Antimicrobial Susceptibility Testing – Frequently Asked Questions

Please check the EUCAST website has a comprehensive list of frequently asked questions about EUCAST methodology.

EUCAST have a specific FAQ section for rapid AST direct from blood cultures

A list of recent AST questions arising from laboratories in the UK can be found below. Please click on the banner for a list of related questions.

If you don’t find the answer to your question here, please contact Mandy Wootton.

Quality Control

Q. Is it OK for me to use third derivative control organisms?

No, both EUCAST and UKAS/CPA state that 2nd generation derivatives should be used for all QC testing, including the verification (20 day qc).

If you keep your control strains in the freezer, they should be 1st generation cultures.

If you are happy to receive 2nd generation cultures from a manufacturer, which will be 3rd generation by the time they are plated out, then you can only use them as they arrive (i.e. stored in fridge usually) and buy more in when run out, you will not be able to store them in the freezer.

Q. Is there any guidance from EUCAST regarding appropriate time frames for reading disc diffusion zones of inhibition (Can I remove AST plates from the incubator at 18hrs but read at 24hrs?

No, there are no specific guidelines relating to if plates can be stored prior to reading; it is assumed that plates are read after the appropriate time of incubation. Ideally, plates would be read at this time but if you needed to delay reading for whatever reason, refrigerate the plates after incubation and read within 4 hours.

Q. How do we subculture fastidious QC organisms according to EUCAST guidelines?

EUCAST guidance is: “For fastidious organisms that will not survive on plates for five to six days, subculture the strain daily for no more than one week. “

Although the guidance is at odds with UKAS recommendations for use of quality control organisms this is part of the EUCAST disc diffusion method and has been rigorously tested in multiple laboratories.

Q. When performing daily IQC for sensitivity testing what procedures do we follow when values are out of range?

EUCAST does not give advice regarding what to do with clinical test results when control organism results are out of range some general principals can be used (from BSAC disc diffusion guidelines archived). Necessary actions would need to be decided locally and may depend upon which antimicrobial agent had failed, some are less important than others.

When the QC is out of range, inspect the out of range test to make sure the growth of the organism and correct discs have been used, plus the incubation temperature and duration is correct. Take note of whether the zone diameter is too large or small. Then check to see if the same lot of media, discs plus environment have been used to achieve within range results. A repeat of the IQC usually sorts the discrepancy but take care that the QC target is what is wanted not just in the range, if you are consistently getting zone diameters on the edge of the range then something is wrong and would require further investigation.

Giotto2 Template

Q. Run-time error ‘S‘ Invalid procedure call or argument

What to do when you get the error [Run-time error ‘S‘ Invalid procedure call or argument] when clicking on some (but not all) of the options in the list.

If it’s only occurring with certain options in the list, navigate to the folder that holds Giotto2 and open the *.GIO files using something like Notepad.  If you compare those that work with those that don’t, you should spot the problem.

For instance, in an example the ‘Oxoid_6_Staphylococcus_aureus_1_181018.gio’ file contained a carriage return, which put part of the comment on a line on its own, so was without a ‘prefix=’ part to the line. Removing the carriage return fixed the issue.

Still experiencing problems with this type of error? There is a workaround to a known problem with one of the Windows updates. Please follow the instructions in the How to fix Runtime Error 5 question section.

Q. How To Fix Runtime Error 5

Follow these instructions:

  1. Click Start, and then click Run.
  2. In the Open box, type regedit and then click OK.
  3. Locate and then click the following registry key: HKEY_CURRENT_USER\Software\VB and VBA Program Settings\fpecomm.
  4. On the Edit menu, click Delete.
  5. On the File menu, click Exit to quit the Registry Editor.
  6. Restart Your Computer
Q. Giotto templates in the zip folders don‘t seem to open properly for a particular manufacturer

Keep trying! Perhaps try another computer and see if the problem continues. On the BSAC website there is only one installation program.  When this installs, there will be a number of files including the one that runs the program.  The others (e.g. the Help file) may look tempting but can only be opened from within the program.

Q. Is there a 64 bit version of this program?

There is only one version of Giotto but it does seem to load on most machines/versions of Windows. Rather than a specific issue running on a 64bit PC, this problem can occur if the computer is a missing dll file.

Q. What to do if a DLL file is missing

If you see the following error [Class not registered. You need the following file to be installed on your machine MSSTDFMT.DLL], please contact Mandy Wootton and ask for the MSSTDFMT.DLL file if you need a copy of the missing file.

Q. Are there any other programs available form creating templates other than Giotto?

There are currently no other freely available programs available for creating templates. If you are having difficulty, please refer to the following document [How to add templates to controlled document], available on the Giotto Template Program page of the Susceptibility Testing Tools page of the BSAC website.

Q. Giotto2 crashes with unregistered class errors

If it looks like COMDLG32.OCX and MSSTDFMT.DLL files have been deregistered or removed from the machine with some form of update, this will results in the Giotto2 program crashing with unregistered class errors.

These DLLs are not Giotto files and are likely to be missing from Windows. In the past, on the few occasions this had occurred, the problem had been fixed by downloading and registering them.

It is possible to download a VisualStudio 6 Redistributable pack that includes the required files directly from Microsoft. They need to be extracted from their respective .cab’s and installed using regsvr32 running in a Command window.

Web address is https://www.microsoft.com/en-US/Download/confirmation.aspx?id=9183 or Google Visual Studio 6 redistributable.

Q. How do you set up a template for 12 discs on a round 150 mm plate?

The Giotto template was created to support BSAC/EUCAST methods, which are based on 6 or 7 discs on a 90mm plate. Unfortunately 12 disc templates using large (150mm) round plates are not available. However 12 and 16 disc templates using 150mm square plates are available.

Q. How do you create zone sizes of 50mm?

The Giotto2 program currently use has a maximum zone size of 45 mm but there is no problem with the new 50mm zone sizes. The only problem that you may have is with one of the new Pseudomonas zone diameters where we have the new 50mm plus an ATU, but there is away around this.

The Giotto2 program can display the new 50mm zone diameters (EUCAST v10, 2020). Just add 50mm in the S box.

Requirements for MIC testing using broth microdilution

Q. What options are available for colistin broth dilution testing?

There are commercial microbroth dilution systems available. These comprise a strip of wells and only require addition of an inoculum. The cost is similar to a gradient strip.

Q. Would you recommend we set up microdilution on all bone and joint isolates if using long term vancomycin or teicoplanin?

One option would be to use MIC gradient strips in the first instance. For any MIC of ≥2mg/L, extra tests can be used to detect the presence of glycopeptide resistance (heteroresistance usually), such as Macro gradient strip test or Glycopeptide Resistance Detection MIC strip.

Uncertainty of measurement (UoM)

Q. What is the Uncertainty of Measurement in terms of AST?

The UoM for the EUCAST disc diffusion method has already been calculated for you. All agents and QC organisms in the EUCAST guidance have had rigorous testing in multiple laboratories. The zone diameter ranges for each agent are determined from these results and so are the UoM. However you do need to estimate the UoM for reading QC plates (see next Question).

Q. How do I report Uncertainty of Measurement on antibiotic zone readings?

You need to get more than 3 staff members to read the same set of all QC plates. Add the readings to an Excel spreadsheet and calculate the standard deviation OR just state that all readings were within 1 or 2mm and that all were within the range given for the particular species.

Further information and tools about UoM can be found on the Susceptibility Testing Tools page

Resistance Mechanisms

Q. What is the difference between ‘derepressed’ and ‘hyper-produced’ AmpC?

AmpC expression is governed by three genes (ampC, ampR and ampD). AmpC produces the ampC enzyme, ampR produces a repressor protein that sits on the ampC gene and stops it from expressing in normal circumstances and ampD has a role in cell wall turnover.

In the case of inducible AmpC resistance, upon exposure to the B-lactam antibiotic, ampD is overwhelmed by the abundance of cell wall components and ampR (repressor) lifts off the ampC gene to help out. This means the ampC will now be expressed. This can be seen in susceptibility testing when there is a flattened zone around the cefoxitin disc on the side against the cefopodoxime or other 3rd genetaion cephalosporin.

In the case of derepressed AmpC resistance, the ampD gene is mutated and therefore non‑functional. The ampR gene now acts in place of the non-functional ampD and the ampC gene will be expressing all the time. This can be seen in susceptibility testing when there is no zone to cefoxitin – a classic sign of ampC activity.

Q. Can you get an ESBL-positive Hafnia alvei?

Yes, H. alvei can have ESBLs. They also have intrinsic AmpC.

Q. Do mecillinam-resistant, amoxicillin-susceptible E. coli exist?

Yes, it is possible for isolates to be susceptible to amoxicillin but resistant to mecillinam. The resistance mechanisms for amoxicillin resistance is mainly B-lactamase-mediated, whereas mecillinam resistance is probably due to mutations in the cysB gene (not B-lactamase or PBP alteration).

Does piperacillin/tazobactam have any effect on meropenem activity? Piptaz is next to meropenem on our first line pseudomonas dispenser and we sometimes observe a corrupted zone on pip/taz.

Pseudomonas species all produce ampC B-lactamases, meropenem is a strong inducer of this ampC. More of this ampC B-lactamase means the organism can withstand growing in the presence of piperacillin – and so grows closer to the pip/taz disc – don’t forget tazobactam has no effect on ampC B-lactamases.

Q. What causes the interaction between mecillinam and trimethoprim when the two discs are placed next to one another one?

This is a synergistic reaction between trimethoprim and mecillinam. Mecillinam disrupts the cell wall and in doing so aids access of trimethoprim inside the cell to reach its target.

Q. What is the mechanism that causes in-vitro synergy between clindamycin and gentamicin in Staphylococcus aureus?

Synergy between clindamycin and gentamicin has been seen in both staphylococci and streptococci; however, it is very strain specific, with other strains showing antagonism. The synergistic mechanism is unknown. It is more likely that this combination is antagonistic as the antibiotics target different regions of the ribosome (clindamycin 50s ribosomal subunit and gentamicin 30s ribosomal subunit), which means the molecules might get in the way of each other. Clinically, there has been no evidence of synergy between these two agents but that is a not to say the agents might not act synergistically if the isolates show synergy in-vitro.

Q. Does Listeria monocytogenes have intrinsic resistance to ciprofloxacin?

No, Listeria monocytogenes is not inherently resistant to ciprofloxacin, although many isolates have the gyrA mutation. Listeria can acquire ciprofloxacin resistance via an efflux pump, which increases the MIC.

Q. Are macrolides and lincosamides two different classes of antibiotic and is it possible for either drug to be sensitive or resistant independently from one another?

Macrolides and lincosamides have completely different structures; nevertheless, the drugs have overlapping rRNA targets so will be affected by the same resistance mechanisms. Acquired erm genes add a methylase critical adenine on the ribosome, blocking the actions of both drug classes. This system is induced strongly by macrolides, but not by lincosamides.  So, if the gene remains inducible, only macrolide resistance is obvious.  Lincosamides are antagonised if erythromycin is added to the susceptibility test and lincosamide resistance is obvious if the erm/MLSb becomes constitutive owing to a mutation.  Although the erm gene is the commonest resistance mechanism in staphylococci and streptococci, acquired mef genes, mostly in streptococci, confer macrolide resistance, but have no effect on lincosamides. Acquired lnu genes, which are rare, mostly in staphylococci, encode lincosamide nucleotidyl transferase, inactivating lincosamides (& conferring resistance) but not affecting macrolides.

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