Exploring comparative analysis of antibiotic resistance patterns in E.coli bacterial strains through the agar plate method
Authors: Waleed Mohamed Marzok A , Shinfar B , Inyat T 
Published: November 2024

Abstract

Introduction
Antibiotic effectiveness varies across classes and bacteria, complicating the fight against infections. Utilising E. coli as a model organism offers insights into the microbial response to antibiotics. The agar plate method is crucial for visualising and assessing antibiotic resistance efficacy1. The looming threat of antibiotic resistance, with a potential death toll of 10 million by 2050, emphasises the urgency2. Investigating E. coli's role in gut health and its interaction with antibiotics via agar plate assays informs the selective use of broad-spectrum and narrow-spectrum antibiotics, vital for medical and agricultural applications3.

Aim
This study aims to explore the differential efficacy of broad-spectrum and narrow-spectrum antibiotics, including ampicillin, chloramphenicol, cefoxitin, and erythromycin, against E. coli using the agar plate method. The study aims to evaluate the effects of different antibiotics on E. coli to identify patterns of resistance and susceptibility, providing insights into the bacterial response to these drugs.

Methods
To maintain aseptic conditions, all equipment was sterilised, and a sterile workspace was established, as illustrated. The agar plates were carefully labelled with the specific bacterial strains and antibiotics slated for evaluation. Employing sterile techniques, the E. coli strains were swabbed onto these agar plates to ensure an even distribution. Antibiotic discs were then methodically placed onto the agar, spaced appropriately to prevent overlap, and gently pressed to ensure contact with the medium. These plates were subsequently incubated at 37°C for 24-48 hours, allowing for bacterial growth and antibiotic diffusion. Post-incubation, the diameters of the inhibition zones around each antibiotic disc were meticulously measured. Following the experimental procedures, biohazard materials were disposed of in accordance with safety protocols, and equipment was re-sterilised. To uphold the experiment’s validity and reliability, it was repeated over four weeks, with the average results compiled and analysed.

Results
This study evaluates the antibacterial effectiveness of four antibiotics over four weeks, measuring the area of inhibition against E. coli. Ampicillin's inhibition varied significantly, with a peak of 2358.58 mm² in Week 3 after a drop to 1468.46 mm² in Week 2. Chloramphenicol also showed variability, peaking at 897.27 mm² in Week 3. Cefoxitin displayed a consistent increase in effectiveness, with an inhibition area of 2317.44 mm² in Week 2. Erythromycin showed no inhibitory effect throughout the study. The data emphasises the dynamic bacterial response to these treatments.

Discussion or Conclusions
This study provides valuable insights into the relationship between antibiotic use and bacterial resistance in E. coli strains, utilising the agar plate method to assess antibiotic effectiveness and predict future resistance trends. By analysing inhibition zone patterns over time, the study identifies emerging resistance, suggesting this method’s utility in forecasting bacterial evolution. However, the approach is limited by its focus on in vitro conditions, which may not fully capture in vivo complexities, and it does not consider genetic mutations or environmental influences. These limitations highlight the need for more comprehensive research. The study advocates for integrated global strategies, combining judicious antibiotic use with novel antimicrobial developments.

References
1. O’Neill, J. Tackling Drug-resistant Infections Globally: Final Report and Recommendations. Arch. Pharm. Pract., 2016, 7(3), 110.
2.  Nwobodo, D.C., Ugwu, M.C., Anie, C.O., Al-Ouqaili, M.T.S., Ikem, J.C., Chigozie, U.V., & Saki, M. Antibiotic resistance: The challenges and some emerging strategies for tackling a global menace. J. Clin. Lab. Anal., 2022, 36(9).
3.  Martinson, J.N.V. & Walk, S.T. Escherichia coli residency in the gut of healthy human adults. EcoSal Plus, 2020, 9(1).