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.

Setting epidemiological cut-off values for Vibrio harveyi relevant to MIC data generated by a standardised microdilution method.

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.

Epidemiological cut-off values for Vibrio anguillarum MIC and disc diffusion data generated by standardised methods.

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.

Characterization of a novel picornavirus isolated from moribund gilthead seabream (Sparus aurata) larvae.

Gilthead seabream represents a species of importance in Mediterranean aquaculture. The larval stage is particularly sensitive and frequently impacted in suboptimal environmental or sanitary conditions. In the present study, investigations were carried out in a seabream hatchery following an unusual mortality reaching 70% among 50-day post-hatching. Anorexia, loss of appetite and abnormal swimming behaviour were observed in absence of parasites or pathogenic bacteria. Proliferation of rod-shaped bacteria in the gut lumen was associated with focal degeneration in the intestinal mucosa. Cytopathic effects on an EK-1 cell line after 21 days of culture at 14°C and 20°C in contact with homogenized affected larvae revealed the presence of a viral agent. Molecular characterization by high-throughput sequencing showed a typical picornavirus genome organization with a polyprotein precursor of 2276 amino acids sharing 46.3% identity with that of the Eel Picornavirus-1. A specific real-time PCR confirmed the presence of the viral genome in affected larval homogenate and corresponding cell culture supernatant. We propose the name Potamipivirus daurada for this novel species within the genus Potamipivirus. The etiological role of this virus remains uncertain at this time, and future studies will be necessary to investigate its prevalence in natural and aquaculture-reared populations as well as its ability to cause diseases in gilthead seabream.

Agreement between the categorization of isolates of Aeromonas salmonicida and Yersinia ruckeri by disc diffusion and MIC tests performed at 22℃.

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.

Draft Genome Sequences of Four Aeromonas salmonicida subsp. achromogenes Strains, 23051, 23053, 23055, and 23056, Isolated from Senegalese Sole (Solea senegalensis).

The bacterial species Aeromonas salmonicida officially has five subspecies. A large majority of the currently available sequences come from Aeromonas salmonicida subsp. salmonicida, which causes furunculosis in salmonids. We present the genomic sequences of four Aeromonas salmonicida subsp. achromogenes strains. This will help increase the robustness of genomic analyses for this subspecies.