Yersinia ruckeri is a Gram-negative bacterium that causes enteric redmouth disease. It commonly infects farmed salmonid fishes, often requiring antimicrobial treatment. Studies on antimicrobial susceptibility patterns of Y. ruckeri have found isolates with decreased susceptibility but have lacked internationally harmonized criteria, known as epidemiological cutoff values (ECVs), which provide consensus on identifying antimicrobial resistance. To address this need, we gathered minimal inhibitory concentration (MIC) testing data for 9 antimicrobials, generated at 22°C for 24-28 and/or 44-48 h using the standard broth microdilution testing method published by the Clinical and Laboratory Standards Institute (CLSI). The dataset includes MIC data from up to 431 isolates from 7 independent laboratories. Data for each antimicrobial were analyzed with the programs Normalized Resistance Interpretation (NRI) and ECOFFinder to compare the means and standard deviations for the 2 timepoints, and to calculate wild-type cutoff values. The parameters for the timepoints were very similar, which confirmed cutoffs were only needed for the 24-28 h incubation. The cutoff values for 8 of the 9 antimicrobials are potential ECVs that have been proposed to CLSI's Subcommittee on Veterinary Antimicrobial Susceptibility Testing. Calculated cutoffs for the final antimicrobial, erythromycin, were off-scale because the MIC values clustered at higher drug concentrations. The resulting new ECVs will be included in the next edition of the VET04 supplement, which will help clinicians and researchers advance antimicrobial resistance surveillance of this bacterium.

This work was performed to generate the data needed to set epidemiological cut-off values for inhibition zone data of 8 antimicrobial agents against Vibrio parahaemolyticus determined using standardised disc diffusion protocols with incubation at 35 ± 2°C for 16 to 20 h and at 28 ± 2°C for 24 to 28 h. The zone diameter data aggregated from 3 to 5 laboratories were analysed by the normalised resistance interpretation algorithm. Cut-off values calculated from data obtained at 35 and 28°C were ≥23 and ≥24 mm for ceftazidime, ≥18 and ≥20 mm for enrofloxacin, ≥28 and ≥29 mm for florfenicol, ≥14 and ≥14 mm for gentamicin, ≥29 and ≥32 mm for meropenem, ≥17 and ≥18 mm for oxolinic acid, ≥22 and ≥24 mm for oxytetracycline, as well as ≥19 and ≥21 mm for trimethoprim/sulfamethoxazole. The influence of the incubation temperature on inhibition zone sizes was investigated by calculating the difference between the zones obtained at 35 and 28°C for a specific antimicrobial agent with a particular isolate by an individual laboratory. The mean of the differences calculated for 1314 paired observations was 0.38 mm with a standard deviation of 2.68 mm. The data generated in this work will be submitted to the Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing for consideration in their setting of internationally agreed-upon epidemiological cut-off values for V. parahaemolyticus that are essential for interpreting antimicrobial susceptibility testing data of this species.

This study analysed published data on the distributions of minimum inhibitory concentrations of a group of freshwater isolates classified as Aeromonas spp. with the aim of establishing whether they provided any evidence that epidemiological cut-off values set from these data would be unreliable. This group contained 233 isolates and included members of at least 11 species. The standard deviations (SDs) of the wild-type distributions for 10 antimicrobial agents were calculated for this multi-species group using the ECOFFinder and normalised resistance interpretation (NRI) algorithms. These were compared to the SDs of 110 distributions established for individual species published by the European Committee on Antimicrobial Susceptibility Testing. Fifty-one of these distributions had been generated by multiple laboratories and 59 by single laboratories. When the ECOFFinder algorithm was used to calculate the SDs, the mean for the multi-species group was 0.63 log2 µg ml-1, and the 51 individual species and multiple-laboratory groups were 0.68 and 0.65 log2 µg ml-1, respectively. When the NRI algorithm was used, the mean for the multi-species group was 0.79 log2 µg ml-1, and the 51 individual species and multiple-laboratory groups were 0.79 and 0.76 log2 µg ml-1, respectively. These comparisons indicate that the heterogeneity in the susceptibility to antimicrobial agents within the multi-species group of Aeromonas is not significantly different from that recorded for individual species. This analysis, therefore, suggests that epidemiological cut-off values designed to be applied to all members of the genus Aeromonas would not be inherently unreliable.

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 .

The Clinical and Laboratory Standards Institute has published epidemiological cut-off values for susceptibility data generated at 22°°C and read after 44-48 h for florfenicol, oxolinic acid and oxytetracycline against Aeromonas salmonicida. The cut-off values for the minimum inhibitory concentration (MIC) and disc diffusion were derived from data obtained by 1 laboratory and 2 laboratories respectively. The present work reports the generation of susceptibility data from additional laboratories and the calculation of provisional cut-off values from aggregations of these data with previously published data. With respect to MIC data, the provisional cut-off values, derived from aggregations of the data from 4 laboratories, were ≤4 µg ml-1 for florfenicol, ≤0.0625 µg ml-1 for oxolinic acid and ≤1 µg ml-1 for oxytetracycline. For disc diffusion data, the provisional cut-off values derived from aggregations of the data from 5 laboratories were ≥30 mm for florfenicol, ≥32 mm for oxolinic acid and ≥25 mm for oxytetracycline. In addition, a cut-off value of ≥29 mm for ampicillin was derived from the aggregation of data from 4 laboratories.

This work was performed to generate the data needed to set epidemiological cut-off values for minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against Vibrio parahaemolyticus determined using standardised broth microdilution protocols. Eight laboratories performed broth microdilution tests with incubation at 35°C for 16 to 20 h, and 7 also performed tests on the same isolates with incubation at 28°C for 24 to 28 h. Data were analysed by the ECOFFinder and normalised resistance interpretation algorithms. The cut-off values calculated for ceftazidime, florfenicol and trimethoprim/sulfamethoxazole, 1, 1 and 0.25/4.75 µg ml-1, respectively, were the same when calculated from data obtained at both temperatures. The cut-off values calculated from data obtained at 35°C and from data obtained at 28°C were 0.25 and 0.5 µg ml-1 for enrofloxacin, 2 and 4 µg ml-1 for gentamicin, 0.5 and 1 µg ml-1 for oxolinic acid and 2 and 1 µg ml-1 for oxytetracycline, respectively. The influence of incubation temperature on MIC values was investigated by comparing MICs obtained at 35 and 28°C for a specific antimicrobial agent with a particular isolate by an individual laboratory. Results showed that 56% of 1473 of these paired MIC values were identical, while 38% differed from one another by not more than 1 dilution step. The data generated in this work will be submitted to the Clinical and Laboratory Standards Institute for consideration in their setting of internationally agreed epidemiological cut-off values for V. parahaemolyticus that are essential for interpreting antimicrobial susceptibility testing data of this species.

This work aims to generate the data needed to set epidemiological cut-off values for minimum inhibitory concentration (MIC) and disc-diffusion zone measurements of Vibrio anguillarum. A total of 261 unique isolates were tested, applying standard methods specifying incubation at 28°C for 24-28 h. Aggregated MIC distributions for a total of 247 isolates were determined in 9 laboratories for 11 agents. Data aggregations of the disc zone for the 10 agents analysed contained between 157 and 218 observations made by 4 to 7 laboratories. Acceptable ranges for quality control (QC) reference strains were available for 7 agents and the related multi-laboratory aggregated data were censored, excluding the data of a laboratory that failed to meet QC requirements. Statistical methods were applied to calculate epidemiological cut-off values. Cut-off values for MIC data were calculated for florfenicol (≤1 µg ml-1), gentamicin (≤4 µg ml-1), oxytetracycline (≤0.25 µg ml-1) and trimethoprim/sulfamethoxazole (≤0.125/2.38 µg ml-1). The cut-off values for disc zone data were calculated for enrofloxacin (≥29 mm), florfenicol (≥27 mm), gentamicin (≥19 mm), oxolinic acid (≥24 mm), oxytetracycline (≥24 mm) and trimethoprim/sulfamethoxazole (≥26 mm). MIC and disc-diffusion zone data for the other agents where not supported by QC, thus yielding only provisional cut-off values (meropenem, ceftazidime). Regardless of whether QC is available, some of the aggregated MIC distributions (enrofloxacin, oxolinic acid), disc zone (sulfamethoxazole), and MIC and disc-diffusion distributions (ampicillin, chloramphenicol) did not meet the statistical requirements. The data produced will be submitted to the Clinical Laboratory Standards Institute for their consideration in setting international consensus epidemiological cut-off values.

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.

The lack of internationally harmonised criteria for interpreting the data generated by standardised susceptibility testing methods presents a serious obstacle for the development of prudent use of antimicrobials in aquaculture. The data required to set epidemiological cut-off values for minimum inhibitory concentrations for antibiotic agents against Vibrio harveyi was determined using a standard microdilution method that specified the use of cation-adjusted Mueller Hinton broth and incubation at 28°C for 24 to 28 h. In total, 120 observations were made in 4 independent laboratories from 109 unique isolates. The aggregated data from these laboratories were analysed by the normalised resistance method and by ECOFFinder to calculate epidemiological cut-off values. The data for chloramphenicol, meropenem and sulfamethoxazole were not considered as suitable for analysis. The data for ampicillin indicated that this species is innately resistant to this agent. No acceptable ranges for quality control strains have been set for ceftazidime and, therefore, only provisional cut-off values could be generated for this agent. The epidemiological cut-off values were, however, calculated for the other 6 agents. These values were ≤0.5 µg ml-1 for enrofloxacin, ≤1 µg ml-1 for florfenicol, oxolinic acid and oxytetracycline, ≤4 µg ml-1 for gentamicin and ≤0.5/9.5 µg ml-1 for trimethoprim/sulfamethoxazole. Evidence is presented demonstrating that the data for these 6 antimicrobial 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 V. harveyi.

The European Commission requested EFSA assess antimicrobial-resistant bacteria responsible for animal transmissible diseases, with a view to listing such pathogens for European Union action. This Scientific Opinion addresses the ad hoc method developed: (i) to give a global state of play as regards resistant animal pathogens that cause transmissible animal diseases, (ii) to identify the most relevant bacteria in the EU and (iii) to summarise their actual or potential animal health impact, and to perform their assessment for being listed and categorised according to the criteria of Articles 7, 5, 9 and 8 within the Animal Health Law (AHL) framework. An extensive literature review is carried out to give the global state of play of selected resistant bacteria that constitute a threat to animal health (i). An expert judgement procedure, based on the outcome of the literature review, is applied to identify which among those bacteria subjected to the literature review are the 'most relevant' in the European Union (ii). Their animal health impact in the European Union and their assessment for being listed and categorised according to the AHL framework will follow the 'ad hoc method for the assessment on listing and categorisation of animal diseases within the framework of the Animal Health Law' that EFSA has developed in the past (iii). The assessment of (i) and (ii) is addressed in distinct scientific opinions that are published separately by animal species of interest (dogs and cats, horses, pigs, poultry, cattle, small ruminants, rabbits and aquatic animal species). The assessment of (iii) is addressed in distinct scientific opinions and published separately by the animal pathogen. (c) 2021 European Food Safety Authority. EFSA Journal published by John Wiley and Sons Ltd on behalf of European Food Safety Authority.

Standard disc diffusion and MIC test procedure were used to investigate the susceptibility of two hundred and fifty-one isolates collected from infected fish in France to florfenicol, oxolinic acid and tetracycline. The tests were performed at 22 ± 2℃ and for the 177 Yersinia ruckeri they were read after 24-28 hr incubation and for the 74 Aeromonas salmonicida isolates they were read after 44-48 hr. Applying epidemiological cut-off values to the susceptibility data generated in these tests, the isolates were categorized as wild-type or non-wild-type. The agent-specific categories into each isolate were placed on the basis of the data generated by the two methods were in agreement in 98% of the determinations made. It is argued that, with respect to categorising isolates, disc diffusion and MIC methods can be considered as equally valid at this temperature and after both periods of incubation.

A multi-laboratory study was performed to investigate the most suitable incubation time for susceptibility tests of fish pathogens Vibrio anguillarum and Vibrio vulnificus performed at 28 degrees C. An isolate set consisting of 30 V. anguillarum and 26 V. vulnificus was used by four participating laboratories in Denmark, France, Sweden, and the Netherlands. Inhibition zone diameters were determined by agar disc diffusion for eight agents and Minimum Inhibitory Concentration (MIC) values were determined for seven agents using the standard CLSI testing protocols for non-fastidious organisms that specify 24-28 h incubation. In this work an additional set of readings was made after 48 h incubation. In total, 1120 paired zone sizes and 399 paired MIC observations were made at the two incubation times. Examination of the data demonstrated that incubation time had a small but statistically significant effect on the numerical values of susceptibility measures. However, the effects of incubation time on the precision of the data sets and the categorisation of isolates based on the application of epidemiological cutoff values were slight and statistically non significant. These analyses suggest that the susceptibility of these Vibrio species could be established using protocols that specify either 24-28 h or 44-48 h incubation. This study does not provide evidence that prolonged incubation to 48 h improves the quality of data generated by the tests. Therefore, it is recommended that the existing standard CLSI protocols with 24-28 h at 28 degrees C should be adopted for susceptibility testing of V. anguillarum and V. vulnificus.

The World Health Organization, the World Organisation for Animal Health, and the Food and Agriculture Organization of the United Nations recognises that the threat posed by the emergence of resistance to antimicrobials must be addressed using a One Health approach.To quantify the contribution of aquaculture to this global problem, it is essential that we generate data on the antimicrobial susceptibility of bacteria isolated from aquatic animals that is of sufficient quality.This paper presents a review of the quality of the data presented in 182 published papers on the antimicrobial susceptibility of various non-cholera Vibrio species isolated from aquatic environments.This review revealed that serious shortcomings either in the performance of the susceptibility tests or in the reporting of those tests occurred with an alarmingly high frequency.The majority of studies failed to provide sufficient details of the testing protocols they used and only a small percentage of the studies provided explicit evidence that they had used standardised susceptibility protocols.Although 203 studies reported frequencies of resistance in the isolates they studied, 185 of them either did not provided the criteria they used to determine resistance, used criteria that had not been validated or used criteria that were inappropriate.As a result of these shortcomings, it is difficult or impossible to compare the data that these papers have presented.It is argued that adopting a few simple rules in the design and reporting of susceptibility studies would, at little cost or effort, result in the production of papers that could make a significant contribution to our understanding of the issues involved in the use of antimicrobial agents in aquaculture.

The susceptibility test protocols used in 186 published papers that reported 203 studies of the antimicrobial susceptibility of non-cholera Vibrio spp were examined. Only a very small percentage of the studies provided explicit evidence that they had used standardised susceptibility protocols. Although all 203 reported frequencies of resistance in the isolates they studied, 185 of them either did not provide the criteria they used to determine resistance, used criteria that had not been validated or used criteria that were inappropriate. Less than 10% of the studies used internationally-harmonised consensus-based interpretive criteria that were appropriate to their data. These shortcomings in the performance and/or the reporting of susceptibility had results such that it was difficult or impossible to compare the data generated in the various studies. It is argued that compliance with the recommendations of OIE Aquatic Animal Code for the performing and reporting of susceptibility studies would be a major step towards limiting the frequency of these shortcomings in the scientific literature.

The methods for antimicrobial susceptibility testing of bacteria, although relatively simple, are not robust. As a consequence, if the data generated in such tests are to be commensurate, all susceptibility tests must be performed using standard protocols. A review of the published literature of antimicrobial susceptibility testing of bacteria from aquatic animals revealed a frequent occurrence of significant errors, particularly with regard to testing methodology, quality control and the use of appropriate interpretive criteria in the performance and reporting of susceptibility tests. This opinion piece provides a set of rules that, if followed, would help authors to avoid these frequently detected shortcomings.

The data that support the findings of this study are available from the corresponding author upon reasonable request.

The antimicrobial susceptibilities of Turkish F. psychrophilum isolates were investigated using a Clinical and Laboratory Standards Institute (CLSI) standard microdilution testing protocol. The significance of the minimum inhibitory concentrations was determined by applying empirically and statistically determined epidemiological cut-off values calculated by normalized resistance analysis. Twenty-five F. psychrophilum isolates made over four years from three different trout species in five different regions were examined. All isolates were fully susceptible to amoxicillin and florfenicol, but 88% of our isolates were categorized as having reduced susceptibility to the two quinolones, oxolinic acid, and enrofloxacin. Eighty percent of the isolates were categorized as showing reduced susceptibility to oxytetracycline. Sequence analysis demonstrated that the reduced susceptibility to the quinolones was associated with chromosomal mutations in the gyrA and parE genes. PCR analysis demonstrated that the genes tetA, tetB, tetC, tetE, tetH, tetL, and tetM were not associated with the reduced susceptibility to oxytetracycline and the significance of this observation is discussed. PCR analysis also showed the presence of sul2 in six isolates and floR in one isolate. This study is the first report of these genes in F. psychrophilum. It was noted, however, that the JUR was detected in an isolate categorized as fully susceptible to florfenicol, but the reasons for its lack of expression in the phenotype was not investigated. It is argued that the Turkish trout industry, in common with those of many countries world-wide, is critically dependent on the continued susceptibility of F. psychrophilum to florfenicol.

Two quite different disc contents are used for antimicrobial susceptibility testing of two fluoroquinolone drugs, flumequine and enrofloxacin, in the disc diffusion test, 30 and 5 µg, respectively. Using the SRA method, single-strain regression analysis, we studied the impact of disc content when testing two relevant bacterial species, Aeromonas sobria and Vibrio anguillarum . There were no major differences between the antimicrobial regression lines for the two species. Wild-type strains produced acceptable zones of inhibition over a wide range of disc contents. The flumequine 30 µg disc should be lowered in its drug content. No rational reasons for choosing so different disc contents for the two antimicrobials were apparent. At present, the choice of disc content for new antimicrobials are outside the realm of clinical microbiologists. It is recommended that reference authorities, such as EUCAST, CLSI and USCAST, are consulted for the choice of disc contents in the future.