Our on-site laboratory has recently acquired a new microbiology analyser, the Vitek 2 Compact, and we have prepared the following update explaining why it is an improvement on previous methodology, and giving a brief overview of how it works and how to interpret the new results.
Microbiology update: New Analyser & MIC Sensitivity Testing
Why is the VITEK better than the old way?
Identification
In May we started using our new machine, the VITEK 2 Compact (V2C), to enable us to provide you with more accurate & hopefully faster results. The V2C provides us with a standardised, automated method to identify pure isolates. Each isolate has an individual card that performs 64 biochemical tests to identify the isolate, using a combination of turbimetric, fluorescence and colorimetric analysis for identification. The machine analyses each isolate every 15 minutes, enabling fast turnaround times for identification.
Susceptibility Testing / Antibiogram
The V2C provides us with MIC (Minimum Inhibitory Concentration) values for a range of antibiotics for all of our isolates. The MIC values are monitored every 15 minutes, each antibiotic completing individually once the MIC value has been determined. Once all of the antibiotics have been tested, the values are assigned with ‘Sensitive / Intermediate / Resistant’ classifications. The V2C then performs ‘Advanced Expert System’ analysis on each isolate. This allows detection of any resistance mechanisms the organism may have (E.G. ESBL in enterobacteriacae, mecA in Staphylococcus etc). This method enables superior speed and accuracy than the old method & provides a more useful result, as it allows determination of the ‘most sensitive’ antibiotic.
What are MICs and how can they be useful?
MICs are the Minimum Inhibitory Concentration. This is defined as: the lowest concentration of a chemical which prevents visible growth of a bacterium.MICs are particularly useful compared with our old method (Kirby Bauer Disk Diffusion), as they provide numerical data for each antibiotic, which can be compared with the data for other antibiotics available for use.
MICs are determined by using a serial dilution method; each well (in a 64-well card) has a serially-diluted concentration of antibiotic which is tested against the same concentration of the bacteria. Susceptibility is determined by growth or no-growth at each concentration of antibiotic. In this instance growth is determined by turbidity. The concentration of antibiotic in each well is known before the test starts, and is measured in ug/ml.
Breakpoints: breakpoints are pre-determined and are set by international governing bodies (we use CLSI – Clinical Laboratory Standards Institute & EUCAST (European Committee on Antimicrobial Susceptibility Testing). They classify the MIC value as Sensitive / Intermediate / Resistant for each organism.
Each different antibiotichas a different breakpoint for each organism. For example the breakpoints forAmpicillinfor the following organisms differ significantly:
| Organism | Sensitive | Intermediate | Resistant |
| Staph pseudintermedius | <=0.25 | >=0.5 | |
| E. coli | <=8 | 16 | >=32 |
This is particularly important to remember as it means MIC values for different organisms cannot be directly compared.
So even if the MIC value were the same for both/all isolates, the interpretation of the result (S/I/R) can be very different.
How are Sensitive / Intermediate / Resistant interpretations reached?
The breakpoints are calculated using the recommended dosage of each antibiotic as per the formulary. The MIC value is directly related to the concentration of antibiotic.
A sensitiveresult indicates that, at the recommended dosage of a specific antibiotic, antimicrobial therapy should have a successful outcome. The lower the MIC value (away from the breakpoint), the lower the concentration of antibiotic required to successfully treat the bacterial infection.
An intermediateresult occurs when the MIC value is close to the breakpoint, but is not determined as resistant. This means that at the recommended dosage of a specific antibiotic it is uncertain if the antibiotic will be effective. An intermediate result is not classified as resistant as at a higher-than-recommended concentration of the antibiotic, the outcome is usually successful.
A resistantresult occurs when the concentration of the antibiotic required to be reached at the site of infection, for the outcome of treatment to be successful, is too high to be safely used in the target species. So it is the concentration at which the drug becomes toxic or dangerous for use in the animal.
Interpreting MICs
With the MIC values, the lower the value’s number compared with the breakpoint, the more ‘Sensitive’ the isolate. MIC values can be useful as they allow you to see which antibiotics are ‘most sensitive’ for each organism. It is important to note that the lowest MIC value of all the antibiotics is usually NOT the most sensitive overall. The antibiotic that is most sensitiveis the one that is the ‘most’ number of serial dilutions below the sensitive breakpoint value.
Please see the below example:
Organism: E. coli
Results:
Antibiotic MIC Interpretation
Ampicillin <= 2 S
Amoxicillin/Clav <= 2 S
Marbofloxacin <= 0.5 S
Ceftiofur <= 1 S
Gentamicin <= 1 S
Trimethoprim-Sulf <= 20 S
E.coli Breakpoints
Antibiotic MIC Breakpoint
Sensitive Intermediate Resistant
Ampicillin <=8 >=16
Amoxyclav <=8 >=16
Marbofloxacin <=1 2 >=4
Ceftiofur <=2 4 >=8
Gentamicin <=2 4 >=8
Trimethoprim-Sulf <=40 >=80
In this example, this is how may ‘serial dilutions’ away from the breakpoint the E. coli’s MIC value is:
Antibiotic Number of dilutions ‘away from breakpoint’
Ampicillin 2
Amoxyclav 2
Marbofloxacin 1
Ceftiofur 1
Gentamicin 1
Trimethoprim-Sulf 1
Therefore ampicillin and amoxyclav are equally the ‘most sensitive’ & should be picked for the best chance of a successful treatment outcome. The lowest MIC value is for marbofloxacin, BUT this is not the most sensitive antibiotic.
So the breakpoints must be used to determine which antibiotic is most likely to give a successful treatment outcome. It is important to remember that for each organism the breakpoints are different. So, if the case has 2 or more isolates, the best antibiotic for each organism cannot be decided from one of the sets of breakpoints.
For each organism in each case, an individual set of breakpoints will be provided in the comments section of the report – please use the different breakpoints that are relevant for each organism isolated.
Results Received Without MIC Values
Occasionally, some of the results received will either NOT have MIC values, or contain results where some antibiotics have MIC values, and some do not. This occurs when:
- There are NO validated MIC values for the organism in question, but there are validated Kirby-Bauer Disk Diffusion zone sizes (and so the old method is used to determine susceptibility).
- The result has been inferred from the MIC value for another antibiotic (as approved by international governing bodies) G: for Streptococcus canis: amoxicillin, ampicillin, cephalexin, Cefoxitin, cefotaxime & piperacillin can be inferred from the Benzylpenicillin susceptibility result (CLSI)
- The organism in question is intrinsically resistant to an antibiotic or class of antibiotic (further information on intrinsic resistance can be found at: http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Expert_Rules/Expert_rules_intrinsic_exceptional_V3.1.pdf)
- The V2C does not offer MIC testing for particular antibiotics (for example there are limited topical antibiotics available for testing, and so the majority of topical antibiotics are tested using the Kirby Bauer Disk Diffusion method)
If you have any questions, please do not hesitate to contact Jade Denham on 01638 572900, micro@dickwhitereferrals.comor jade.denham@dwr.co.uk