Cefiderocol Disk Diffusion Instructional Video

I’m Sam Simon, the Associate Director of Field Microbiology and Infectious Diseases at Shionogi Inc. This video focuses on the determination of zones of inhibition using disk diffusion as a method to determine the susceptibility of isolates to cefiderocol.

Cefiderocol is a cephalosporin antibiotic that contains an iron-binding catechol moiety that enables the compound to utilize bacterial iron uptake transporters to gain cell entry. It can also enter the cell through passive diffusion through porin channels. This dual entry mechanism necessitates a tailored approach to susceptibility testing to assess cefiderocol’s activity.

Cefiderocol leverages the iron-uptake systems expressed by bacteria at the infection site of the host, where the amount of free iron is limited. To mimic these conditions during susceptibility testing, iron-depleted cation-adjusted Mueller-Hinton broth is required for broth microdilution. Alternatively, for disk diffusion, standard Mueller-Hinton agar can be used, since the agar itself is thought to naturally sequester iron, creating an iron-depleted environment, necessary for promoting expression of bacterial iron-uptake systems.

Disk diffusion testing is a standard laboratory technique used to determine bacterial susceptibility to antimicrobial agents. It involves placing antibiotic-infused paper disks onto a bacterial lawn grown on agar. The antibiotic diffuses outward from the disk, inhibiting bacterial growth in the surrounding area.

When cefiderocol susceptibility testing is performed using the disk diffusion method, clear zones of inhibition are usually seen. However, different growth patterns can sometimes occur with certain species, particularly Acinetobacter baumannii. These patterns include zones with inner colonies and zones with hazy areas or blurred edges.

Knowing how to correctly interpret each of these patterns is essential for accurate susceptibility reporting.

This video will walk through some examples of how to determine the cefiderocol zone of inhibition associated with different growth patterns.

In most cases, the zone of inhibition refers to the clear, circular area surrounding the cefiderocol disk where bacterial growth is prevented. The diameter of this zone – measured in millimeters using a caliper or ruler – is used to determine whether an isolate is susceptible, intermediate, or resistant to cefiderocol.

In the example shown on the screen, the diameter of the clear zone of inhibition surrounding the cefiderocol disk is approximately 38 mm.

In this next example, there is no clear zone of inhibition surrounding the cefiderocol disk, so we would just measure the width of the disk itself, which is 6 mm.

The appearance of colonies within the clearance zone surrounding the cefiderocol disk, which is seen at times, complicates reading. Colonies appearing within the clearing should not be ignored, and the inner zone – free from any colonies – should be used as the zone of inhibition.

In this example, we can see multiple colonies around the periphery of the clearance zone, some of which are pointed out on the screen. Focusing just on the inner zone free from colonies, the zone of inhibition is measured at approximately 20 mm.

This example shows denser growth of inner colonies within the clearance zone. However, if we focus just on the inner zone completely free of any colonies, the zone of inhibition is measured at approximately 10 mm.

Instead of discrete inner colonies, some tests may show a more diffuse halo within the clearance zone. Similar to the preceding examples, the hazy growth should not be ignored, and the inner zone free from any bacterial growth should be used as the zone of inhibition.

Applying this approach to the example shown here, the zone of inhibition is approximately 24 mm.

In this second example, with a thicker hazy ring, the zone of inhibition is measured at approximately 24 mm.

It should be noted that use of different brands of commercial Mueller-Hinton agar for disk diffusion can result in discrepancies in antimicrobial susceptibility test interpretation. BD BBL agar provides the highest MIC correlation when using CLSI breakpoints. Shionogi recommends avoiding Bio-Rad and Hardy agar, when possible, due to larger zones of inhibition.

With regard to disks, any brand of cefiderocol disk can be used, although it should be noted that Oxoid disks can produce larger zone diameters, thereby possibly overestimating susceptibility.

Keep in mind that certain agar brands, in combination with certain disks, may produce larger zones of inhibition. Shionogi recommends avoiding agar-disk combinations that produce larger zones of inhibition, specifically Oxoid disks with Bio-Rad Mueller-Hinton agar.

Remember, when determining the cefiderocol zone of inhibition by disk diffusion, always measure the diameter of the circular area where there is an absence of any bacterial growth. In other words: Where it's clear, measure here.

For additional questions about cefiderocol susceptibility testing or cefiderocol more broadly, please visit the Cefiderocol Microbiology Resource Center and the Shionogi US Medical website.