Virulence of Subspecies Biovar and Phenotypic Change during Serial Passages on Artificial Media.

() is the etiological agent of the zoonotic disease tularemia. subspecies biovar has rarely been isolated in Japan and is considered to have moderate virulence, although the biological properties of fresh isolates have not been analyzed in detail. Here, we analyzed the virulence of two strains of subspecies biovar (NVF1 and KU-1) and their phenotypic stability during serial passages in Eugon chocolate agar (ECA) and Chamberlain's chemically defined medium (CDM) based agar (CDMA). C57BL/6 mice intradermally inoculated with 10 colony-forming units of NVF1 or KU-1 died within 9 days, with a median time to death of 7.5 and 7 days, respectively. Both NVF1 and KU-1 strains passaged on ECA 10 times had comparable virulence prior to passaging, whereas strains passaged on ECA 20 times and on CDMA 50 times were attenuated. Attenuated strains had decreased viability in 0.01% HO and lower intracellular growth rates, suggesting both properties are important for virulence. Additionally, passage on ECA of the KU-1 strains altered lipopolysaccharide antigenicity and bacterial susceptibility to β-lactam antibiotics. Our data demonstrate strain virulence in Japan and contribute to understanding phenotypic differences between natural and laboratory environments.