Durham, Mallard, Perez 1

Experimental Evolution of Bacterial Resistance to Glyphosate

Durham L, Mallard M, Perez I

Dept. of Biological Sciences, University of Idaho

Abstract

The extensive use of glyphosate in agriculture has led to a population resistance in

weeds and plants. This is becoming a problem because glyphosate is used, most commonly in

Roundup, as an herbicide. However, if glyphosate is no longer having any effect on weeds, a

solution will have to be discovered to control the evolutionary growth of not only weeds, but

harmful bacteria as well. In our experiment, we used Proteus mirabilis in a large plastic tub to

simulate a petri dish of glyphosate to test the hypothesis that the bacteria will evolve and grow

through the glyphosate. We found that using different concentrations of glyphosate, the Proteus

mirabilis was resistant to only the 1% concentration, leaving the 1.5%, 2%, and 2.5%

concentrations untouched, although we found other organisms had grown in the higher

concentrations.

Background

This experiment tests the evolution of bacteria when faced with chemicals that should

potentially kill the bacteria. A fundamental problem in biology is the evolutionary transition from

single cells to multicellular life forms (Szathmáry and Smith). Although there is much theory,

there are few empirical studies (Rainey and Rainey).

Glyphosate accounts for 11% of the world’s herbicide sales. The extensive use of

glyphosate in agriculture has led to a population of resistant weeds and plants. One study

showed how following 15 years of successful use, glyphosate failed to control a population of

widespread grass weed rigid ryegrass in Australia...The presence of glyphosate resistance in a

major weed species indicates a need for changes in glyphosate used patterns (Powles et al.).

Proteus mirabilis is a gram-negative bacterium known for its ability to swarm across surfaces in

a bulls’-eye pattern. This organism is found in the urinary tract, especially in patients with
 Durham, Mallard, Perez 2

catheters (Duke). Proteus mirabilis is also well-known in clinical laboratories as the species that

swarms across agar surfaces. It is useful because Proteus mirabilis overtakes other species

present in the process” (Duke). This is why Proteus mirabilis is the optimum bacteria for us to

use in our experiment. When P. mirabilis is added to an agar surface, the bacteria grow in

place

for a time, then differentiate into swarmer cells and move forward as a population (first noted by

Hauser in 1885). DNA replication without septation occurs during swarm cell formation,

resulting in very long, polyploid cells. After a period, the bacteria differentiate into swarmer

cells. These bacteria move across many media surfaces in a repeated process of swarming and

consolidation, resulting in a bull's-eye pattern (Duke).

We expect after inoculating the first section of agar, containing no glyphosate, with our

bacteria, the bacteria will grow just fine until it hits the barrier containing the 2% glyphosate agar

mix. At this point, the bacteria will adapt to be able to continue growing in the 2% solution. It

should hit another barrier at the 3% glyphosate-agar mix boundary, until once again it adapts to

grow, and should continue this process up to and through the 5% solution.

Materials and Methods

Proteus mirabilis was used as the test subject because of its’ antibiotic resistant properties and

ability to undergo morphological change (Flores-Mireles et al.). Using a streak plate of the P.

mirabilis, the bacteria was grown in broth. TSA broth was used as a growth medium after

consultation with Dr.’s Ederer and Hartzell. However, LB agar was used as well.

We used different concentrations of glyphosate in TSA broth to test P. mirabilis’s initial

resistance to glyphosate. To do this, bacteria was taken from a streak plate and grown in broth

to use for spot plating. In 8, 5mL test tubes, the bacteria grew for later use. Each of the test

tubes were placed into the tube rotator for 96 hours for the bacteria to grow.

Five TSA agar plates were used at different volumes of glyphosate to create different

concentrations for spot plating. A stock solution of 10% glyphosate was diluted to a maximum
 Durham, Mallard, Perez 3

concentration of 1%. The range of glyphosate concentrations used included a 1% solution

containing 1.25 mL of glyphosate, 0.5% solution with 625µL of glyphosate, 0.25%, with 313µL,

0.1% with 125µL, and 0.0% concentration with 62.5µL glyphosate.

Once the bacteria finished growing in test tubes, serial dilutions, ranging in dilutions from

undiluted to 1:1,000,000, were spot plated. After 48 hours, the spot plates were placed in the

refrigerator to stop growth. Upon evaluation of the spot plates, 1% Glyphosate-agar solution had

no significant effect on the growth of the bacteria. We also realized that it would be impossible

to make 100x, and 1000x lethal limit concentrations of Glyphosate-agar because it would make

too much glyphosate and the agar would just be glyphosate and a small amount of agar

powder. Adding this much glyphosate could also alter the pH of the agar and prevent it from

setting into a usable gel.

For this reason, the parameters of the experiment had to be altered. Five 2%

Glyphosate-agar solutions were made by adding 25 mL of the 10% stock glyphosate to 100 mL

of LB agar. LB agar was used because it was readily available. Once the glyphosate-agar set,

spot plates were made with the serial dilution. We will allow the bacteria to grow, or not, and

evaluate from there what our new large petri dish concentrations will be. As of right now, we are

planning to use 2% through 5%.

Using a large plastic bin, tin-foil wrapped cardboard dams were constructed and placed

evenly so as to separate the Glyphosate concentrations. Using a 40X Glyphosate solution, 1%,

1.5%, 2%, and 2.5% agar solutions were made. They were poured, and, once set, a soft agar

was then poured on top to allow the bacteria to spread through as well as to allow the

Glyphosate to diffuse into the soft agar. The 0% Glyphosate was inoculated into the bacteria

once the soft agar set and observation began.

Results

The overall objective was to see how quickly P. mirabilis could evolve and grow in lethal

concentrations of glyphosate, starting in 0% glyphosate and working up to 2.5% glyphosate. We

 Durham, Mallard, Perez 4

expected the bacteria to grow into viewable colonies, but instead grew in a sheet across the

agar. From there we expect it to take 2-3 days for the colonies to completely cover the 1st agar

section, then break the barrier into the 1% glyphosate-agar. It ended up taking 4 days to cover

the 0% agar and 5 days to break through the barrier into the 1% agar solution. From there we

expected another 2-3 days for the bacteria to grow and cover the 2nd agar section, and

continue this pattern until it eventually covers the 5th agar section containing 2.5% glyphosate.

It took around 9 days total for the bacteria to cover the 1% agar solution. It slightly broke

through into the 1.5% but did not continue growing after that. These predictions were based off

of the experiment conducted at Harvard where bacteria and an antibiotic were used, which took

11 days for the bacteria to break a 1000x lethal limit of antibiotic.

Expected vs Actual Growth Rates of P. mirabilis.

Glyphosate Concentration # of Days Until Growth (Exp) # of Days Until Growth (Act)

0% 1 2

1% 3 5

1.5% 6 9

2% 9 N/A

2.5% 12 N/A
Fig 1 The bacteria took longer to grow than originally anticipated and was not able to
successfully adapt to a 1.5% concentration.

Figure 2
 Durham, Mallard, Perez 5

Fig 2 represents the adaptive daily rate of bacterial growth through increasing concentrations of
Glyphosate.

Discussion

The original hypothesis stated that the bacteria would be able to develop a resistance to the

glyphosate and continue growing into lethal concentrations. The results of the experiment

demonstrated that with P. mirabilis, this was not the case. From spot plating, it was evident that

it could survive in a 1% Glyphosate solution, which was shown in the experiment, but it was not

able to adapt to higher concentrations. Some post experimental observations that could have

improved the process include using a dye in the agar to more easily view the bacterial growth,

using video cameras to capture a time lapse of the growth, creating a better Glyphosate

gradient by tilting the agar while it was setting, and possibly continuing to observe the bacteria

past 3 weeks.
 Durham, Mallard, Perez 6

Literature Cited

Duke, Stephen O. “The History and Current Status of Glyphosate.” Pest Management Science,

vol. 74, no. 5, May 2018, pp. 1027–34. Web of Science, doi:10.1002/ps.4652.

Flores-Mireles, Ana L., et al. “Urinary Tract Infections: Epidemiology, Mechanisms of Infection

and Treatment Options.” Nature Reviews Microbiology, vol. 13, no. 5, May 2015, pp.

269–84. Crossref, doi:10.1038/nrmicro3432.

Powles, Stephen B., et al. “Evolved Resistance to Glyphosate in Rigid Ryegrass (Lolium

Rigidum) in Australia.” Weed Science, vol. 46, no. 5, 1998, pp. 604–07.

Rainey, Paul B., and Katrina Rainey. “Evolution of Cooperation and Conflict in Experimental

Bacterial Populations.” Nature, vol. 425, no. 6953, Sept. 2003, pp. 72–74.

www.nature.com, doi:10.1038/nature01906.

Szathmáry, E., and J. M. Smith. “The Major Evolutionary Transitions.” Nature, vol. 374, no.

6519, Mar. 1995, pp. 227–32. PubMed, doi:10.1038/374227a0.