Isolation and Characterization of Host-Independent Bdellovibrios

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Abstract
A reliable method has been developed for the isolation of host-independent (H-I; i.e., “saprophytic”) strains of Bdellovibrio from host-dependent (H-D; i.e., “parasitic”) cultures. The technique involves growing streptomycin-resistant (Smr) H-D cultures on streptomycin-susceptible (Sm8) host cells. A lysate containing large numbers of the Smr H-D cells and some remaining Sm8 host cells is transferred to a selection medium which contains the antibiotic. The Sm8 host cells in the lysate are killed, and the Smr H-I strains develop in broth within 3 to 6 days. By use of this method, it has been possible to isolate H-I strains from 16 different H-D Bdellovibrio strains studied. The frequency of occurrence of host independence is in the range of one H-I colony per 106 to 107 plaque-forming units of H-D bdellovibrios. The H-I cultures are nonfermentative, do not reduce nitrate, are strongly proteolytic, are oxidase-positive, and do not utilize 14 different carbon compounds as sources of energy for growth. Most H-I cultures are catalase-positive upon initial isolation from H-D lysates, but some cultures lose this enzyme upon subsequent transfers through host-free media. Most H-I bdellovibrios are pleomorphic, consisting of vibrio- to spiral-shaped cells typically measuring 0.3 to 0.4 μm in width and 1 to 10 μm in length. All H-I bdellovibrios have a cytochrome a and c component (H-I A3.12 differs from the other strains in the location of the peaks of the cytochrome spectrum). All are sensitive to oxytetracycline and (except for strain H-I A3.12) to the vibriostatic pteridine 0/129; most bdellovibrios, except for H-I A3.12, are generally uniformly resistant or susceptible to a given antibiotic. Bdellovibrio and Vibrio spp. have common cytochrome difference spectra and susceptibilities to oxytetracycline and to the vibriostatic pteridine 0/129. All H-I bdellovibrios examined produce an exocellular protease which digests heat-killed host cells. Bdellovibrios possessing predatory and bacteriolytic properties could be reselected from H-I bdellovibrio cultures growing in the presence of living host cells. Attempts to select for bacteriolytic isolates from Vibrio and Spirillum spp. were unsuccessul.