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ASIARESIST Report Summary

Project ID: ICA4-CT-2001-10028
Funded under: FP5-INCO 2
Country: Belgium

SOP-ASIARESIST-ABR, for antibiotic susceptibility testing by disc duffusion

A Standard Operating Procedure (SOP) was defined for the antibiotic susceptibility testing of project isolates using the disc diffusion (DD) method (SOP-ASIARESIST-ABR). The final version of the SOP was obtained following intensive discussions among partners and was made available to all partners during the lifetime of the project via the restricted access area of the project website. DD testing was performed on Iso-sensitest Agar (ISA) to minimize interactions between medium components and antibiotic gradients as much as possible.

In order to increase the relevance of the outcome of the harmonization study with regard to the recommendations of Alderman & Smith on standard methods for susceptibility testing of bacteria associated with fish diseases [Aquaculture 196:211-243 (2001)], a panel of reference strains was designed as follows: Aeromonas hydrophila subsp. hydrophila, Stenotrophomonas maltophilia, Salmonella enteritidis, Escherichia coli, Acinetobacter junii, Aeromonas salmonicida subsp. salmonicida, Yersinia ruckeri, Vibrio alginolyticus, Vibrio anguillarum, and Photobacterium damsela subsp. piscicida.

These strains were stored using the cryobeads system in each partner country and, following harmonization testing in each partner lab, a strain verification including purity and recovery check and control of the taxonomic authenticity of the strains by fatty acid analysis was performed. The value of this strain verification control was highlighted by the fact that some subcultures yielded poor recovery or lacked purity, upon which new subcultures were requested from the laboratories concerned.

The analysis of the final data set was based on the determination of the arithmetic mean and its standard deviation for each of the 60 possible strain-disk combinations [i.e. 10 control strains tested for 6 antibiotic disks: ampicillin (25 ug), tetracycline (30 ug), oxytetracycline (30 ug), oxolinic acid (2 ug), streptomycin (25 ug), and chloramphenicol (30 ug)]. Ten repeated determinations by the five partners delivered 50 data points per combination, or a total of 300 data sets (i.e. 60 combinations tested in five labs). Overall, the intralaboratory performance of SOP-based DD testing was highly satisfactory as evidenced by a standard deviation (SD) range of 0-2 mm when 79% of all recorded data sets were considered.

It was agreed that this range can be defined as the tolerance limit that is acceptable for DD susceptibility testing of the chloramphenicol resistant isolates in the course of the project. In these determinations, E. coli strain LMG 8223 (= ATCC 25922 = NCIMB 12210) was included as a control strain in each analysis batch. In 8% of the data sets, the SD value exceeded 4 mm which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distributed in one of the labs.

As could be expected, the SD range at the interlaboratory level was somewhat broader ranging from 0-5 mm for 81% of the SD values based on global averaging of 50 data points per strain-disk combination. This SOP thus provides a validated guide to conduct susceptibility studies at an inter-laboratory level. The results of the harmonization study were integrated in a joint manuscript for the peer-reviewed journal Diseases of Aquatic Organisms 66:197-204 (2005).

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