With great dosage comes greater

resistance
Evolution, in general, happens over a timescale of millions of years. However, in lower and simpler
organisms such as bacteria, we could observe evolution occurring within shorter spans of time, thanks to
their rapid reproduction and shorter life-span. To study such an evolution, we subject E. coli to antibiotic
environments of varying degree which act as agents of selection for antibiotic resistance. We then assay
our final cultures with antibiotics of different concentrations and confirm if evolution has taken place
yielding antibiotic-resistant bacteria.

1. A brief note on Evolution

The theory of Evolution, as proposed by Darwin, is the creation of new species formed out of descent with
modification. Traits are passed on from one generation to another, with slight modifications that might arise
due to mutation or other factors. These modifications/ changes accumulate over time in a population, and
the populations change over time.
When a population in a niche is subjected to a change in environment, a few individuals in the population
with a trait that could tolerate such change will survive. If such individuals were a fraction of the initial
population, their share in the next generation changes and subsequently increases in the population over
many generations. This process is called natural selection. For example, in the population of finches in the
Daphne Islands in the Galapagos, the finches with smaller beaks could not survive the dry spell of 1977,
which left smaller seeds scarce. This change led to the increase in the population of finches with larger
beaks which could eat large seeds in subsequent generations, thus demonstrating natural selection, and in
such a short period.
Evolution of new species or new traits in complex organisms takes a very long time. This timescale is often
a limitation when faced with the problem of experimental verification of evolution. It takes about 10-20
Million years for evolutionary changes in fish to be observed. However, in smaller or lower organisms, due
to smaller generations, evolution can be seen in action.

2. Bacterial evolution and antibiotic resistance

Bacteria being prokaryotes, are much simpler organisms and have much smaller lifespans and replication
times, and hence could evolve at a much higher pace than complex organisms. For example, E. coli has a
replication time of 20 minutes under optimum conditions. This means hundreds of generations of E. coli
fall within one day. Given this fact, it is clear that within one generation of humans, bacteria go through
millions of generations and evolve at a faster pace. This is evident from the fact that antibiotic strains of
bacteria evolve within a person’s lifetime and often heavier doses of medication are prescribed between a
person’s birth and death. Bacteria hence have the upper hand in the evolutionary race when compared with
humans (letting us know who the “big” guy is!). At this evolutionary pace, variation in a given population
is certain and abundant.
Antibiotics are chemicals that kill or stop the growth of bacteria. Within a few decades from the first usage
antibiotics, bacteria growing resistant became a menace. Overuse, abuse, and inappropriate prescription
were common causes for the emergence of resistant bacteria. These factors drew the existing bacterial
populations to populations that could resist a higher dosage of antibiotics.
In a given population, some individuals can be resistant to an antibiotic or up to a concentration of the
antibiotic. The lowest concentration of the antibiotic that completely inhibits the visible growth of the
bacteria is called the Minimum Inhibitory Concentration (MIC).
In a bacterial population exposed to a concentration of an antibiotic, a few individuals resistant to that
concentration will survive. Due to variations in the population, some individuals may resist concentrations
even beyond the (earlier) MIC. These individuals survive and reproduce, and their numbers increase in
successive generations. This leads to a shift in the MIC, which is a sign of evolution.

3. Materials and Methods

LB medium was taken in two separate tubes, one for control and one for test. To prevent the growth of
other bacteria, we select kanamycin-resistant bacteria for studying and add kanamycin to our test and
control tubes. 20uL of inoculum is added to both tubes. We initiate the test with a concentration of antibiotic
(definite values of either of Norfloxacin, Ampicillin, Streptomycin, and Rifampicin).
Every 24 hours, both the tubes are sub-cultured by extracting 200uL and adding them to new test and control
tubes respectively, containing 5ml LB and 5uL kanamycin and, test containing the antibiotic solution. This
is done for a week. The concentration of antibiotic is increased every three days till it reaches MIC.
Assay
Twelve wells for test and control each were filled with nutrient. The antibiotic concentrations in the wells
ranged from MIC/50 to 10MIC with steps of 1MIC after MIC. One well had no antibiotic. The expectation
is for bacteria in the control wells to stop growth at MIC and in the test wells to continue to grow beyond
MIC. Such a shift in the MIC for the test bacteria is indicative of evolution for antibiotic resistance.
A cross test was also done with

Schematic representation of the assay.

x=MIC and shaded wells indicate
growth. Dr. Neelesh Dahanukar, BIO221
Class White Board.
 4. Results
The assays were performed, and the results are tabulated as follows:

Batch NO 6 8 9 10
I AB MIC/50 MIC 2MIC 3MIC 4MIC 5MIC MIC 7MIC MIC MIC MIC
Mo 1 Cont + - - + - - - - - - - -
Norfloxacin + + + + + + - - - - - -

Mo 2 Cont + + - - - - + - - - - -
Rifampicin + + + + + + + + + + + +

Mo 3 Cont + + + + + + + + + + + -
Ampicillin + + + + + + + + + - - -

Mo 4 Cont + - - - - - + - - - - -
Streptomycin + + + + + + + + + + + +

Batch NO 6 8 9 10
II AB MIC/50 MIC 2MIC 3MIC 4MIC 5MIC MIC 7MIC MIC MIC MIC
Tu 1 Cont + - - - - - - - - - - -
Norfloxacin + + + + + - - - - - - -

Tu 2 Cont + + + + - - - - - - - -
Rifampicin + + + + + + + + + + + +

Tu 3 Cont + + + + + + + + + + + +
Ampicillin + + + + + + + + + + + +

Tu 4 Cont + - - - - - - - - - - -
Streptomycin + + + + + + + + + + + +

Batch NO 6 8 9 10
III AB MIC/50 MIC 2MIC 3MIC 4MIC 5MIC MIC 7MIC MIC MIC MIC
We 1 Cont + - - - - - - - - - -
Norfloxacin + + + + + - - - - - - -

We 2 Cont + - + - - - - - - - - +
Rifampicin + + + + + + + + + + + +

We 3 Cont + + + + + + + + + + + +
Ampicillin + + + + + + + + + + + +

We 4 Cont + - - - - - - - - - - -
Streptomycin + + + + + + + + + + + +
Batch NO 6 8 9 10
IV AB MIC/50 MIC 2MIC 3MIC 4MIC 5MIC MIC 7MIC MIC MIC MIC
Th 1 Cont + - - - - - - - - - - -
Norfloxacin + + + + + - - - - - - -

Th 2 Cont + - - - - - - - - - - -
Rifampicin + + + + + + + + + + + +

Th 4 Cont + - - - - - - + - - - -
Streptomycin + + + + + + + + + + + +

NO 10 14 16 18
AB MIC/50 MIC 2MIC 4MIC 6MIC 8MIC MIC 12MIC MIC MIC MIC
Th 3 Cont + + + + + + + + + + + +
Ampicillin + + + + + + + + - - - -

Key
+ Growth
- No growth
Non-reliable
results

It is evident that there is a shift in MIC in all cases, and hence the E. coli bacteria have evolved for antibiotic
resistance.

5. Discussion
A shift in MIC is evident in the tests for all antibiotics except Ampicillin, which we will discuss later.
In Norfloxacin, the control consistently dies out in the lowest concentration of antibiotic (MIC/50) which
implies that the taken initial value of MIC was much higher than the actual MIC of our culture. The
Norfloxacin tests withstand up to 4MIC, at least 3MIC in most cases. There is a three-fold difference in the
tolerance to Norfloxacin between the control and the test. The bacterial population hence shifted towards
resistance to Norfloxacin over the gradual increase in selection stress.
In Rifampicin, the control cultures show resistance too, with cultures three tolerating up to MIC/50, MIC,
and 2MIC. A small population of the bacteria was hence resistant to Rifampicin. On selection, we see the
test cultures survive till 10MIC. This is a huge leap, and the population of bacteria did have many
individuals resistant to a higher concentration of Rifampicin, which was selected out in the test and hence,
showed resistance till 10MIC.
Streptomycin controls showed consistent death at MIC/50 unable to resist any amount of antibiotic.
Streptomycin tests, however, develop resistance till 10MIC. This is a much greater shift of MIC that in the
case of Norfloxacin. Thus, the cultures in our experiment did contain some individuals resistant to higher
concentrations of Streptomycin which were selected by gradually increasing the concentration during sub-
culturing.
However, our test cultures were kept in a selective pressure of only 1MIC at maximum. When the bacteria
were exposed to antibiotic during the experiment, the bacteria that are resistant to higher concentrations
survive and increase their number in successive subcultures. This implies that the fraction of population
that are adaptive to higher concentrations of antibiotic in the test are higher than in the control because they
are selected out. Thus, the growth of test cultures in higher concentrations is facilitated by even a lower
selection pressure because the fraction of resistant bacteria to higher concentrations is larger even on
application of a weak selection pressure, and the population of such resistant bacteria continues to increase
in successive generations whereas the fraction of resistant bacteria remain low in the control.
Existence and survival of resistant bacteria in a population is completely random. A very small fraction of
resistant bacteria could appear and disappear any time in a population of bacteria. Apart from the
explanation that the MIC we assumed was inaccurate (being instructional and not determined by an assay
in the lab), this randomness could also explain the fact that control bacteria could withstand a few times the
MIC in the assay. A very small fraction of the bacterial population could have a resistance which would’ve
visibly grown at higher concentrations in the assay.
The ‘non-reliable’ results could also be attributed to such randomness in the appearance of resistant bacteria
in the population, and when assayed, any small fraction could be resistant to a higher concentration by
chance. This also has to do with the fact that the culture is not uniform with resistant bacteria distributed
throughout the culture and hence extraction also does play a role.
On selective pressure of subjecting a population to a concentration of antibiotic and gradually increasing
the concentration, this randomness is eliminated, and the individuals that survive are ones with resistance
to at least the given concentration. This could contain bacteria resistant to higher concentrations as well,
and the fraction of such bacteria increases and is higher than when no selection is applied, and randomness
prevails. This leads to a ‘unidirectional’ evolution of bacteria to achieve higher resistances, which does not
happen in the case of the control, where the growth is completely random. The term ‘unidirectional’
however is only applicable in the case of our experiment where resistance is artificially selected out,
whereas in nature, often the selection is completely random and not towards any particular trait.
The Ampicillin anomaly
Ampicillin shows anomalous results with the control culture surviving way beyond MIC in every case.
This is because the MIC we took was based on instruction and wasn’t determined by an assay during the
experiment. Hence, the actual MIC could be much greater than the value it was assumed to be. A possible
explanation for the growth being observed throughout the control whereas not as much in the test could be
that exposure to the antibiotic, in some manner reduced the fitness of the test culture and hence they could
not survive beyond a certain concentration. However, due to unconstrained growth, the fitness of the control
bacteria is unchanged and could survive beyond 18MIC (assumed MIC).
Summary of the Inferences

 The test bacteria in Rifampicin, Norfloxacin and Streptomycin clearly showed a shift in the MIC,
hence validating natural selection and evolution.
 Ampicillin control withstood higher concentrations than the test, and this is due to our assumption
of the MIC without performing a test, and possibly due to the decrease in fitness/ survival
probabilities of the test due to the exposure to antibiotics.
 Our experiment also demonstrates how random traits (resistance to antibiotics) exist in a population
and are selected through some pressure.
 A small fraction of resistant bacteria that arose randomly in a population is selected under strain of
antibiotic exposure, and their numbers increase in successive generations, which does not take place
in the case of control cultures where the population’s composition is random across generations
and the fractions of resistant bacteria would remain constant.

6. References

1. https://www.thoughtco.com/descent-with-modification-129878
2. The antibiotic resistance crisis: part 1: causes and threats. P T. 2015;40(4):277-83.
3. https://www.sciencedirect.com/topics/immunology-and-microbiology/minimum-inhibitory-
concentration
4. Experimental Evolution of Antibiotic Resistance in Bacteria. Amy C. Krist and Sasha A. Showsh.
The American Biology Teacher Feb 2007 : Vol. 69, Issue 2, pg(s) 94- 97