In recent years we have observed a significant increase of antimicrobial resistance with multiple and often combined enzymatic mechanisms affecting an increasing number of microorganisms.
In the past, an “interpretative reading of an antibiogram” has been used to infer resistance mechanisms behind resistant phenotypes, but the “automatic” reading no longer able to adequately identify the mechanisms of resistance.
A microorganism may have multiple coexisting mechanisms of resistance, and this resistance can result from one, or more, point mutations in target genes, or from the acquisition of new genes (plasmids or transposons). A resistance mechanism may be functional (and always expressed), or inducible in the presence of an antibiotic (difficult to detect in vitro). If resistance to an antibiotic emerges from a high frequency mutation, there is a significant risk that the resistance will be selected during a mono-therapy with that drug.
Molecular methods can precisely define the mechanisms of resistance. However, in addition to be expensive and not available in all centers, they are difficult to apply in clinical practice and they still have some limitations.
The antibiogram expresses the susceptibility of a microorganism to several antibiotics, tested in different concentrations. The most used tests are: Kirby Bauer technique, broth micro-dilution (BMD) and gradient method (E-test).
Kirby-Bauer disk diffusion method provides for evaluation of the diameters of the inhibition zones around the disks containing the antibiotic tested; BMD (method that can be automated) is used more frequently, and represent the reference method.
E-test is a manual method, performed in agar, using a paper strips with a gradient concentration of a given antibiotic. Actually, to provide faster reporting of susceptibility results and to face the large numbers of tests that come in centralized laboratories, they use automated system, such as Vitek2 (Biomerieux).
BMD and automated systems allow to obtain the minimum inhibitory concentration (MIC), which is the lowest concentration of antibiotic that is able to inhibit in vitro growth of the microorganism after 18-24 hours of incubation.
The diameters of inhibition zones and MICs must be compared to clinical breakpoints standardized for different organism-antibiotic combinations, in order to obtain the results of susceptibility (sensitive, intermediate or resistant). Breakpoints are established by specific committees, one in Europe (EUCAST) and one in the USA (CLSI) combinations.
EUCAST: what is it?
In 1997 European Committee on Antimicrobial Susceptibility Testing (EUCAST) has unified the different standards for interpreting the antibiogram previously used in six European countries. The EUCAST is a committee jointly organized by ESCMID (European Society for Clinical Microbiology and Infectious Diseases), by ECDC (European Centre for Disease Prevention and Control) and by the six national committees previously active. At now, breakpoints defined by EUCAST are the only officially recognized by the EMA (European Medicines Agency), body authorizing the placing on the market of drugs in the countries of the European Union.
Italian microbiological laboratories have adopted EUCAST Guidelines since 2011. EUCAST documents are free and consultable on web from http://www.eucast.org.
Breakpoints are set according to several parameters: microbiological, pharmacological (relationship between PK/PD indexes and response to treatment) and clinical (best evidence from literature). They are ultimately derived from human clinical studies comparing outcomes with the MICs for the infecting pathogen.
Breakpoints are used to define the clinical category of susceptibility: susceptible (when bacterial strain growth is inhibited in vitro by a concentration of the antibiotic that is associated with a high likelihood of therapeutic success), intermediate (uncertain probability of successful treatment), resistant (high likelihood of therapeutic failure).
For each organism-antibiotic combination there are two breakpoints (in this case there are 3 categories of interpretation: susceptible, intermediate, resistant) or just a breakpoint (so providing for two categories of interpretation: susceptible and resistant).
The aim of clinical breakpoints is to use MIC values to separate strains where there is a high likelihood of treatment success using in vivo the given antibiotic from those whose treatment is more likely to fail due to a resistance mechanism. This is why we talk about “clinical” breakpoint (BC).
EUCAST breakpoints are often lower than those of the CLSI, with more restrictive interpretation of susceptibility, except for those related to carbapenems and Enterobacteriaceae. The EUCAST breakpoints are available, both for Kirby-Bauer disk diffusion method and micro-dilution broth test.
EUCAST, differently from the CLSI, has defined the epidemiological cut-off (ECOFF), which is the concentration value that split wild type isolates (which does not have any acquired and mutational resistance mechanisms) from the population of strains with resistance mechanisms. The epidemiological breakpoints are important in the choice of the antibiotic and the drug dosages.
Often in automated antibiograms MICs are reported equal or less of the minimum concentration tested regarding some antibiotics, but in some cases, for example for Enterobacteria and quinolones, it is important to know the accurate MIC. In those cases where the epidemiological cut-off is different from the clinical breakpoints: to know with accuracy the MIC allows to understand if the germ has already developed some mutation that could let mono-therapy to fail.
Changes on interpretation of susceptibility and changing rules
We must know that the reporting rules of the antibiogram are not immutable over time, but may change in relation for example to the need to put in place new strategies for limiting the spread of resistance or based on new evidence in the literature.
Since 2010, some changes were made in the interpretation of susceptibility, especially for gram negative rods.
In addition to lowering of the breakpoint for some specific microorganism-antibiotic combinations, experts can decide to avoid from reporting some combinations as they are no longer considered adequate in the therapeutic area, or the microorganism is naturally resistant to the drug. Both, CLSI and EUCAST, separately decided to modify breakpoints for oxymino-cephalosporins and Enterobacteria.
Moreover, the new rule recommended, for some combination microorganism-antibiotic, such as Enterobacteriaceae and cephalosporins or carbapenems, reporting MIC results “as found”, regardless of resistance mechanisms and do not search anymore the mechanism of resistance, such as ESBL or carbapenemases, except for epidemiological purposes. ESBL-producing enterobacteria then will be reported as “susceptible” to some cephalosporins (instead as “resistant”) and the interpretation will be based on clinical breakpoints and is no longer required to test for ESBL and consequently change the result, as in the past. This is mainly created to limit the widespread use of carbapenems in the clinical.
Changes on interpretation of susceptibility and changing rules
The interpretation of an antibiogram is much more than the categorization of susceptibility and represents an attempt of phenotypic interpretation of the resistance mechanisms exhibited by the microorganisms that are isolate in clinical practice.
Reading and understanding an antibiogram, and therefore its interpretation, is based on the recognition of the possible mechanism at the base of resistance, which can be extended to non-tested drugs or that lead the change of category obtained in vitro (as for example the presence of oxacillin-resistant staphylococci determines resistance to all beta-lactams, except some new cephalosporins such as ceftaroline or ceftobiprole).
The clinician should keep up to date on antibiotic resistance, to recognize the exceptional resistance phenotypes, natural resistance and combinations of antibiotic and organism for which there is high likelihood of resistance development to simple mutation.
The correlation between the data obtained in vitro (MIC or inhibition zone) and the real clinical efficacy of the drug depends on a complex set of factors, including the site of infection and the ability of that drug to reach the appropriate concentrations in the target site. Dose and effective drug delivery are obviously also important in terms of pharmacokinetics and pharmacodynamics.
For example, it is very important to know the bacteriostatic activity (antibiotics that only inhibit the growth) and bactericidal activity (antibiotics that kill): the mechanisms that allow the bacteriostasis can be different from those of the bactericidal either to the same drug that for the same bacterial species.
The absolute value of the MICs reported on susceptibility testing must not be evaluated “vertically” between the different drugs tested. For example if there is an antibiotic X with a MIC of 0.5 mg/l and breakpoint 2 mg/l, and an antibiotic Y with a MIC of 2 mg/l but breakpoints of 16 mg/l, the drug with a MIC more favorable is the antibiotic Y. this relationship, this “distance” between MIC and the breakpoint, it’s very important in the choise of therapy and would be useful to have the clinical breakpoints values on antibiograms. Clinical breakpoint are available from the site http://www.eucast.org.
– EUCAST European Committee for antimicrobial Susceptibility Testing. URL: http://www.eucast.org.
– Clinical and laboratory Standards Institute. Performance standards for antimicrobial susceptibility testing: nineteenth informational supplement. Document M-100-S19. Wayne PA: CLSI 2010.