phylotypes in samples from initial filter mating and serial growth experiments.
Fig. 3: Persistent genera or phylotypes at the end of the serial growth experiment.
Fig. 4: Richness of permissive phylotypes relative to all recipient phylotypes over time.
Fig. 5: Plasmid effect of permissive phylotypes during the serial growth experiments.

References

  1. 1.

    Soucy SM, Huang J, Gogarten JP. Horizontal gene transfer: building the web of life. Nat Rev Genet. 2015;16:472–82.

  2. 2.

    Holmes AH, Moore LSP, Sundsfjord A, Steinbakk M, Regmi S, Karkey A, et al. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet. 2016;387:176–87.

  3. 3.

    Crofts TS, Gasparrini AJ, Dantas G. Next-generation approaches to understand and combat the antibiotic resistome. Nat Rev Microbiol. 2017;15:422–34.

  4. 4.

    Merlin C, Bonot S, Courtois S, Block JC. Persistence and dissemination of the multiple-antibiotic-resistance plasmid pB10 in the microbial communities of wastewater sludge microcosms. Water Res. 2011;45:2897–905.

  5. 5.

    Cairns J, Ruokolainen L, Hultman J, Tamminen M, Virta M, Hiltunen T. Ecology determines how low antibiotic concentration impacts community composition and horizontal transfer of resistance genes. Commun Biol. 2018;1–8. https://doi.org/10.1038/s42003-018-0041-7.

  6. 6.

    Ronda C, Chen SP, Cabral V, Yaung SJ, Wang HH. Metagenomic engineering of the mammalian gut microbiome in situ. Nat Methods. 2019;16:167–70.

  7. 7.

    Bergstrom CT, Lipsitch M, Levin BR. Natural selection, infectious transfer and the existence conditions for bacterial plasmids. Genetics. 2000;155:1505–19.

  8. 8.

    Levin BR, Stewart FM. The population biology of bacterial plasmids: a priori conditions for the existence of mobilizable nonconjugative factors. Genetics. 1980;94:425–43.

  9. 9.

    Harrison E, Brockhurst MA. Plasmid-mediated horizontal gene transfer is a coevolutionary process. Trends Microbiol. 2012;20:262–7.

  10. 10.

    Pinto UM, Pappas KM, Winans SC. The ABCs of plasmid replication and segregation. Nat Rev Microbiol. 2012;10:755–65.

  11. 11.

    San Millan A, MacLean RC. Fitness costs of plasmids: a limit to plasmid transmission. Microbiol Spectr. 2017;5:1–12.

  12. 12.

    Harrison E, Guymer D, Spiers AJ, Paterson S, Brockhurst MA. Parallel compensatory evolution stabilizes plasmids across the parasitism-mutualism continuum. Curr Biol. 2015;25:2034–9.

  13. 13.

    Dionisio F, Conceicao IC, Marques AC, Fernandes L, Gordo I. The evolution of a conjugative plasmid and its ability to increase bacterial fitness. Biol Lett. 2005;1:250–2.

  14. 14.

    Loftie-Eaton W, Bashford K, Quinn H, Dong K, Millstein J, Hunter S, et al. Compensatory mutations improve general permissiveness to antibiotic resistance plasmids. Nat Ecol Evol. 2017;1:1354–63.

  15. 15.

    Lundquist PD, Levin BR. Transitory derepression and the maintenance of conjugative plasmids. Genetics. 1986;113:483–97.

  16. 16.

    Bahl MI, Hansen LH, Sørensen SJ. Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5 in bacterial populations. FEMS Microbiol Lett. 2007;266:250–6.

  17. 17.

    Lopatkin AJ, Meredith HR, Srimani JK, Pfeiffer C, Durrett R, You L. Persistence and reversal of plasmid-mediated antibiotic resistance. Nat Commun. 2017;8:1689.

  18. 18.

    Hall JPJ, Harrison E, Lilley AK, Paterson S, Spiers AJ, Brockhurst MA. Environmentally co-occurring mercury resistance plasmids are genetically and phenotypically diverse and confer variable context-dependent fitness effects. Environ Microbiol. 2015;17:5008–22.

  19. 19.

    Heuer H, Ebers J, Weinert N, Smalla K. Variation in permissiveness for broad-host-range plasmids among genetically indistinguishable isolates of Dickeya sp. from a small field plot. FEMS Microbiol Ecol. 2010;73:190–6.

  20. 20.

    Porse A, Schønning K, Munck C, Sommer MOA. Survival and evolution of a large multidrug resistance plasmid in new clinical bacterial hosts. Mol Biol Evol. 2016;33:2860–73.

  21. 21.

    Heuer H, Fox RE, Top EM. Frequent conjugative transfer accelerates adaptation of a broad-host-range plasmid to an unfavorable Pseudomonas putida host. FEMS Microbiol Ecol. 2007;59:738–48.

  22. 22.

    De Gelder L, Ponciano JM, Joyce P, Top EM. Stability of a promiscuous plasmid in different hosts: No guarantee for a long-term relationship. Microbiology. 2007;153:452–63.

  23. 23.

    Hall JPJ, Wood AJ, Harrison E, Brockhurst MA. Source–sink plasmid transfer dynamics maintain gene mobility in soil bacterial communities. Proc Natl Acad Sci. 2016;113:8260–5.

  24. 24.

    Klümper 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 J. 2015;9:934–45.

  25. 25.

    Li L, Dechesne A, He Z, Madsen JS, Nesme J, Sørensen SJ, et al. Estimating the transfer range of plasmids encoding antimicrobial resistance in a wastewater treatment plant microbial community. Environ Sci Technol Lett. 2018;5:260–5.

  26. 26.

    Jacquiod S, Brejnrod A, Morberg SM, Abu Al-Soud W, Sørensen SJ, Riber L. Deciphering conjugative plasmid permissiveness in wastewater microbiomes. Mol Ecol. 2017;26:3556–71.

  27. 27.

    Gstalder ME, Faelen M, Mine N, Top EM, Mergeay M, Couturier M. Replication functions of new broad host range plasmids isolated from polluted soils. Res Microbiol. 2003;154:499–509.

  28. 28.

    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 α, β, and δ sub-groups. Plasmid. 2007;58:31–43.

  29. 29.

    Klümper U, Dechesne A, Smets BF. Protocol for evaluating the permissiveness of bacterial communities toward conjugal plasmids by quantification and isolation of transconjugants. In: McGenity TJ, Timmis KN, Balbina N, editors. Hydrocarbon and lipid microbiology protocols: genetic, genomic and system analyses of communities. Berlin Heidelberg: Springer; 2014. p. 275–88.

  30. 30.

    OECD. Test No. 303: simulation test – aerobic sewage treatment. In: OECD guidelines for the testing of chemicals, section 3. France, Paris: OECD Publishing; 2013. p. 50.

  31. 31.

    Yu Y, Lee C, Kim J, Hwang S. Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng. 2005;89:670–9.

  32. 32.

    Sundberg C, Al-Soud WA, Larsson M, Alm E, Yekta SS, Svensson BH, et al. 454 pyrosequencing analyses of bacterial and archaeal richness in 21 full-scale biogas digesters. FEMS Microbiol Ecol. 2013;85:612–26.

  33. 33.

    Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: high-resolution sample inference from Illumina amplicon data. Nat Methods. 2016;13:581–3.

  34. 34.

    Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, et al. The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools. Nucleic Acids Res. 2013;41:590–6.

  35. 35.

    Schliep KP. Phangorn: phylogenetic analysis in R. Bioinformatics. 2011;27:592–3.

  36. 36.

    Wickham H. ggplot2—elegant graphics for data analysis. 2nd ed. New York, NY: Springer; 2016.

  37. 37.

    Oksanen J, Blanchet FG, Friendly M, Kindt R, Legendre P, McGlinn D, et al. vegan: community ecology package. R package version 2.5-4. 2019. https://CRANR-project.org/package=vegan.

  38. 38.

    Janda JM, Abbott SL. The genus aeromonas: taxonomy, pathogenicity, and infection. Clin Microbiol Rev. 2010;23:35–73.

  39. 39.

    Bradley DE. Determination of pili by conjugative bacterial drug resistance plasmids of incompatibility groups B, C, H, J, K, M, V, and X. J Bacteriol. 1980;141:828–37.

  40. 40.

    Bradley DE. Characteristics and function of thick and thin conjugative pili determined by transfer-derepressed plasmids of incompatibility groups I1, I2, I5, B, K and Z. Microbiology. 1984;130:1489–502.

  41. 41.

    Sota M, Top EM. Host-specific factors determine the persistence of IncP-1 plasmids. World J Microbiol Biotechnol. 2008;24:1951–4.

  42. 42.

    Bottery MJ, Wood AJ, Brockhurst MA. Temporal dynamics of bacteria-plasmid coevolution under antibiotic selection. ISME J. 2018;13:559–62.

  43. 43.

    Enne VI, Bennett PM, Livermore DM, Hall LMC. Enhancement of host fitness by the sul2-coding plasmid p9123 in the absence of selective pressure. J Antimicrob Chemother. 2004;53:958–63.

  44. 44.

    Zhang T, Zhang X-X, Ye L. Plasmid metagenome reveals high levels of antibiotic resistance genes and mobile genetic elements in activated sludge. PLoS ONE 2011;6:e26041.

  45. 45.

    Schlüter A, Szczepanowski R, Pühler A, Top EM. Genomics of IncP-1 antibiotic resistance plasmids isolated from wastewater treatment plants provides evidence for a widely accessible drug resistance gene pool. FEMS Microbiol Rev. 2007;31:449–77.

  46. 46.

    Bahl MI, Burmølle M, Meisner A, Hansen LH, Sørensen SJ. All IncP-1 plasmid subgroups, including the novel ε subgroup, are prevalent in the influent of a Danish wastewater treatment plant. Plasmid. 2009;62:134–9.

  47. 47.

    Vogwill T, Maclean RC. The genetic basis of the fitness costs of antimicrobial resistance: a meta-analysis approach. Evol Appl. 2015;8:284–95.

  48. 48.

    Kottara A, Hall JPJ, Harrison E, Brockhurst MA. Variable plasmid fitness effects and mobile genetic element dynamics across Pseudomonas species. FEMS Microbiol Ecol. 2018;94:1–7.

  49. 49.

    Olaniran AO, Nzimande SBT, Mkize NG. Antimicrobial resistance and virulence signatures of Listeria and Aeromonas species recovered from treated wastewater effluent and receiving surface water in Durban, South Africa. BMC Microbiol. 2015;15:234.

  50. 50.

    Popowska M. Occurrence and variety of β-lactamase genes among Aeromonas spp. isolated from urban wastewater treatment plant. Front Microbiol. 2017;8:1–12.

  51. 51.

    Hedges RW, Smith S, Brazil G. Resistance plasmids of aeromonads. J Gen Microbiol. 1985;131:2091–5.

  52. 52.

    Piotrowska M, Popowska M. The prevalence of antibiotic resistance genes among aeromonas species in aquatic environments. Ann Microbiol. 2014;921–34.

  53. 53.

    Ye L, Zhang T. Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing. Environ Sci Technol. 2011;45:7173–9.

Download references

Acknowledgements

This work was supported by a Joint Programming Initiative-Antimicrobial Resistance grant (JPI-AMR; DARWIN project #7044-00004B) to BFS; and a fellowship under the H.C. Ørsted Postdoc programme co-funded by the Marie Skłodowska-Curie Actions awarded to LL.

Author information

Correspondence to Barth F. Smets.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Li, L., Dechesne, A., Madsen, J.S. et al. Plasmids persist in a microbial community by providing fitness benefit to multiple phylotypes. ISME J (2020). https://doi.org/10.1038/s41396-020-0596-4

Download citation