This study determined the minimum inhibitory concentrations (MICs) for oxytetracycline hydrochloride (OTC) and erythromycin (Em), along with the a-glucosidase (a-glu) activities in 110 Nocardia seriolae strains isolated in Miyazaki and Kagoshima prefectures in 2008-2009. The strains were examined for the presence of the tet(K), tet(L), tet(M), tet(O), tet(S), erm(A), erm(B), mph(A), mef(A), and msr(D) genes. All the a-glu-positive strains (n = 15) were OTC resistant and Em sensitive, with MICs of 32-64 and \0.125 lg/ml, respectively. All the aglu-negative strains (n = 95) were OTC sensitive, with MICs of 2-4 lg/ml, and most of them (93 of 95) were Em resistant, with MICs of [128 lg/ml. The MICs for Em in the remaining 2 a-glu-negative strains were \0.125 lg/ml. The 15 OTC-resistant strains possessed the tet(K) and/or tet(L) gene(s), and all of the 93 Em-resistant strains possessed both the mef(A) and msr(D) genes. The relationship between a-glu activity and drug sensitivity of the N. seriolae strains may explain the difference in prevalence of each phenotype. Nevertheless, the relationship should be further explored using N. seriolae isolates collected from more prefectures and farms.

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Microplate resazurin assay was applied to investigate the in vitro activity of four essential oils (EOs); cinnamon (Cinnamomum zeylanicum), thyme (Thymus vulgaris), lemongrass (Cymbopogon flexuosus) and tea tree (Melaleuca alternifolia) oils against 80 clinical isolates of the fish pathogen Nocardia seriolae. The checkerboard test was then used to determine the possible synergistic effect of EOs combination against reference type strains of fish nocardiosis. All tested EOs had antibacterial activity against N. seriolae isolates. Among the tested EOs, cinnamon and thyme oils both exhibited the lowest minimum inhibitory concentrations (MICs) with 5-160 and 10-160 μg/ml, respectively. The activities of lemongrass and tea tree EOs were noted to be less effective with MICs of 20-640 and 160->5120 μg/ml, respectively. The checkerboard panel of cinnamon-thyme EOs combination against N. seriolae ATCC43993 demonstrated a synergistic effect with a fractional inhibitory concentration (FIC) index of 0.75. For N. salmonicida ATCC27463, the combination panel showed an additive effect with an FIC index of 1.0. For N. asteroides ATCC19247, the combination panel demonstrated an indifference effect with an FIC index of 1.125. These results indicate that thyme and cinnamon oils alone or the combination of them at a given ratio has a promising potent clinical significance in the treatment of fish nocardiosis. Despite the promising results given by our in vitro studies, the clinical benefits of these EOs combinations can only be determined through carefully designed in vivo experimental studies.