Antibiotics: what quantity should we use against bacteria?

Antibiotics: what quantity should we use against bacteria?

Everywhere, while you can’t see them, micro-organisms surround us and are part of our lives. Microbiology is the science that study these microorganism such as bacteria, fungus, protist, microscopics algae or plants and plankton. Researchers study all these organisms in order to understand their metabolism, and the medical part of microbiology focus mainly on pathogen bacteria and how to eliminate them. Indeed, some bacteria are the source of infections that can be deadly for humans. This is why we use antibiotics, in order to stop the development of pathogens bacteria and kill them. But antibiotics have one major weakness: some bacteria can survive to antibiotics by developing a resistance to it, and from these survivors are formed populations of bacteria with the same resistance of antibiotics called antibioresistance. In hospital and laboratory, there is a defined concentration of antibiotics that we have to use in order to kill bacteria called the efficient concentration, but I asked myself if a lower concentration of antibiotics could stop efficiently the growth of bacteria. Indeed, a lower efficient concentration of antibiotic could prevent the apparition of antibioresistant bacteria, and could allow to use higher concentration to fight tough and dangerous infections.

My project was about the effect of gentamicin, a well known antibiotic, on Pseudomonas aeruginosa, a species of bacteria usually use to study antibioresistance. Knowing that the efficient concentration of gentamicin is 50 µg/mL, I tested 4 concentrations: 50 µg/mL, 25 µg/mL, 10 µg/mL and 5 µg/mL. I prepared samples for each concentration with a defined quantity of bacteria and liquid media to permit bacteria to growth. I measured each hour the absorbance of each sample: when the number of cells increase, the absorbance increase so we can connect this value to the growth of the population and draw a growth curve.

The growth curves I obtained: each concentration as one attributed colour.

The result of my experiment are synthesized thanks to this graphics. For each color, there is an associated concentration of gentamicin. Positive controls (in purple) are samples of bacteria and growth media without gentamicin in order to check if growth occurs in normal condition. Negative controls (in green) are samples with only growth media in order to check if there were no contamination.

In one hand, we can see that the purple, the yellow and the black ones respectively for positive controls, concentration of 10 µg/mL and of 5 µg/mL, are closed. These curves reach a plateau called the stationary phase: the population of bacteria grows to its maximum.

In the other hand, we observe that the green, the blue and the red curves, respectively corresponding to negatives controls, concentration of 25 µg/mL and 50 µg/mL, are closed: they stay at low stationary values, and except for one green curve (for which we suspect there have been contamination) we can see there is no growth.

What can we conclude from this? Well, we can say that concentration of 5 µg/mL and 10 µg/mL of gentamicin are not enough to stop the Pseudomonas aeruginosa growth. However we see that, as we already known, a concentration of 50 µg/mL stop the growth, but a concentration half smaller have the same effect: gentamicin at 25 µg/mL is as efficient as the concentration used in the everyday life.

This experiment shows that it is pertinent to think about reducing the amount of antibiotics in efficient concentration. But before that, it is important to verify if a lower concentration is really efficient to eliminate bacteria. So it would be interesting to test in other ways how a concentration of 25 µg/mL of gentamicin affects development of bacteria, by example by observing the effect of gentamicin on colonies and cells, and to test if a diminution of efficient concentrations is conceivable for other type of antibiotics against other species of bacteria.

If you want to know more!

Scientific Papers

1 - Wiegand, Irith, Kai Hilpert, and Robert EW Hancock. "Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances." Nature protocols 3.2 (2008): 163-175.

2 - Müsken, Mathias, et al. "A 96-well-plate–based optical method for the quantitative and qualitative evaluation of Pseudomonas aeruginosa biofilm formation and its application to susceptibility testing." Nature protocols 5.8 (2010): 1460-1469.

3 - Kaya, Ibrahim, et al. "Comparison of the efficiency of different antibiotic irrigation solutions in decontamination of allografts contaminated with Staphylococcus aureus." Acta orthopaedica et traumatologica turcica 47.4 (2012): 281-285.

4 - Potel, G., et al. "Impact of dosage schedule on the efficacy of gentamicin, tobramycin, or amikacin in an experimental model of Serratia marcescens endocarditis: in vitro-in vivo correlation." Antimicrobial agents and chemotherapy 35.1 (1991): 111-116.

Websites

4 - Kansas State University, Antibiotics Usage, Tuesday 30 August 2016, https://www.k-state.edu/hermanlab/protocols/AntibioticUsage.html

5 - Altlanta Biological, Inc., Gentamicin 10 mg/mL, Tuesday 30 August 2016, https://atlantabio.com/catalog/antibiotics-supplements-&-reagents/gentamicin-10-mg-ml

6 - Thermo Fisher Scientific, Gentamicin 50 mg/mL, Tuesday 30 August 2016, https://atlantabio.com/catalog/antibiotics-supplements-&-reagents/gentamicin-10-mg-ml

Books

7 - BARKER Kathy, At the bench, a Laboratory Navigator, I.K. International Publishing House Pvt. Limited, 2000.

8 - HAUNER Hans, SKURK Thomas & WABTISCH Martin, Adipose Tissue Protocols, Gérard Ailhaud, 2001, p. 239 - 247

The draw is mine.
All gifs come from giphy.com.
Original videos:  https://www.youtube.com/watch?v=gEwzDydciWc & https://www.youtube.com/watch?v=xZbcwi7SfZE&feature=youtu.be