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The determination of minimum inhibitory concentrations by broth microdilution is recommended as method of choice for susceptibility testing of veterinary bacterial pathogens. Accordingly, broth microdilution is used in veterinary routine diagnostic laboratories at a progressive rate. To reduce the costs of susceptibility testing, it is reasonable to develop widely accepted uniform microtitre plate layouts that are produced in large quantities. Such microtitre plate layouts have already been developed and published for the susceptibility testing of pathogens from food-producing animals. However, a microtitre plate layout, especially designed for the testing of bacteria from dogs and cats, should be available, too. The choice of the antimicrobial agents or combinations of antimicrobial agents to be included in a suitable layout should be based on the following criteria: (1) the approval and availability of an antimicrobial agent or combination of agents, (2) known cross-resistances, and (3) availability of approved clinical breakpoints. The latter point is of particular importance for the choice of the numbers of concentrations per antimicrobial agent tested and the range of test concentrations. Taking into account these aspects, a science-based layout proposal for microtitre plates, which are suitable for routine testing of bacteria from dogs and cats, is presented and discussed.

Phenotypic resistance of veterinary pathogens to more than one antimicrobial agent (multi-resistance) may be caused by intrinsic resistance to the antimicrobial agents, acquired cross-resistance, or acquired co-resistance. Known cross-resistances allow to select so-called "representative substances" which are tested and the results of which can also be regarded as being valid for other members of the same class of antimicrobial agents. In general, a limitation in the number of antimicrobial agents to be tested in routine diagnostics is necessary because of capacity and cost efficiency. This is of particular relevance when the broth microdilution method - recommended as the method of choice - with 96-well microtiter plates is used. The knowledge about the relationship between different resistance phenotypes and the corresponding resistance mechanisms is of major value for both, the laboratory personnel and the veterinary practitioner. This review explains how "representative substances" for the most relevant classes of antimicrobial agents used in veterinary medicine are chosen on the basis of known cross-resistances.

In accordance with NCCLS guideline M31-A2, the DVG working group "antimicrobial resistance" developed a standard operating procedure (SOP) for the determination of the minimal inhibitory concentration (MIC) of antimicrobial agents by broth microdilution. This SOP was evaluated for its fitness for use in a national interlaboratory test. A total of 32 participating laboratories tested five strains (including two internationally accepted reference strains and three field strains representing in total three different bacterial species) three times at a one week interval each, using uniform microtitre plates. In 31 of the 32 laboratories more than 80% of MIC determinations performed yielded values in the expected range. In total 94.0% of the results were reproducible, with a lesser deviation of 4.0% from the expected values for laboratories performing MIC determination as a matter of routine (46.9%), compared to 7.9% for laboratories without such routine (53.1%). Comparing the consistency of results on the basis of the tested strains, a higher reproducibility of the results was observed for reference strains (96.1%) than for field strains (92.6%). In particular results obtained for the Streptococcus uberis field strain were afflicted with a higher error ratio (98 deviations from the expected values). Among the tested antimicrobial agents, a higher variability of results was recorded only for gentamicin with 16.7% divergent MIC determinations (mean value 6.0%). The high reproducibility of the results confirmed by this interlaboratory study underlines the robustness of the developed SOP as well as broth microdilutions as the method of choice for MIC determina tion.

Two layouts for microtitre plates, which should serve for in-vitro susceptibility testing in routine diagnostics, have been set up by the working group "Antibiotic resistance" of the German Society for Veterinary Medicine. One of these layouts was designed for the testing of bacteria from cases of mastitis and the other for bacteria from infections in large food-producing animals. The choice of the antimicrobial agents and their concentrations to be included in these layouts were based on (1) the bacteria frequently associated with the respective diseases/animals, (2) the antimicrobial agents licensed for therapeutic use in these diseases/animals, (3) the currently available breakpoints, and (4) cross-resistances between the antimicrobial agerts so far known to occur in the respective bacteria.

In vitro susceptibility tests are performed to receive information for selecting the most suitable antibacterial agent. As result of in vitro susceptibility tests, the minimum inhibitory concentration (MIC) indicates bacteria as resistant or sensitive. To determine MIC, therapeutically relevant breakpoints have to be defined. Microbiological criteria, chemical and physical characteristics as well as pharmacokinetic and toxicological (tolerance) properties of the antimicrobial compounds have to be considered in the selection of the therapeutic agent in addition to clinical experiences. Using some concentration and time dependent antibiotics as examples, it is demonstrated that the above mentioned criteria are not sufficiently considered in currently defined breakpoints.

In-vitro susceptibility testing provides valuable informations for choosing the most suitable antimicrobial agent for the control of bacterial infections in animals. Different diffusion and dilution methods, as conducted according to various approved performance standards, can be used to determine the in-vitro susceptibility of bacterial pathogens. In the present article, problems are discussed which arise from the use of different methods and the difficulty to interpret such results. While most approved performance standards were designed for testing of bacteria from human sources, the NCCLS document M31-A2 exclusively focusses on susceptibility testing of bacteria isolated from animals and--in contrast to all other standards--includes veterinary specific breakpoints for a number of antimicrobial agents used in veterinary medicine. Therefore, performance of in-vitro susceptibility testing of veterinary pathogens should follow the recommendations given in the NCCLS document M31-A2. The microdilution method is recommended as the method of choice for susceptibility testing. The result of a microdilution test is given as the minimum inhibitory concentration (MIC). This value provides a quantitative result which precisely indicates the degree of susceptibility of the tested bacterial strain and in return gives the veterinarian a clear guidance whether therapeutic intervention with the antibiotic in question will be successful.