University of Toronto Journal of Scientific Innovation Volume 1: Issue 1

Determining the Minimum Inhibitory Concentration (MIC) of Different

Antibiotics Against a Drug-Resistant Bacteria
Eleni Pitsiniaga, Year 2
Dr. Ruby May A. Sullan
University of Toronto Scarborough

Introduction

Antibiotics play a crucial role in many fields including pharmacy, agriculture, and

surgery. Their broad application arises from the properties that their molecules have in terms of

killing bacteria or preventing bacterial growth. Although the invention of antibiotics significantly

increased the quality of healthcare, there is currently a rapid emergence of antibiotic-resistant

bacteria in all organisms on a global scale (Ventola, 2015). To help mitigate this issue, the

Mechano-microbiology lab at the University of Toronto Scarborough is developing

nanomaterial-based antimicrobial platforms which can be loaded with antibiotics to more

effectively deliver and target sites of infections (i.e., biofilm-related infections) which can

prevent off-target effects. To achieve this goal, different antibiotics were tested against a model

bacterium, a drug-resistant laboratory strain of E. coli (Tetracycline-resistant AR3110).

Specifically, the Minimum Inhibitory Concentrations (MIC) of each drug against E. coli AR3110

were determined. MIC is a parameter used in assessing an organism's susceptibility to

antibiotics, defined as the lowest concentration required to prevent observable bacterial growth

following incubation (Andrews, 2001). MIC is therefore a useful parameter in assessing the

efficacy of antimicrobials.

This report explores the effect of three different protein synthesis inhibitor antibiotics

(Chloramphenicol (CHL), Kanamycin (KAN), and Erythromycin (ERY)) on the growth of E.

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coli AR3110. Bacteria replicate through the process of protein synthesis, which is facilitated by

ribosomal proteins. Any disruptions in the production of these proteins will prevent protein

synthesis and ultimately inhibit bacterial growth (Champney & Burdine, 1995). These antibiotics

were chosen as each has a different inhibition mechanism. Chloramphenicol inhibits protein

synthesis in the prokaryotic cell by its ability to bind to the 50S ribosomal subunit of the 70S

ribosome. Chloramphenicol is largely recognized as an antibacterial agent of choice for the

treatment of various bacterial infections, despite the concerns about toxicity and restricted usage

in developed countries (Xaplanteri, et al. 2003). Kanamycin binds the 30S ribosomal subunit,

causing severe mistranslation and preventing protein synthesis translocation. It is a wide-ranged

aminoglycoside antibiotic that is commonly used on humans and animals. It has significant

importance, as listed in the World Health Organization (WHO) list of Essential Medicines

(Bashir & Cho, 2016). Erythromycin effectively terminates cell translocation by adhering to the

23S ribosomal RNA in the 50S ribosomal subunit. This inhibits polypeptide chain elongation,

while also targeting the cellular structure of the ribosome, and destroying bacteria by blocking

this structure formation (Champney & Miller, 2002). The precise mechanisms by which this

antibiotic inhibits protein synthesis is not fully understood (Champney & Burdine, 1995). By

monitoring the growth of E. coli in the presence of increasing concentrations of these antibiotics,

their Minimum Inhibitory Concentration (MIC) against E. coli AR3110 is obtained.

Materials and Methods

Preparing E. coli for overnight incubation

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A single colony of bacterial strain E. coli AR3110 with mCherry was first inoculated

using 5 mL of Luria-Bertani medium (LB broth) and 16 μL of Tetracycline. The bacteria cells

were incubated overnight (~20 hours) at 37 ºC and 250 rpm.

Serial Dilution of Antibiotic and Optical Density (OD) measurement

Using the stock solution of the antibiotics (30 µg/mL CHL, 30 µg/mL KAN, 50 µg/mL

ERY), twelve different concentrations were prepared in Phosphate-buffered saline (PBS) via

serial dilution. For each antibiotic, twelve epi-tubes were filled with 500 µL of LB broth.

Following the serial dilution, the incubated bacteria were diluted in a cuvette with LB broth to

obtain an absorbance reading. Bacteria were then diluted in a fresh falcon tube with LB to

prepare a 5 mL stock solution with an absorbance of 0.02 a.u.

Loading of bacterial solutions to microplate and microplating

A 96-well microplate was loaded from rows B to F and columns 1 to 12. Rows B, C, D

each represent separate replicates, meaning each trial had three replicates. These rows were

prepared from pipetting 100 µL of the diluted bacteria with 100 µL of the diluted antibiotics. In

each trial, column 1 indicated the most concentrated antibiotic solution, while 12 had no

antibiotic (0 µg/mL). Row E consisted of 200 µL of LB broth in the first three columns, while

row F included 100 µL of the diluted antibiotics, much like rows B, C, and D, however instead

of adding 100 µL of bacteria, 100 µL of LB was added so to serve as a control. The microplate

was then run in a microplate reader at 37 ºC, 600 nm for 20 hours, while absorbance readings

were recorded every 30 minutes. Absorbance and standard deviation readings from the relevant

rows were averaged using Microsoft Excel. Using these average absorbances, growth curves for

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each antibiotic were plotted and Igor Pro analysis software (WaveMetrics Inc.) was used to

normalize the data.

Results

Chloramphenicol (CHL)

A) B)

C)

Figure 1. Effect of Chloramphenicol (CHL) on the growth of E. coli AR3110. (A) Optical density (OD) at 600 nm as a function
of time. (B) Maximum average absorbance (i.e., OD at 20 hours from 1A) at different CHL concentrations. (C) Normalized data
from (B) fitted with a modified Gompertz equation (red line) for MIC calculation. (A-C) Each point is the average of 3 separate
trials, with the standard deviation as the error bars.

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Kanamycin (KAN)

A) B) 0.8

Maximum Average Absorbance

0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
-0.1 0.1 1 10 100 1000
Log Kanamycin Concentration (µg/mL)

C)

Figure 2. Effect of Kanamycin (KAN) on the growth E. coli AR3110 (A) Optical density (OD) of E. coli solution monitored at
600 nm as a function of time. (B) Maximum average absorbance (i.e., OD at 20 hours from 1A) at different KAN concentration.
(C) Normalized data from (B) fitted with a modified Gompertz equation (red line) for MIC calculation. (A-C) Each point is the
average of 3 separate trials, with the standard deviation as the error bars.

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Erythromycin (ERY)

A) B)

Figure 3. Growth curves for the first (3A) second trial (3B) using ERY. Each series indicates the growth of bacteria under the
influence of the antibiotic concentrations, according to the legend. The error bars represent the average standard deviation of the
sample.

Chloramphenicol (CHL)

Figure 1A shows the average absorbance (n=3) at 600 nm plotted against time (hours).

Figure 1A demonstrates that as the concentration of chloramphenicol is increased, there is an

observable decrease in the growth rate, as well as maximal growth of E. coli AR3110.

The maximum absorbance at 20 hrs, representing the maximum growth of the bacteria,

was then plotted with respect to Chloramphenicol concentration (Figure 1B). This plot

demonstrates that at lower antibiotic concentrations (<1μg/mL), E. coli growth is barely affected

by the presence of the antibiotic. However, as the concentration of Chloramphenicol increased (>

1μg/mL), E. coli growth decreased until no more growth was observed, starting at 8 μg/mL of

antibiotic. In order to determine the MIC of Chloramphenicol against E. coli AR3110, the data

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from Figure 1B was normalized then fitted with the modified Gompertz equation, Equation (1)

(Figure 1C) (Lambert & Pearson, 2000):

𝐵(𝑥−𝑀)
𝑓(𝑥) = 𝐴 + 𝐶𝑒 −𝑒 (1)

where A is the lower asymptote of the normalized absorbance, f(x); B is a slope

parameter; C is the distance between the upper and lower asymptote, and M is the log

concentration of the inflection point. Once the data is fitted (red line, Figure 1C), MIC was

calculated from the fitted parameters using Equation (2) (Lambert & Pearson, 2000):

𝑀𝐼𝐶 = 10𝑀 + 1/𝐵 (2)

Igor Pro software was used to fit the data in Figure 1C, giving the following fitted

parameters and MIC:

(1): A = 0.0046 ± 0.118; B = 5.97 ± 3.22; C = 1.21 ± 0.15; M = 0.52 ± 0.085

(2): MIC= 10^((0.51569 +1/5.9696)) = 4.87 ± 1.39 μg/mL

The calculated MIC value for Chloramphenicol is very similar to what was reported for

this antibiotic against other strains of E. coli, 4 μg/mL – 8 μg/mL. (Andrews, 2001; Kidsley, et

al. 2018).

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Kanamycin (KAN)

The data shows that growth inhibition of E. coli by Kanamycin is also concentration-

dependent, as shown in Figure 1B, similar to what was observed with Chloramphenicol. As with

Chloramphenicol, the MIC of Kanamycin against E. coli AR3110 was also obtained from fitting

the normalized data shown in Figure 2C: MIC = 55.33 ± 15.18 µg/mL.

The calculated MIC falls within the reported MIC values of KAN against several strains

of E. coli, 2–64 µg/mL (Cheng, et al. 2010).

Erythromycin (ERY)

For the first trial, the highest concentration was 64 µg/mL. Figure 3A shows that even

though the bacteria grew less in the presence of 64 µg/mL Erythromycin, it wasn’t enough to

completely prevent growth. Hence, in the second trial (Figure 3B) there was an increase in the

antibiotic concentration to 256 µg/mL. However, this still did not completely prevent E. coli

growth, rendering MIC determination impossible. More trials are needed, with much higher

concentrations of Erythromycin. Though this data was not able to estimate the MIC for this

antibiotic, it suggests that E. coli AR3110, although engineered to be only resistant to

Tetracycline, may be resistant to Erythromycin.

Discussion

The goal of this paper was to calculate the MIC for Chloramphenicol, Kanamycin, and

Erythromycin against Tetracycline-resistant E. coli AR3110. According to Andrews (2001) and

Kidsley, et al. (2018), the expected MIC for Chloramphenicol ranges from 4 µg/mL to 8 µg/mL.

After the fourth trial, the calculated MIC of this antibiotic was 4.87 ± 1.39 μg/mL. The

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calculated MIC agrees with the range suggested by the literature; however, it is noted that it falls

slightly outside this range when looking at the lower boundary. For Kanamycin, the MIC value

obtained was 55.33 ± 15.18 µg/mL, which falls within the expected range (2–64 µg/mL),

although this value is typically 32 µg/mL (Cheng, et al. 2010). The associated error for this MIC

value falls slightly out of range when looking at the higher boundary. However, for

Erythromycin, there was not enough data to calculate the MIC. E. coli AR3110 seems resistant to

this antibiotic, i.e., MIC >256 μg/mL. For other strains of E. coli, MIC for Erythromycin ranges

from 16 µg/mL to >1,024 µg/mL (Nguyen, et al. 2009). These data, although still requiring

further experiments, seem to suggest that Chloramphenicol is the most effective antibiotic

against E. coli AR3110, followed by Kanamycin. This strain of E. coli appears more resistant to

Erythromycin.

Once these values are successfully determined and confirmed, the MIC values can be

used to inform nanomaterial-based antimicrobial platform development in the group, especially

platforms requiring antibiotic loading. MIC may be used as a starting point in optimizing the

antibiotic concentration required to block observable bacterial growth. This will allow for higher

efficiency in the targeted delivery of antibiotics to sites of infection, reducing off-target effects.

Future studies may use these MIC to inform studies working on antibiotic resistance, especially

in the realm of targeted delivery. Continued research and discussion are needed to mitigate the

rapid emergence of resistant bacteria, which pose a significant threat to the health of all

organisms and the quality of human life.

The high error associated with Figure 1A was the biggest challenge in this investigation.

Indeed, this error contributed to a repeat of Chloramphenicol trials a total of four times. The

results of the first three trials are not included. In the future, practicing the aseptic technique may

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lead to less error, as well as practicing the serial dilution technique. Perhaps error occurred

during the loading of the bacterial suspension and antibiotic solution on the microplate (some

wells may have been loaded with more or less antibiotics or bacteria). As Chloramphenicol was

the first antibiotic tested, the minimized error in Kanamycin and Erythromycin may be attributed

to the fact that the Chloramphenicol trials informed subsequent trials. There is a downward trend

of error as more trials are conducted (error bars kept steadily decreasing (Figure 1 vs. Figures 2

and 3). More trials are necessary to precisely obtain the MIC of Kanamycin against E. coli

AR3110. Generally, more trials should be conducted to validate results.

A weakness of the investigation was that optical density was used as a measure of

bacterial growth. This may not always be an accurate metric, as optical density measurement

does not distinguish between alive and dead bacteria. It may be beneficial to quantify bacterial

populations through serial dilution plating upon the creation of growth curves. Future studies

may also look at the effect of initial bacterial concentration on the MIC. Additionally, future

experiments should look at Erythromycin’s effect on the resistance of the bacterial strain in

concentrations between > 256 µg/mL and >1,024 µg/mL, to successfully obtain an MIC reading.

Lastly, the synergistic or antagonistic properties of all these antibiotics against E. coli AR3110

may be explored in future experiments. For example, combining two of the three antibiotics may

result in lower MIC values if synergistic effects are observed or higher MIC values for

antagonistic effects. Ultimately, these MIC values will be used in future work on nanomaterial-

based delivery systems to increase targeting efficiency to sites of bacterial infection and

minimize off-target effects.

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Conclusion

This paper investigated the specific MIC of Chloramphenicol (CHL), Kanamycin (KAN),

and Erythromycin (ERY) against Tetracycline-resistant E. coli AR3110 strain. The calculated

MIC was: CΗL = 4.87 ± 1.39 µg/mL and KAN = 55.33 ± 15.18 µg/mL, while ERY was

undetermined due to the apparent resistance of AR3110 to this antibiotic. Future studies may

look at confirming the MIC of Chloramphenicol, as well as repeating more trials for Kanamycin.

New trials for Erythromycin should be conducted where concentrations between >256 µg/mL

and >1,024 µg/mL may be tested to ensure that accurate MIC values have been obtained. These

values are imperative to calculate and confirm to better inform the development of future

nanomaterial-based antibiotic delivery systems.

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