Standardization of a broth microdilution susceptibility testing method to determine minimum inhibitory concentrations of aquatic bacteria.

A multiple laboratory study was conducted in accordance with the standards established by the Clinical and Laboratory Standards Institute (CLSI), formerly the National Committee for Clinical Laboratory Standards (NCCLS), for the development of quality control (QC) ranges using dilution antimicrobial susceptibility testing methods for bacterial isolates from aquatic animal species. QC ranges were established for Escherichia coli ATCC 25922 and Aeromonas salmonicida subsp. salmonicida ATCC 33658 when testing at 22, 28 and 35 degrees C (E. coli only) for 10 different antimicrobial agents (ampicillin, enrofloxacin, erythromycin, florfenicol, flumequine, gentamicin, ormetoprim/sulfadimethoxine, oxolinic acid, oxytetracycline and trimethoprim/sulfamethoxazole). Minimum inhibitory concentration (MIC) QC ranges were determined using dry- and frozen-form 96-well plates and cation-adjusted Mueller-Hinton broth. These QC ranges were accepted by the CLSI/NCCLS Subcommittee on Veterinary Antimicrobial Susceptibility Testing in January 2004. This broth microdilution testing method represents the first standardized method for determining MICs of bacterial isolates whose preferred growth temperatures are below 35 degrees C. Methods and QC ranges defined in this study will enable aquatic animal disease researchers to reliably compare quantitative susceptibility testing data between laboratories, and will be used to ensure both precision and inter-laboratory harmonization.

Streptococcus didelphis sp. nov., a streptococcus with marked catalase activity isolated from opossums (Didelphis virginiana) with suppurative dermatitis and liver fibrosis.

beta-Haemolytic, catalase-positive, Gram-positive cocci that formed chains in broth media but did not react with Lancefield group antisera were isolated from skin lesions, spleen, liver and lungs of nine opossums, including eight from a research colony and one from a wildlife rehabilitation organization. The isolates had vigorous catalase activity that was retained on initial passage on non-blood-containing media, but this activity was lost in subsequent passages. The use of standard phenotypic tests did not lead to satisfactory identification of these organisms beyond the genus level, even if the aberrant catalase reaction was not considered. The 16S rRNA gene sequence of the isolates was most similar (96%) to Streptococcus dysgalactiae, but distinct from that species as 16S rRNA gene similarity of different strains of S. dysgalactiae was > 99%. Characterization of biochemical reactions and cell-wall fatty acid profiles also revealed significant differences between the opossum isolates and all other known Streptococcus spp., thus it is proposed as a new species with the name Streptococcus didelphis, sp. nov. The type strain is ATCC 700828T.