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TITLE: Evolution of Bacteria towards Plasmid Addiction

Hypothesis:
Coevolution of bacteria and plasmids that confer antibiotic resistance can cause a new bacterial
host to become plasmid-addicted. These plasmid-addicted bacteria can have a greater fitness than
the same strain evolved without a plasmid.
Introduction and Rationale for the Project
The World Health Organization and Centers for Disease Control and Prevention (CDC) agree that
one of the largest global medical threats we currently face is the growing number of antibiotic
resistance genes in pathogenic bacteria (World Helath Organization 2014)(Center for Disease
Control 2013). Antibiotic resistance is an especially pressing issue because modern treatments for
infectious diseases often require the application of antibiotics, and with the advent of multidrug
resistant bacterial strains, some of these treatments may become impossible to perform. The spread
of antibiotic resistance is broadly shaped by the horizontal transfer of genes allowing bacteria to
rapidly acquire new resistances to antibiotics. Plasmids play a key role in horizontal transfer of
antibiotic resistance. They are mobile genetic elements that may confer multiple antibiotic
resistance traits to their host and allow the spread of antibiotic resistance genes across distantly
related bacteria. Many plasmids are self-transmissible, which means they carry all the genes that
code for the machinery which allows their transfer from one bacterium to another.
Plasmids can have benefits to their host bacteria when they
confer traits that are useful in the bacterias environment.
However, it has long been thought that unused plasmids are a
burden (or costly) to their host. This is especially true when a
plasmid is newly acquired by a host (De Gelder et al. 2008). Due
to this fitness cost, the number of generations a plasmid carrying
strain can undergo in a specific period of time is lower than that
of its plasmid-free counterpart (see b in Fig. 1) (Znd et al. 1980).
Therefore, in an environment which does not select for the
plasmid, plasmid-free bacteria would be expected to take over
the population, wiping out plasmid-containing cells. If we
consider this, and that antibiotic resistance gene carrying
plasmids may not always be under positive selection, then
antibiotic resistance containing plasmids should be lost and
eventually head towards extinction. This is not the case and
Fig. 1: Each arrow represents a
plasmids seem to persist in populations even in the absence of
generation. Plasmid loss occurs at
certain rates (a), the plasmid cost selection (Stalder et al. 2017). Pathogens that carry self-
lowers the host fitness/growth rate transmissible plasmids encoding multiple resistance genes are
(b), and plasmids can conjugate more and more commonly isolated from pathogens. This
(c). Bacteria evolved towards phenomenon has been named the Plasmid Paradox (Harrison et
improved plasmid persistence may
al. 2012) and represents a knowledge gap regarding factors
have changes in any of these
parameters (dashed red arrows). which allow for the continued persistence and conjugation of
plasmids in bacterial populations. One possible explanation is

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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.

Methods and Scientific Approach:

We are proposing to test our hypothesis by measuring the fitness costs of three plasmids coevolved
in three different hosts. We will use a similar approach as described above where each plasmid-
containing strain will be challenged by the evolved control strain in a competition assay. A
competition assay is a test where two populations of bacteria are grown from known initial densities
in the same culture. After a set period, the mixed culture is then plated, and the densities of the
final populations can be compared to the initial densities to assign values of relative fitness to each
strain (Fig. 3.).
We will evaluate relative cost of plasmids
after coevolution by performing
competition assays to elucidate the
differences in fitness of bacteria evolved
across 600 generations with and without
plasmids. Each evolution for a given
strain is performed within the same
growth environment, whether or not the
host has a plasmid, with the exception of
a selection agent for the plasmid in those
Fig. 3 Representation of the competition assay technique. evolutions with the plasmid present. A
host evolved with plasmid will be
competed against a host evolved without plasmid. All three costs will be compared to the original
costs which will be identified by a competition assay between the ancestral strain with a plasmid
and the ancestral strain without. The Top Lab already has several strains pre-evolved to 600
generations in at least two of the three configurations needed for testing, which eliminates some
difficulty in this project. The strains are from previous projects in the Top Lab, or those of our
affiliates and include:
- E. coli MG1655 with and without pALTS28 evolved for 600 generation
- Klebsiella pneumoniae with and without CI 08 ( (Porse et al. 2016) for 600 generation
- Pseudomonas sp. H2 with and without RP4 (Loftie-Eaton et al. 2015) for600 generation
The plasmid pALTS28 was isolated from biosolids during an earlier project studying the transfer
of antibiotic resistance gene plasmids to pathogens from bacteria found in wastewater treatment
biosolids used as fertilizers. It is already well characterized both in terms genomes and fitness. This
plasmid is multidrug resistant and was captured in pathogenic bacteria and are thus relevant to the
aims of this proposal.

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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.

Competition assay (proposed protocol)

- Each strain to be competed will be grown from the freezer in the appropriate liquid culture
media for 16h at 30C. If needed, the culture media will be supplemented with the appropriate
antibiotic for plasmid selection.
- Bacterial cells will be harvested by centrifugation (10000g, 3min, RT) of 1.5mL of the cell
culture in 2mL microcentrifuge sterile tubes to remove the spent media and antibiotics.
- Cells will be resuspended in 1.5mL of sterile PBS, and an equal volume of each competitor
(100L + 100L) will be mixed in a 2mL microcentrifuge sterile tube.
- The mixture will be used to inoculate a fresh liquid culture media by diluting the inoculum
1024 times and incubated in a plate shaking incubator at 30C for 24h. We are proposing to run
in parallel 6 replicates for each competition assay in a total volume of 100L using 96-well
plates.
The use of multichannel pipettes can substantially increase the speed and reproducibility of the
experimental manipulations. This will allow us to test more conditions (e.g. competitor ratios,
strains and replicates) bringing more strength to the analysis. To control for potential cross-
contamination between wells each set of assays will be separated by a row of sterile liquid
culture media.
-This step will be repeated two times every 24h (when the mixture grown to saturation),
enabling more generations to grow and compete.
- We will determinate the proportion of plasmid-containing and plasmid-free cells in the initial
mixture 0h and in the mixture after 24h, 48h and 72h by spreading on the appropriate solid
culture media a known volume (50L) of several serial-dilutions of each inoculated broth (10 fold
serial-dilutions in sterile PBS).
This will allow us to determine the total bacterial concentration (cfu/ml) and the proportion of
plasmid-containing and plasmid-free cells in the initial mixture of the assay (T0). For this later
measure, we are proposing to replicate 384 individual colonies grew on the agar plates (after 24h
of growth at 30C) on solid culture media with and without the antibiotic selecting for the
plasmid. To achieve such a high-throughput scale we are proposing to use the new RoToR HDA
robot recently acquired by the department of biological sciences. After 24h of growth at 30C the
number of colonies growing on each culture media will be recorded.

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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.

MA = NA(f) / NA(i) = PCA(f) * DF / PCA(i)

MB = NB(f) / NB(i) = PCB(f) * DF / PCB(i)

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

Product Quantity or Volume Price*

Culture media (LB) 500g $ 50.00
Agar for solidifying culture media 100g $ 25.00
Phosphate buffer saline (PBS) 1L $ 20.00
Large Petri Dishes 100 $ 90.00
Plate for SBS-format PlusPlate with lids 200 $ 330.00
Tetracycline 5g $ 40.00
Spreaders 100 $ 50.00
Inoculating loops 250 $ 80.00
Microcentrifuge tubes 500 $ 30.00
Poster print for a presentation 1 $ 30.00
Multichannel pipette (8 channels) 1 $ 755.00
TOTAL $ 1,500.00

*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.