IntroductionE. coli is clinically significant because of its pathogenic mechanisms and the development of antibiotic resistance in various strains (Hassan, Ojo & Abdulrahman, 2021). As a gram-negative bacterium, E. coli possesses an outer membrane rich in lipopolysaccharides that limits antibiotic penetration, while efflux pumps and enzymes like beta-lactamases expel or degrade antibiotics before they can reach their targets (Alves et al., 2017; Kakoullis et al., 2021; Nasrollahian, Graham & Halaji, 2024). As a result, E. coli is one of the most prevalent antibiotic-resistant bacteria in humans (Alves et al., 2017; Pormohammad, Nasiri & Azimi, 2019). Hence, studying its antibiotic response is essential, as it has significant implications for morbidity and mortality (Bezabih et al., 2021).

Aminoglycoside antibiotics, such as kanamycin and gentamicin, block bacterial protein production by binding to the 30S ribosomal subunit, thereby inhibiting translation and causing bacterial cell death (Grayson et al., 2017). This mode of action, combined with their ability to penetrate the outer membrane via porin channels, makes them particularly effective against gram-negative bacteria such as E. coli (Gauba and Rahman, 2023). Among aminoglycosides, kanamycin and gentamicin were selected due to their clinical relevance and proven effectiveness against E. coli (Grayson et al., 2017). Despite their widespread use, direct comparative data on their effective MICs against E. coli remains limited. MIC values are essential for guiding clinical decisions and optimizing dosing strategies to prevent under-dosing of antibiotics, which contributes to the rise of resistant strains (Kowalska-Krochmal & Dudek-Wicher, 2021). This experiment aimed to determine the MICs of kanamycin and gentamicin on E. coli by analysing the area of inhibition zones at different concentrations, hypothesizing that gentamicin, with its broader spectrum of activity, may show greater inhibitory effect at lower concentrations (Grayson et al., 2017). Identifying MICs for specific antibiotics helps ensure more targeted and effective treatment methods while also addressing the growing global health threat posed by antibiotic-resistant E. coli (Bezabih et al., 2021).

Methods

This experiment aimed to determine the MIC of the aminoglycoside antibiotics kanamycin and gentamicin on E. coli by assessing the area of inhibition zones at four different concentrations using the disc diffusion method. A quantitative design was used under standardised conditions, and the area of the inhibition zones served as a measurable indicator of the antibiotics' bactericidal effects.

A laboratory strain of E. coli grown in liquid culture was used as the test bacterium. An inoculum was prepared by diluting the culture using sterile nutrient broth based on OD600 measurements. Kanamycin and gentamicin sulfate, both at an initial stock concentration of 1 mg/mL, were serially diluted fivefold with sterile deionised water to yield four working concentrations: 750 µg/mL, 150 µg/mL, 30 µg/mL, and 6 µg/mL. The positive control was 1 mg/mL of gentamicin, selected due to its proven effectiveness against E. coli (Grayson et al., 2017), and the negative control was sterile deionised water. Nutrient agar (pH ~ 7.0) was used as the growth medium. The experiment was conducted using 12 agar plates, with three replicate plates for each of the 4 antibiotic concentrations (kanamycin and gentamicin) and controls. Antibiotic assay discs were placed on each plate to accommodate the different antibiotic concentrations and controls. Standard aseptic techniques were followed throughout the procedure using a Bunsen burner to create a sterile field, and ethanol was used to sterilise the forceps, and glass spreaders. A Gilson pipette with sterile tips was used for the accurate transfer of liquids.

100 µL of diluted E. coli culture was pipetted onto each agar plate and evenly spread using a sterilised glass spreader. After the plates had dried for approximately ten minutes, the underside of each agar plate was divided into four sections to indicate the positions of kanamycin, gentamicin, positive and negative controls. Antibiotic assay discs were aseptically placed in each section using sterilised forceps. Then, 10 µL of the corresponding antibiotic concentration and control solution were added to each disc. The same procedure was followed for each concentration and replicated three times (see Appendix A for an image of the plate showing this setup). The plates were incubated overnight at 37°C.

On the following day, the diameter of each zone of inhibition was measured in millimetres using a ruler. The area of inhibition zones was calculated using the formula A = πr², and the results were then analysed with a two-way ANOVA to assess the effects of antibiotic type and concentration. Microsoft Excel was used for initial data recording and calculations, while R programming software was used to perform the statistical calculations and visualise the results.