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ABSTRACTEdwardsiellaspp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as Edwardsiella tardaactually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each Edwardsiellaspecies using matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the Edwardsiellaspp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of Edwardsiellaspp. and advantages of employing alternative single-copy genes gyrBand sodBfor molecular identification and classification of Edwardsiellawere demonstrated. Last, sodBsequencing confirmed that isolates previously defined as typical motile fish-pathogenic E. tardaare synonymous with Edwardsiella piscicida, while atypical nonmotile fish-pathogenic E. tardaisolates are equivalent to Edwardsiella anguillarum. Fish-nonpathogenic E. tardaisolates are consistent with E. tardaas it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of Edwardsiellaisolates in clinical and research settings.

Flavobacterium columnareand F. psychrophilumare major fish pathogens that cause diseases that may require antimicrobial therapy. Choice of appropriate treatment is dependent upon determining the antimicrobial susceptibility of isolates. Therefore we optimized methods for broth microdilution testing of F. columnareand F. psychrophilumto facilitate standardizing an antimicrobial susceptibility test. We developed adaptations to make reproducible broth inoculums and confirmed the proper incubation time and media composition. We tested the stability of potential quality-control bacteria and compared test results between different operators. Log phase occurred at 48 h for F. columnareand 72–96 h for F. psychrophilum, confirming the test should be incubated at 28°C for approximately 48 h and at 18°C for approximately 96 h, respectively. The most consistent susceptibility results were achieved with plain, 4-g/L, dilute Mueller–Hinton broth supplemented with dilute calcium and magnesium. Supplementing the broth with horse serum did not improve growth. The quality-control strains, Escherichia coliATCC 25922 and Aeromonas salmonicidasubsp. salmonicidaATCC 33658, yielded stable minimal inhibitory concentrations (MIC) against all seven antimicrobials tested after 30 passes at 28°C and 15 passes at 18°C. In comparison tests, most MICs of the isolates agreed 100% within one drug dilution for ampicillin, florfenicol, and oxytetracycline. The agreement was lower with the ormetoprim–sulfdimethoxine combination, but there was at least 75% agreement for all but one isolate. These experiments have provided methods to help standardize antimicrobial susceptibility testing of these nutritionally fastidious aquatic bacteria. Received June 24, 2015; accepted October 2, 2015.

Mycobacterium shottsiiand M. pseudoshottsiiare recently described mycobacteria commonly isolated from Chesapeake Bay striped bass Morone saxatilis. However, their distribution in striped bass outside of the Chesapeake region and their ability to infect alternative hosts have not been described. Mycobacteria identified as M. shottsii(based on fatty acid methyl ester analysis and multigene sequencing) were isolated from striped bass collected in Albemarle Sound, North Carolina, and white perch Morone americanain the Rhode River, Maryland, and detected in striped bass from the New York Bight off Long Island, New York. Mycobacterium pseudoshottsiiwere isolated from white perch in the Rhode and Corsica rivers, Maryland, and detected in striped bass in the New York Bight. This work demonstrates that these mycobacteria can be found outside of the Chesapeake Bay as well as in hosts other than striped bass.