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that after plasmid acquisition, coevolution of the plasmid and its host rapidly result in compensation
for the costs of the plasmid. Several studies demonstrated that after a short number of generations
of plasmid-host coevolution the relative cost of the plasmid decreased (Loftie-Eaton et al.
2017)(Harrison et al. 2015)(San Millan et al. 2014)(De Gelder et al. 2008) as represented in Fig. 1
by the red vertical arrows.
Interestingly, two recent studies (Loftie-Eaton et al. 2017) (Starikova et al. 2013), one of which
was conducted in the laboratory of Dr. Eva Top, suggested that plasmid-host coevolution can do
more than just compensate for the cost of the plasmid, but rather make the host addicted to the
plasmid (from here on briefly referred to as plasmid addicted): after coevolution the host with
plasmid showed a higher fitness than the same strain without the plasmid. Fitness is measured in
competition assays as the relative difference in offspring (here increase in cell numbers) of two
competitors after a specific period of growth in the same environment. Even more interestingly, a
recent experiment (Fig. 2) performed in the lab aiming at gaining insight into this phenomenon
showed that bacteria evolved with plasmid are now fitter than their counterparts who were evolved
for the same period of time without plasmid. We have called these control strains control-evolved
hosts, or here briefly control strains. These control strains were evolved from the same ancestor
as the plasmid-bearing evolved populations, in the same environment, expect for the absence of
the antibiotic that selected for the plasmid. The cost of the plasmid was tested by competing the
coevolved plasmid-host (HevPev in Fig. 2) against the control strains (Ac, Bc, Cc in Fig. 2). This
experiment is novel, because all studies on plasmid addiction prior to this compared the coevolved
plasmid host with a derivative cured from its plasmid. This means that the competitions were
between cell types that had both evolved with a plasmid. In contrast, our recent competition
experiments compared cell types that had evolved with and without a plasmid. In this recent
experiment, the fitness of plasmid-containing cells was calculated relative to the plasmid-free
counterpart after 24h of coculture, in which the coevolved plasmid-hosts and control strains were
initially mixed together. All the plasmid-containing strains had higher fitness than the strains
evolved without the plasmid in the 24h assay (Fig. 2).
Fig. 2: Relative fitness of various strains of hosts evolved with a plasmid present (HevPev)
competed against control cells evolved without a plasmid present (Ac, Bc, Cc). Unpublished data
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These results support the hypothesis that through coevolution, plasmids can become beneficial
for the host. If this is the case and common in nature it would explain why pathogens carrying
large and supposedly costly multidrug resistance plasmids remain in circulation when not
selected for. That in turns sheds light on why many multidrug resistant plasmids are currently
found in pathogens.
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Below is a sample of a protocol adjusted to use the newly acquired departmental RoToR HDA
robot which can be used to obtain much higher replicate numbers on certain steps. This will allow
for much larger sample sizes and therefore more accurate statistical analysis.
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Expected Results
We will gather data based upon the replica plating results. The relative fitness values will be
calculated based on the numbers obtained from the above protocol, which describe the colony
counts of plasmid-bearing vs plasmid free bacteria. The relative fitness is then calculated upon the
calculations used by the Barrick Lab.
The relative fitness (W) of strains A relative to strand B is the ratio of their Malthusian
parameters (MA and MB) over the course of a representative growth cycle.
N = cell number.
PC = plate count on TA.
DF = dilution factor of all transfers combined.
i and f are the initial and final time points.
W = log(MA) / log(MB)
Plasmid cost is then calculated as c =1-W. Thus if W < 1 (typical for a costly plasmid), then cost
c will be large than 0. If the case of plasmid addiction, W > 1 and the cost becomes negative
which indicates a benefit of the plasmid for the host.
We expect to observe a higher number of colonies growing on antibiotic plates after evolution than
before evolution, and potentially a higher fitness in plasmid-containing bacteria than their plasmid-
free equivalents.
If we see a higher number of colonies growing on antibiotic plates, and calculate a plasmid cost
where W<1 and c is negative, we will have data that supports our hypothesis that coevolution
plasmids and bacteria can lead to plasmid-addiction. Once plasmid-addiction is present in a system,
it would explain why plasmids persist even in the absence of selection.
Anticipated Difficulties and Possible Solutions
Given the fact that the method used for this project are routinely performed in the Top lab, and that
some of the strains used are already evolved, we do not foresee technical difficulties with regards
to the strains other than potential contamination issues. These can be detected, and potentially
resolved using adding spacer wells in our assays as mentioned in the methods section. The place
where we will likely have the most trouble is using the RoToR HDA robot, where we may find
some issues that we are not aware of, but will likely be relatively easily solved. Sometimes there
are issues with measuring small differences between the plasmid-containing and plasmid-free cells,
and can require several trials before one can measure a difference. We hope that the high throughput
of the robot will give a better resolution and reproducibility to the assays.
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Citations
- Centers for Disease Control, Antibiotic resistance threats in the United States, 2013, U.S
Department of Health and Human Services
- Gelder, L. D., Williams, J. J., Ponciano, J. M., Sota, M., & Top, E. M. (2008). Adaptive
Plasmid Evolution Results in Host-Range Expansion of a Broad-Host-Range
Plasmid. Genetics, 178(4), 2179-2190. doi:10.1534/genetics.107.084475
- Harrison, E., & Brockhurst, M. A. (2012). Plasmid-mediated horizontal gene transfer is a
coevolutionary process. Trends in Microbiology, 20(6), 262-267.
doi:10.1016/j.tim.2012.04.003
- Harrison, E., Guymer, D., Spiers, A. J., Paterson, S. & Brockhurst, M. A. (2015). Parallel
compensatory evolution stabilizes plasmids across the parasitism-mutualism continuum.
Current Biology, 25, 20342039.
- Loftie-Eaton, W., Suzuki, H., Bashford, K., Heuer, H., Stragier, P., Vos, P. D., Top, E. M.
(2015). Draft Genome Sequence of Pseudomonas sp. nov. H2. Genome
Announcements, 3(2). doi:10.1128/genomea.00241-15.
- Loftie-Eaton, W. (2016). Evolutionary paths that expand plasmid host-range: implications
for spread of antibiotic resistance. Molecular Biology Evolution, 33, 885897.
- Loftie-Eaton W, Bashford K, Quinn H, Dong K, Millstein J, Hunter S, Thomason MK,
Merrikh H, Ponciano JM, Top EM. (2017). Compensatory mutations improve general
permissiveness to antibiotic resistance plasmids. Natural Ecolology and Evolution, Sep
1(9), 1354-1363.
- Porse, A., Schnning, K., Munck, C., & Sommer, M. O. (2016). Survival and Evolution
of a Large Multidrug Resistance Plasmid in New Clinical Bacterial Hosts. Molecular
Biology and Evolution, 33(11), 2860-2873. doi:10.1093/molbev/msw163.
- Millan, A. S., Pea-Miller, R., Toll-Riera, M., Halbert, Z. V., Mclean, A. R., Cooper, B.
S., & Maclean, R. C. (2014). Positive selection and compensatory adaptation interact to
stabilize non-transmissible plasmids. Nature Communications, 5, 5208.
doi:10.1038/ncomms6208.
- Stalder, T., Rogers, L. M., Renfrow, C., Yano, H., Smith, Z., & Top, E. M. (2017).
Emerging patterns of plasmid-host coevolution that stabilize antibiotic
resistance. Scientific Reports, 7(1). doi:10.1038/s41598-017-04662-0.
- Starikova, I., Al-Haroni, M., Werner, G., Roberts, A. P., Sorum, V., Nielsen, K. M., &
Johnsen, P. J. (2013). Fitness costs of various mobile genetic elements in Enterococcus
faecium and Enterococcus faecalis. Journal of Antimicrobial Chemotherapy, 68(12),
2755-2765. doi:10.1093/jac/dkt270
- Antimicrobial resistance: global report on surveillance. (World Health Organization,
2014).
- Znd, P & Lebek, Gerhard. (1980). Generation time-prolonging R plasmids: Correlation
between increases in the generation time of Escherichia coli caused by R plasmids and
their molecular size. Plasmid. 3. 65-9. 10.1016/S0147-619X(80)90034-7.
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Budget
*Prices have been rounded up to the higher decimal to account for shipping costs. Test tubes and
96-well plates are reusable and already owned by the laboratory of Dr. Eva Top.