Normalised Resistance Interpretation Research Articles

Antimicrobial Susceptibility and Local Epidemiological Cut-Off Values of Vibrio anguillarum Isolated From Fish Farms in Turkey.

Studies on preventing antimicrobial resistance in aquaculture emphasise the need to responsibly and prudently use antimicrobials, selecting those most effective in controlling and/or reducing mortalities caused by vibriosis. In this study, the distribution of the antimicrobial susceptibility of 28 Vibrio anguillarum isolates from Turkish fish farms was determined using the broth microdilution minimum inhibitory concentration (MIC) test according to the Clinical and Laboratory Standards Institute (CLSI) guideline. The epidemiological cut-off (COWT) values of the V. anguillarum isolates of florfenicol (FLO), tetracycline (TET), doxycycline (DOX), oxolinic acid (OXO), enrofloxacin (ENR) and trimethoprim/sulfamethoxazole (SXT) were calculated using the normalised resistance interpretation (NRI) and ECOFFinder methods. Isolates were categorised as belonging to the fully susceptible wild-type (WT) or non-wild-type (NWT) populations. Calculated COWT values (in μg mL-1) were ≤ 1.0 for FLO and DOX, ≤ 0.5 for TET, ≤ 0.016 for ENR, ≤ 0.032 for OXO, and ≤ 4.0 for SXT using the NRI analysis. Percentages of V. anguillarum isolates categorised as belonging to the NWT population were small for the antimicrobials FLO (10.7%) and SXT (0%), whereas they were higher for the antimicrobials OXO (39.3%) and ENR (39.3%). This is the first study to estimate the local COWT values for antibiotics used in the control of V. anguillarum isolates recovered from farmed fish in Turkey.

Epidemiological cut-off values for Yersina ruckeri disc diffusion data generated by a standardised method.

In order to establish the meaning of data generated in antimicrobial agent susceptibility tests, it is necessary to develop internationally harmonised interpretive criteria. Currently, such criteria have not been developed for data generated in studies of the susceptibility of the fish pathogen Yersinia ruckeri. This work generated the data that would be required to set epidemiological cut-off values for the susceptibility data of this species that had been generated using a standardised disc diffusion method that specified the use of Mueller Hinton agar and incubation at 22°C for 24-28 h. Using this method, sets of inhibition zones data for 4 antimicrobial agents were generated by 3 independent laboratories. The data from these laboratories were aggregated and analysed using the statistically based normalised resistance interpretation. For ampicillin, florfenicol, oxytetracycline and trimethoprim-sulfamethoxazole the cut-off values calculated by this analysis were ≥16, ≥23, ≥24 and ≥30 mm, respectively. Evidence is presented demonstrating that the data for these 4 agents was of sufficient quantity and quality that they could be used by the relevant authorities to set internationally harmonised, consensus epidemiological cut-off values for Y. ruckeri.

Epidemiological cut-off values for non-O1/ non-O139 Vibrio cholerae disc diffusion data generated by standardised methods

This work generates the data needed to set epidemiological cut-off values for disc-diffusion zone measurements of Vibrio cholerae. The susceptibility of 147 European isolates of non-O1/non-O139 V. cholerae to 19 antibiotics was established using a standardised disc diffusion method which specified incubation of Mueller Hinton agar plates at 35°C. Epidemiological cut-off values were calculated by analysis of the zone size data with the statistically based normalised resistance interpretation method. Cut-off values for 17 agents were calculated by analysis of the aggregated data from all 4 laboratories participating in this study. The cut-off values calculated were ≥18 mm for amoxicillin/clavulanate, ≥18 mm for amikacin, ≥19 mm for ampicillin, ≥27 mm for cefepime, ≥31 mm for cefotaxime, ≥24 mm for ceftazidime, ≥24 mm for chloramphenicol, ≥31 mm for ciprofloxacin, ≥16 mm for erythromycin, ≥ 27 mm for florfenicol, ≥16 mm for gentamicin, ≥23 mm for imipenem, ≥25 mm for meropenem, ≥29 mm for nalidixic acid, ≥28 mm for norfloxacin, ≥13 mm for streptomycin and ≥23 mm for tetracycline. For the other 2 agents the data from 1 laboratory was excluded from the censored aggregation because the data from that laboratory was considered excessively imprecise. The cut-off values for these 2 agents calculated for the aggregation of the data from 3 laboratories were ≥23 mm for trimethoprim and ≥24 mm for trimethoprim/sulfamethoxazole. These zone size data will be submitted to the Clinical Laboratory Standards Institute (CLSI) and European Committee for Antimicrobial Susceptibility Testing (EUCAST) for their consideration in setting international consensus epidemiological cut-off values for non O1/non-O139 V. cholerae.

Influence of incubation temperature and time on the precision of MIC and disc diffusion antimicrobial susceptibility test data

Normalised resistance interpretation was applied to measure the effects of incubation temperature and times on the precision of the data generated by MIC and disc diffusion antimicrobial susceptibility tests performed at 35 °C for 16 h, 28 °C for 24 h, at 22 °C for 24 h, at 22 °C for 48 h and <19 °C for >96 h. Analysis of 151 MIC data sets and 141 disc diffusion data sets demonstrated the precision of MIC data sets was not affected by the incubation conditions but that the precision of the disc data decreased significantly as the incubation temperature decreased and the time increased. For MIC data sets the standard deviation of the normalised distribution of the log2 transformed wild-type observations, which were independent of the numerical values of those sets, were applied as measures of the relative data set precision. The mean values of these standard deviations for 59 data sets generated at 35 °C, 27 data sets generated at 28 °C, 28 data sets generated at 22 °C and 37 data sets generated at 18–19 °C were 0.76 ± 0.22 log2 μg ml−1, 0.76 ± 0.26 log2 μg ml−1, 0.77 ± 0.20 log2 μg ml−1and 0.76 ± 0.19 log2 μg ml−1 respectively. For the disc diffusion data sets the relative precision was quantified by calculating the standard deviation of the normalised distribution of the wild-type observations. The mean values of the standard deviation for 40 data sets generated at 35 °C, 50 data sets generated at 28 °C, 25 data sets generated at 22 °C and 26 data sets generated at ≤18 °C were 2.15 ± 0.61 mm, 2.65 ± 0.74 mm, 4.56 ± 1.09 mm and 6.70 ± 1.70 mm respectively. Because the incubation times increased as the temperatures decreased it was not possible in this work to determine whether changes in either or both were responsible for the reduction in precision observed in the disc zone data.The low precision of disc diffusion data obtained after prolonged incubation at temperatures below 22 °C suggests, that for bacteria that require these conditions, preference should be given to MIC susceptibility testing methods.

Epidemiological cut-off values for disc diffusion data generated by standard test protocols from Edwardsiella tarda and Vibrio harveyi

The susceptibility of 52 isolates of Edwardsiella tarda and 54 isolates of Vibrio harveyi to 12 antibiotics was established using internationally standardised disc diffusion protocols. Normalised resistance interpretation was employed to generate statistically valid epidemiological cut-off values for these susceptibility data. For three of these 24 species/antibiotic data sets, there were insufficient fully sensitive strains to allow analysis and for an additional two there were sufficient fully susceptible isolates to allow only a provisional estimate to be made. The data sets for a further two species/antibiotic combinations were considered too diverse to allow the calculation of a valid cut-off value. Valid epidemiological cut-off values were, therefore, calculated for 17 species/antibiotic combinations. It is argued that these cut-off values will greatly facilitate the performance of studies designed to monitor the consequence of antibiotic use in aquaculture. They will also facilitate the prudent and rational choice of antibiotics to be applied therapeutically in commercial farming of aquatic animals.

How many strains are required to set an epidemiological cut-off value for MIC values determined for bacteria isolated from aquatic animals?

Normalised resistance interpretation was applied to the estimation of epidemiological cut-off values from data sets that contained >150 observations from fully susceptible wild-type (WT) strains for six bacterial groups/antibiotic combinations. A series of 20 subsets containing 10, 20, 30 and 40 WT observations were randomly generated from these six larger data sets. The epidemiological cut-off values estimated using these 480 small data sets were compared with those estimated from their larger, parental data sets. Eighty-seven per cent of the 360 epidemiological cut-off values estimated from subsets that contained ≥20 WT observations were in agreement with those estimated from their respective larger sets. The remaining 13 % differed only by one doubling dilution. These calculations suggest that small strain sets can be used to set epidemiological cut-off values for MIC data without unacceptable loss of precision. It is concluded that the design of experiments intended to generate the data necessary to set epidemiological cut-off values should aim to include observations on at least 30 WT observations.

Setting an epidemiological cut-off value for the single plate protocol from disc diffusion data generated for Aeromonas salmonicida

The single plate protocol was applied to generate susceptibility measures for 50 strains of Aeromonas salmonicida against five antibiotic agents. Analysis of the resultant data demonstrated that a single epidemiological cut-off value could be set that was appropriate for all 5 agents. The classification of strains as wild type or non-wild type by the application of this cut-off value showed complete agreement with the classification produced by applying normalised resistance interpretation generated cut-off values to the data generated by the standard Clinical and Laboratory Standards Institute protocols.Consideration of the design features of the single plate protocol and the published disc diffusion data for bacteria isolated from aquatic animals suggests that the application of this single cut-off value is valid for many, if not all, agents and species and in all laboratories. It is argued that the existence of a single universal cut-off value adds to the already considerable advantages that the single plate protocol presents for laboratories involved in clinical diagnosis of disease epizootics in aquaculture.

Use of normalised resistance analyses to set interpretive criteria for antibiotic disc diffusion data produce by Aeromonas spp

Normalised resistance interpretive (NRI) analysis was applied to the development of interpretive criteria for antibiotic disc diffusion zones of mesophilic Aeromonas spp. Minimum quantitative and qualitative properties of data sets from which normalised resistance interpretation could generate acceptable cut-off values were established by examining published data that had been used to set the epidemiological cut-off values for bacteria of human and aquatic interest. Applying these criteria to disc diffusion data generated in a previous study of 129 mesophilic Aeromonas spp. isolated from pet fish, demonstrated that normalised resistance interpretation was capable of generating acceptable cut-off values for seven (erythromycin ≥ 8 mm, gentamicin ≥ 18 mm, moxalactam ≥ 32 mm, oxytetracycline ≥ 21 mm, streptomycin, ≥ 20 mm tetracycline ≥ 28 mm, and trimethoprim/sulfamethoxazole ≥ 26 mm) of the agents tested. This analysis demonstrated, with respect to these agents, that a single set of interpretive criteria could be developed for application to data generated from all the Aeromonads studied. Provisional cut-off values were generated for three other agents (chloramphenicol ≥ 35 mm, florfenicol ≥ 35 mm and furazolidone ≥ 23 mm). The distribution of zone sizes for these agents showed an apparent bimodality in the high-zone end. As a result, the question of whether, for these three agents, a single set of interpretive criteria could be validly set for all Aeromonads could not be resolved. With respect to the five quinolone agents studied, a cut-off value could only be estimated for oxolinic acid. Because of the high frequency of resistance to quinolones in the strain set, this value (≥ 32 mm) should be treated as a provisional estimate. With respect to some agents, strains manifesting a low-level resistant phenotype, zones ≤ 8 mm smaller than the wild-type cut-off, represented a significant proportion of the strains classified as non wild-type. The difficulties that these frequencies of low-level resistance would present for attempts to set and apply universal, laboratory-independent interpretive criteria are discussed.

Setting interpretive breakpoints for antimicrobial susceptibility testing using disk diffusion

Antimicrobial susceptibility testing plays a key role in clinical microbiology. The disk diffusion test dates back to the 1940s and became standardised from the 1950s, with the International Collaborative Study (ICS) and National Committee for Clinical Laboratory Standards (NCCLS) as the two major standards. Interlaboratory variation of disk test results was recognised early but has never been dealt with in a satisfactory manner. The error-rate bounded method was described in 1974 and its role is discussed. Species-specific susceptibility interpretation was coined in 1980 for Proteus mirabilis and chloramphenicol. In the late 1970s, more extensive use of species-specific breakpoints was introduced in Lund (Sweden). At the same time, P. Mouton constructed species-specific regression lines and pointed out the difficulties with narrow ranges of minimal inhibitory concentration (MIC) values. A more general use of species-specific regression lines was made possible with single-strain regression analysis, using one well-defined strain tested in disk diffusion with a range of disk contents. This method made it possible to calibrate the disk test in an individual laboratory. Other methods to achieve such calibration are also described. A recent method, ‘MIC-coloured zone diameter histogram-technique’, has proven useful for the validation of species-specific interpretive breakpoints. The microbiological breakpoint proposed by Williams in 1990 has experienced a renaissance with the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological cut-off value (ECOFF). MIC and zone diameter distributions with accompanying ECOFFs for species–antimicrobial combinations are published on the EUCAST website. A method for the reconstruction of wild-type zone diameter populations, namely normalised resistance interpretation, is described. This method can produce resistance figures that are truly comparable between laboratories.

A rapid method of improving the criteria being used to interpret disc diffusion antimicrobial susceptibility test data for bacteria associated with fish diseases

This work presents an analysis of the consequence of using data sets containing small numbers of disc diffusion inhibition zones to calculate epidemiological cut-off values. Twenty replicate data sets that contained 10, 20, 40, 60, 80 and 100 zone size measurements were obtained by randomly sampling a previously published data set that consisted of inhibition zone measurements for discs containing 30 µg florfenicol, 30 µg oxytetracycline and 2 µg oxolinic acid against 217 isolates of Aeromonas salmonicida. Application of normalised resistance interpretation allowed epidemiological cut-off values to be estimated from all of these 360 data sets. The precision associated with the replicate cut-off values was a linear function of the log of the number of data points used to generate them. The frequency with which false sensitive and false resistance errors would have resulted from the application of the cut-off values generated from small data sets using normalised resistance interpretation were compared with those that would have resulted from the application of the breakpoints that have been reported as being currently in use. This comparison demonstrated that most laboratories could improve their interpretation of the disc diffusion data they obtain for any species by performing normalised resistance interpretation of their own data for that species. This would be true even if they possessed inhibition zone data for as few as ten members of that species.

How difficult is it to achieve compliance with the quality control requirements of the Clinical and Laboratory Standards Institute's guideline M42-A?

Data accumulated in a multi-laboratory study of disc diffusion susceptibility data for Escherichia coli ATCC 25922 was analysed with respect to the acceptable ranges for this strain specified in the Clinical and Laboratory Standards Institute's M42-A guidelines. Seven laboratories each generated 60 zone size measurements for discs containing five agents for which acceptable ranges had been published. Of the 35 data sets analysed 37% contained >10% of their measurements outside the acceptable ranges. For each agent, between one and four of the seven data sets obtained contained >10% of the zone sizes outside the acceptable ranges. Only one laboratory obtained data sets that for all agents, contained <10% of the zone sizes outside the acceptable. For the other six, non-compliant data sets were obtained for between one and four of the agents. These data suggest that the adherence to a specified test protocol cannot be assumed to result in the achievement of compliance with the quality control requirements of that protocol. They would further suggest that the need to undertake corrective actions, as specified in M42-A, would be frequently required before a laboratory could claim to be using this protocol and could legitimately apply any cut-off values or breakpoints that are or that might be, associated with it use. As laboratories involved in susceptibility testing of bacteria associated with aquaculture frequently handle only a small number of strains per year, the achievement of compliance with the quality control requirements of a standard protocol might require a disproportionate and unacceptable increase in their work load. The data obtained in this retrospective study also demonstrated that, even if all data sets containing >10% non-compliant measurements were eliminated; significant inter-laboratory variation in mean zone sizes would remain.Laboratory-independent cut-off values will be of value only if inter-laboratory variation in disc diffusion data can be reduced to a minimum. It is argued that the data presented here provides strong grounds for questioning whether, particularly in the case of laboratories with small strain throughputs, inter-laboratory variation can be appropriately resolved by rigorous specifications of test protocols and quality control criteria. Thus, they would rather support the development of cut-off values by the application of normalised resistance interpretation. As the cut-off values generated by this approach are laboratory-specific, they are not influenced by inter-laboratory variation.

The advantages of the use of discs containing single agents in disc diffusion testing of the susceptibility of Aeromonas salmonicida to potentiated sulphonamides

The susceptibilities of 106 strains of Aeromonas salmonicida to trimethoprim/sulfamethoxazole (SFT) were determined in two laboratories using the Clinical and Laboratory Standards Institute's M42-A disc diffusion protocols. The data generated by the use of discs containing 25 μg SFT (SFT25) allowed the strains to be placed into two groups. Strains in one group (17 strains) generated no inhibition zones and the zones obtained from the other 89 strains were distributed over a wide range but showed no natural division into separate sub-classes. A further investigation performed by one of the participating laboratories, of the susceptibility of 91 of these 106 strains used discs containing 100 μg sulfmethoxazole (SFM100) and 5 μg trimethoprim (TMP5). Application of normalised resistance interpretation to these data allowed the estimation of epidemiological cut-off values for WT strains of ≥9 mm for SFM100 and ≥21 mm for TMP5. This investigation demonstrated the presence of three distinct phenotypic classes, one containing strains manifesting wild type susceptibility to both agents, another containing strains manifesting non-wild type susceptibility to both and a third containing strains manifesting wild type susceptibility with respect to TMP but non-wild type with respect to SFM. Analysis demonstrated the inability of SFT25 discs to generate data that allowed the separate identification of strains that were fully susceptible to both TMP and SFM from those that were fully susceptible to TMP but were not fully susceptible to SFM.It is recommended that, in investigation of the susceptibility to potentiated sulphonamides of isolates from diseased fish, separate discs, containing the individual components of the mixture, should be employed.

Application of normalised resistance interpretation to disc diffusion data on the susceptibility of Aeromonas salmonicida to three quinolone agents

The susceptibilities of 106 strains of Aeromonas salmonicida to oxolinic acid (OXA), flumequine (FLU) and enrofloxacin (ENR) were established in two independent laboratories, using the M42-A disc diffusion protocols of the Clinical and Laboratory Standards Institute. The zone sizes obtained for OXA and FLU by both laboratories showed a bi-modal distribution. For OXA there was clear separation of the two modal classes but for FLU the zones for the two classes showed a degree of overlap. In contrast, there was no clear evidence of bi-modality in the data obtained for ENR.Normalised resistance interpretation (NRI) was used to calculate laboratory-specific epidemiological cut-off values for wild type (WT) strains for all three agents. For OXA these cut-off values were 30 mm and 33 mm for the two laboratories and their application resulted in a complete concordance in the classification of the strains by the two laboratories. For FLU the NRI cut-off values were 39 mm and 40 mm and these resulted in an agreement between the laboratories in the classification of 95% of the 106 strains. The NRI cut-off values for ENR were 36 m and 40 mm and resulted in an agreement with the classification of 90% of these strains. When the zone sizes for the two modal classes overlap some strains with non wild type (NWT) phenotypes would generate zones larger than the WT cut-off value set by NRI analysis. Thus, definitely for ENR and possibly for FLU, the use of NRI cut-off values as provisional breakpoints could lead to some NWT strains being reported as sensitive.Analysis of the data obtained in this work demonstrated that the frequency of cross-resistance between the three agents was high and this is consistent with other reports in the literature. This high level of cross resistance provides a method by which the problems with the cut-off values set by NRI analysis can be avoided. It is argued that the use of OXA as a reporter agent represent an effective method of reducing error in reporting susceptibility to ENR and FLU. In this approach all strains shown to be NWT with respect to OXA would also be reported as NWT with respect to the other quinolones. The validity of this approach is dependent on there being a very low frequency of strains that are resistant to ENR and FLU but sensitive to OXA.It is recommended that, at the present state of our knowledge, the most effective method of reducing errors in determining the susceptibility of strains of A. salmonicida to quinolone agents is to employ OXA disc data as a proxy measure for susceptibility to the other agents in this class.

Application of Normalised Resistance Interpretation to the detection of multiple low-level resistance in strains of Vibrio anguillarum obtained from Greek fish farms

Disc diffusion methods were employed to characterise the distributions of susceptibility to oxolinic acid, oxytetracycline, trimethoprim/sulfamethoxazole and furazolidone in 38 strains of Vibrio anguillarum isolated from Greek fish farms. For each of the four agents one strain gave no zone of inhibition and these four strains could be identified, unambiguously, as manifesting non-wild type (NWT) susceptibility. Visual examination of the distributions of zone sizes obtained with oxolinic acid discs for the remaining strains suggested that 4–5 of these had a reduced susceptibility and could also be classified as NWT. The zone sizes obtained for the other three agents were distributed within a single group that, in all cases, showed a significant skew towards lower values. Normalised Resistance Interpretation (NRI) was used to investigate these data and this analysis suggested that for each of the three agents, oxytetracycline, trimethoprim/sulfamethoxazole and furazolidone, there were grounds for classifying further seven strains as non-wild type and, therefore, as manifesting a low-level resistance phenotype. In total, 11 strains were identified as manifesting low-level resistance to at least one of the four agents. Of these 11 strains, 2 were classified as manifesting a low-level resistance to all four agents and 3 showed this phenotype with respect to at least three of the agents. Although this analysis suggests that multiple low-level resistance might be reasonably common in V. anguillarum there are, at present, no data that allow any prediction to be made as to the probable clinical significance of such strains.

Impact of inter-lab variation on the estimation of epidemiological cut-off values for disc diffusion susceptibility test data for Aeromonas salmonicida

Two laboratories investigated the susceptibility of 106 Aeromonas salmonicida strains (from Denmark, France, Ireland, Norway and Scotland) to erythromycin, gentamicin, oxytetracycline and oxolinic acid using the disc diffusion protocols (M42-A) published by the Clinical and Laboratory Standards Institute. In studies of susceptibility to florfenicol an additional 15 Canadian strains were included. Comparison of the data generated by the two laboratories demonstrated that for each disc both detected a similar pattern of distribution but that there was a significant numerical difference in the zone sizes they recorded. Analysis of the extent of this lateral shift between the data generated in two laboratories indicated that the application of a single laboratory-independent epidemiological cut-off value for each disc could result in disagreement between the laboratories as to whether a strain should be classified as wild-type or non wild-type.Normalised resistance interpretation was employed to generate epidemiological cut-off values from the data obtained by each laboratory. The use of these laboratory-specific cut-off values resulted in both laboratories achieving complete agreement as to the classification of all strains to all agents.

Extended antimicrobial resistance screening of the dominant faecal Escherichia coli and of rare resistant clones

Fifty faecal samples from healthy adults were grown on MacConkey agar and three pink colonies were subcultured, identified to species level and their antimicrobial susceptibility determined. Forty-seven samples yielded 141 isolates of Escherichia coli that were susceptible to most antimicrobials. Resistance was noted for ampicillin (30.5%), chloramphenicol (12.1%), tetracycline (23.4%), trimethoprim (24.8%) and co-trimoxazole (22.7%). A direct faecal plating method was used for extended resistance screening with E. coli as the indicator organism. Zone breakpoints were determined using normalised resistance interpretation and gave similar susceptibility results. Eighty-eight isolates of E. coli from within the zones of inhibition revealed four times more antimicrobial resistance. Extended antimicrobial resistance screening both provides the susceptibility profile of the dominant E. coli isolate and detects greater resistance in rare isolates.

Meropenem susceptibility breakpoint for Pseudomonas aeruginosa strains hyperproducing mexB mRNA

Twenty-five isolates of Pseudomonas aeruginosa with different meropenem susceptibilities were subjected to quantitative RT-PCR for analysis of transcription levels of oprD, mexB and mexD, and, in selected isolates, PA3720, which is hyper-expressed in nalC efflux mutants. Regulator genes of efflux pump MexAB-OprM, mexR and PA3721 (putative) were sequenced in selected isolates. The potential for mathematical reconstruction of the ideal susceptible population using normalised resistance interpretation (NRI) was also studied. In three isolates with intermediate susceptibility to meropenem (according to Swedish breakpoints), a reduction in MIC from 4 to 2 mg/L was observed with efflux inhibitor MC-207,110. These isolates would be considered susceptible according to British Society for Antimicrobial Chemotherapy and NCCLS breakpoints. These three isolates had between 4.6- and 5.0-fold increases in mexB transcription. None of these isolates had significant nalB mutations, but an Ala145-->Val mutation was observed in PA3721 in two of the isolates. However, these isolates had moderately increased production of PA3720 only. Single-strain regression analysis did not detect any major biological differences between the different groups. Using NRI, a disk-diffusion susceptibility breakpoint of >/= 28 mm was generated. Isolates with intermediate susceptibility to meropenem, which are considered fully susceptible in many countries, displayed possible low-grade meropenem resistance mechanisms, implying that the susceptibility breakpoint should be reconsidered. The increased transcription of mexB mRNA in such isolates seems unrelated to nalB or nalC mutations.

Determination of the real standard distribution of susceptible strains in zone histograms

The procedure for NRI, a method for normalised resistance interpretation, is presented. By means of its promising autocalibration system the method generates zone breakpoints for resistance in zone diameter histograms for bacterial species–antibiotic combinations. These breakpoints give a normalised interpretation of resistance which is independent of disc test standard chosen, disc potency, medium used, inoculum size, etc. of the individual laboratory as long as the performance shows good reproducibility and precision. The method should be further tested as a tool for setting ‘bacteriological breakpoints’ or ‘epidemiological breakpoints’, for calculating resistance rates for comparative purposes, e.g. in antibiotic resistance surveillance, and for quality control of the disc diffusion test.