AST of bacteria

EUCAST warnings concerning antimicrobial susceptibility testing products or procedures.

The EUCAST disk diffusion development laboratories, a network of laboratories coordinated from the EUCAST development laboratory in Växjö, Sweden, from time to time discover products (disks, media batches, gradient tests or procedures) which are not performing to the expected standard. When this is the case we inform the manufacturer and publish a warning on this page.

We do not systematically test all products so the lack of a warning does not imply that there is no problem with the product in question.

Laboratories which experience problems with a susceptibility test method, and suspect that this may be related to a particular product, may contact EUCAST for advice.


1. Problems with piperacillin-tazobactam gradient tests from two manufacturers. 
Warning partially lifted - see below. 

2. Wide variation in disk quality in 16 disks from nine manufacturers - this warning was issued in 2015 and reiterated 2016 and 2017. There is new information on improved quality November 23, 2017 (see below). 

3. Problems with colistin susceptibility testing and several commercially available products - the warning initially issued July 2016 was updated 26 June, 2017 and is extended and still valid April 2019.

4. Vancomycin susceptibility testing in Enterococcus faecalis and E. faecium using MIC gradient tests – a modified warning; 21 May, 2019.

5. A problem with meropenem in a Thermo Fisher Sensititre panel (DKMGN) was discovered (6 November, 2018). 

6. Variable performance of the itraconazole agar in the azole resistance screening plate from VipCheck


1. Problems with piperacillin-tazobactam gradient tests.

This warning, issued 2015, is partially lifted April 2019.

The recently released new piperacillin-tazobactam Etest from bioMerieux has now been evaluated by the EUCAST Development Laboratory (April 2019) using isolates of E. coli, K.pneumoniae and Ps. aeruginosa with varying MICs to piperacillin-tazobactam (wild-type isolates MICs in the area 1 - 8 mg/L and isolates with MICs of 16, 32 and 64 mg/L). The correlation between broth microdilution and the new Etest MIC values was satisfactory and we therefore lift the warning on Etest piperacillin-tazobactam. However, it is important to remind users of the new test that it was designed and validated for Enterobacteriaceae, Pseudomonas (and Acinetobacter) only. There are no recommendations for use with other species.

Currently we have no information pertaining to piperacillin-tazobactam gradient tests from other manufacturers.


2. Wide variation in disk quality in 16 disks from 9 manufacturers.

The warning was issued 2015 and reiterated in 2016. It will be removed when problem is solved.

During 2014 and 2015, EUCAST has evaluated the disk potency of 16 strategically important antibiotic disks from nine manufacturers of disks for antimicrobial susceptibility testing. Results from the first study were released at ECCMID 2014. Manufacturers were offered an opportunity to supply new disk lots to possibly improve their results. The second round of testing was presented at ECCMID in Amsterdam. Some manufacturers have asked that EUCAST performs further checks on new lots produced to try to right poor quality in previous lots. The EUCAST evaluation was published in CMI 2018.

The study disclosed some good and some poor quality among disks and manufacturers. Disks from a few of them were consistently of a high quality whereas the opposite was true for some manufacturers. Laboratories are warned against the use of some disks and manufacturers are urged to improve their manufacsome disksturing process.


It is the responsibility of manufacturers to provide high quality material BUT it is the responsibility of laboratories to perform quality control to guarantee that the material used performs to the standards of the laboratory and the health care system. The fact that a manufacturers failed on a few disks should not exclude the manufacturer from the market. The fact that a manufacturer performed well in the 2014 and 2017 EUCAST surveys, does not guarantee eternal high quality. Laboratories should ascertain that products meet their expectations whenever a new batch of material is introduced for use. Laboratories should also insist on a clause in purchase contracts which the provider of susceptibility testing material are held resposnible for the quality of the material. Comment added 30 June, 2019.


3. Antimicrobial susceptibility testing of colistin - problems detected with several commercially available products.


This warning was issued July 2016 and it is still valid April 2019. The current text was updated April 2019 and is a summary of the previous warnings and includes the results following further testing of more broth microdilution tests and more organisms. The warning is extended April 2019 to include the colistin gradient test from HiMedia (Colistin EZY MIC Strip), tested by EUCAST 2018/19 and shown to give as unreliable results as other gradient tests. 

Warnings will be modified or removed when issues have been resolved.


Article now available in CMI.


Antimicrobial susceptibility testing of colistin has been fraught with difficulties. A joint EUCAST and CLSI subcommittee recently issued recommendations confirming that broth microdilution (BMD) is so far the only valid method and that disk diffusion does not work because of the poor diffusion of the large colistin molecule. The report did not evaluate gradient tests of colistin, but reports in the literature have questioned the validity of MICs obtained with gradient tests.

We used a collection of clinical isolates (n=75) obtained from international contacts (acknowledge: SENTRY collection (P. Rhomberg, JMI Laboratories, USA), S. Gatermann, Bochum, Germany, R. Henriksen, DTU, Copenhagen, Denmark, Ö. Samuelsen, Tromsö, Norway, J. Vila, Barcelona and L. Martinez-Martinez, Santander, Spain) of Escherichia coli (n= 14), Klebsiella pneumoniae, (n=18 )  Pseudomonas aeruginosa (n=21) and Acinetobacter spp (n=22) without and with various resistance mechanisms and with different levels of resistance to colistin. Several Enterobacteriaceae had the mcr-1 gene. Broth microdilution MIC values of the four species were 0.25 – 128 mg/L.


Summary and conclusion:

  • The broth microdilution tests we have evaluated all seem to give correct results both for susceptible and non-susceptible isolates (see below) although some of them may benefit from further improvements. 
  • Disk diffusion cannot be used for susceptibility testing of colistin. It does not discriminate between susceptible and resistant isolates.
  • Currently available gradient tests underestimate colistin MIC values and undercall resistance, and should be avoided, even when quality control results are within range.
  • We could not systematically evaluate semi-automated colistin methods but by sending isolates with MIC values in the non-susceptible range to colleagues around the world we have disclosed the frequent occurrence of Very Major Errors.
    Users of semi-automated devices should apply rigorous QC (see below) and check with the manufacturer whether or not they are confident that their method for colistin AST gives correct results.
  • Quality control of colistin must be performed with both a susceptible QC strain (E. coli ATCC 25922 or P. aeruginosa ATCC 27853) and the colistin resistant E. coli NCTC 13846 (mcr-1 positive). For E. coli NCTC 13846, the colistin MIC target value is 4 mg/L and should only occasionally be 2 or 8 mg/L.


Summary of results for all commercial BMD methods

Essential agreement (MICs within ± 1 dilution of reference MIC):

  • Sensititre (Thermo Fisher Scientific): 96%
  • MICRONAUT-S (Merlin Diagnostika): 96%
  • MICRONAUT MIC-Strip (Merlin Diagnostika): 99%
  • SensiTest (Liofilchem): 88%
  • UMIC (Biocentric): 82%

Major Errors (false resistance), No of tests of a total of 75

  • Sensititre (Thermo Fisher Scientific): 4
  • MICRONAUT-S (Merlin Diagnostika): 6
  • MICRONAT MIC-Strip (Merlin Diagnostika): 5
  • SensiTest (Liofilchem): 7
  • UMIC (Biocentric): 3

Very Major Errors (false susceptibility), No of tests of a total of 75

  • Sensititre (Thermo Fisher Scientific): 0
  • MICRONAUT-S (Merlin Diagnostika): 2
  • MICRONAT MIC-Strip (Merlin Diagnostika): 2
  • SensiTest (Liofilchem): 1
  • UMIC (Biocentric): 3


The study, now published in CMI, evaluates seven commercial MIC determination products against standard broth microdilution for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp.

The study has, since the publication, been extended to encompass Colistin EZY MIC Strip (from HiMedia). This product produces MIC results on colistin which are as unreliable as those produced by the other tested gradient tests. Broth Microdilution is still the only method for colsitin recommended by EUCAST.


4. Vancomycin susceptibility testing in Enterococcus faecalis and E. faecium using MIC gradient tests – a modified warning 21 May, 2019. 

Original warning was issued 10 July, 2018, against the use of gradient tests for the detection of vanB-positive Enterococci.

Several studies (Norwegian Reference Laboratory, Tromsö, Norway; The EUCAST Development Laboratory, Växjö, Sweden; Robert Koch Institute, Wernigerode, Germany) show that the use of MIC gradient tests with standard inoculum and incubation fail to detect glycopeptide resistance in low-level resistant enterococci (see posters 1754 and 1764, ECCMID 2019). Confirmation of suspected vancomycin resistance with gradient tests, can be significantly improved by the use of a macro method (BHI-medium, McF 2.0 and 48 hours incubation; see poster 1764, ECCMID 2019). Uncertain results should be confirmed with a molecular test for vanA and vanB.


We remind users of the EUCAST standard disk diffusion test for vancomycin in Enterococcus spp. to measure the zone (suspect resistance if <12 mm), and to note whether the zone edge is sharp or fuzzy (suspect resistance if fuzzy) and to take into account any colonies inside the inhibition zone (suspect resistance if colonies in zone). Either of these phenomena indicates glycopeptide resistance and a PCR should be performed to confirm or exclude the presence of vanA and vanB.




5. Meropenem in a broth microdilution panel (DKMGN) from Sensititre.


Meropenem in a broth microdilution panel (DKMGN) from Sensititre (November, 2018).


The reference method for antimicrobial susceptibility testing (AST) is broth microdilution (BMD). Increasing numbers of commercial AST systems, both BMD and others, are available on the market. The EUCAST Development Laboratory (EDL) does not have the capacity to systematically check the quality of these systems. 

EUCAST was recently alerted to a potential problem for meropenem in E. coli and K. pneumoniae on the Sensititre panel DKMGN (batches B8012, B8201A and B8314A) manufactured by Thermo Fisher Scientific. This was confirmed by the EUCAST Development Laboratory and recently also by the company and has now led to a company issued Technical Bulletin addressing the problem. The importance of an adequate inoculum is emphasized.




6. Variable performance of the itraconazole agar in the azole resistance screening plate from VipCheck.

The warning was issued 13 Dec, 2018. It will be removed when problem is solved.


During October-December, 2018, the EUCAST AFST committee has experienced variable performance of two batches of VipCheck plates (screening plate for detection of azole resistance in A. fumigatus). In brief, wild type (wt) clinical Aspergillus fumigatus isolates and the susceptible quality control strain A. fumigatus ATCC 204305 grew on an unacceptable number of itraconazole wells (batches #144914 and #145151). These isolates were confirmed wt and susceptible by EUCAST E.Def 9.3.1 reference testing and target gene sequencing, and failed to grow on itraconazole in house plates prepared according to E.Def 10.1. The concentration of itraconazole has subsequently been compared in VipCheck plates and in house plates and the results suggest a variable itraconazole concentration to be the cause of the problem. This  may lead to erroneus categorization of both susceptible and resistant isolates. It is currently unknown how many batches, and also how many plates in each batch, may be affected. The manufacturer is currently reviewing the protocol for dissolving itraconazole to improve their manufacturing process.

Laboratories are warned against the use of the VipCheck plates until this issue has been resolved. 



Warnings will be removed when the issues have been solved.