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Antimicrobial Susceptibility Testing (AST) Methods

In this video Gunnar Kahlmeter discusses Antimicrobial Susceptibility Testing (AST): Main categories of methods.
Hello, everyone. I’m Gunnar Kahlmeter. I’m a clinical bacteriologist, and I’ve been dealing with antimicrobial susceptibility testing for the better part of my life. I’m going to talk to you about methods. We can divide methods into phenotypic methods, genotypic methods, mechanistic methods, and expert rules methods. Let’s start with phenotypic AST. It really means measuring the activity of the agent against the isolate bug in question. Phenotypic AST is good because it predicts resistance and susceptibility, so you can actually exclude resistance with phenotypic methods. It’s also quantitative, which is a great advantage. You can say things like, this is more susceptible than that. And that way, you can quantitate your possibilities of success. The reference method is broth microdilution.
It has been described both by the American organisation, by the European organisation, and they’ve come together united in an ISO description of how that method should be performed. All other methods are surrogate tests.
Genotypic AST is the detection of the gene– the whole gene or an important part of the genes, which will convey specificity. It predicts resistance, but cannot really guarantee susceptibility. It’s not quantitative to any great extent. Breakpoints are not required, but when you set it up, you need to validate the detection of the gene against clinical susceptibility or resistance. There is so far no reference method available, but this is a volatile field so eventually there will probably be one. Mechanistic AST that is when you detect the end result of the resistance gene if you like.
So if you have a gene that conveys the production of an enzyme, which, in turn destroys the agent, then by detecting the enzyme, you can predict that the isolate will be resistant to that specific agent.
It’s not quantitative and it does not require breakpoints, but again, you need to initially calibrate it against clinical significance if you like. Then you can use something called expert rules, and what it really means is that if you know that something is resistant to one agent, you can deduce that it will also be resistant to another agent, normally in the same class. And you write rules that are formulated in the form of if, then. If resistant to erythromycin, then also resistant to clarithromycin. Not quantitative, and it’s not really trustworthy because there will be changes over time.

In this video, Professor Gunnar Kahlmeter briefly discusses some of the types of AST methods.

Phenotypic AST – based on measuring the activity of the agent against the microorganism, MIC and breakpoints.

Genotypic AST – detection of a resistance gene (mecA, vanA/vanB, etc) in the genome or on transferable genetic elements.

Mechanistic AST – detection of a resistance mechanism – by detecting the product of the resistance gene (enzyme).

Expert rules AST – susceptibility based on empiric knowledge.

Professor Gunnar Kahlmeter goes on to explain the Minimum Inhibitory Concentration (MIC) and its importance.

MIC is the lowest concentration of a drug needed to inhibit cell division in the environment in which the test was performed. It is a broth microdilution and is the basis for all susceptibility tests. All other methods are classed as surrogate methods which are addressed in a subsequent step. Clinical AST should produce a clinical recommendation to determine whether to treat or not and whether standard dosing will suffice or if increased exposure is required.

The MIC is not an absolute value and is affected by factors such as:

  • Inoculum
  • Incubation time
  • The broth used and its composition
  • Atmosphere
  • pH

Testing many isolates of the same species against a defined agent can determine the wildtype distribution of the species for that particular agent. Wildtype isolates are those that lack any resistance mechanisms. Once the wildtype distribution is established and epidemiological cut-off values (ECOFF) determined, the highest MIC of wildtype distribution, then successive isolates can be categorised as wildtype or non-wildtype.

To view the full video, please click here.

Let us know in the comments section below, which type of method have you heard of or used before?

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Introduction to Practical Microbiology

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