Isobologram Analysis

The interaction between peptide and antibiotics was determined using the checkerboard assay and subsequent isobologram analysis as described previously (Hakeem et al., 2019). Briefly, defined combinations of each antimicrobial agent (i.e., IC₅₀ of PinA and IC₅₀ of ciprofloxacin) were added to the wells of a 96-well plate. The following equation was used to calculate the IC amounts:

where F represents the percentage of reduction, H represents the Hill slope from the dose-response curves, and IC₅₀ is the concentration that gives a 50% reduction in bacterial growth.

Two different serial dilutions were made for the checkerboard assay. A two-fold serial dilution was made for one antimicrobial agent from left to right in a 96-well plate. Another two-fold serial dilution was made for the second antimicrobial agent from top to bottom in the same plate. Then, C. jejuni culture was added into each well at a density of 5 × 10⁴ bacteria/well. The plate was incubated with orbital shaking at 220 rpm for 48 h, and growth was determined using a microplate reader at 595 nm (OD₅₉₅). The IC₅₀ of the two antimicrobial substances were plotted on the axes, and the additive line connecting the two IC₅₀s was drawn. The combination of antimicrobial agents (axial point) that produced a 50% reduction in bacterial growth was plotted on the graph. An axial point landing on the additive line indicates an additive effect, under the additive line indicates a synergistic effect, and above the additive line indicates an antagonistic effect.