The description of Massilia timonae, a nonfermentative aerobic gram-negative rod, was based on a single strain. A subsequent report of a second isolate has been recently published. Phenotypic descriptions of these two strains were based primarily on commercial test kit results. We have identified three additional strains as M. timonae by 16S rRNA sequence analysis and have characterized them phenotypically in parallel with the type strain of M. timonae, CIP 105350, by conventional test methods. A fourth strain, designated M. timonaelike, was also characterized. All four strains were isolated from human patients: two were blood isolates, one was isolated from cerebrospinal fluid, and one was isolated from bone. The four strains and the type strain were quite similar phenotypically. However, in contrast to the original description, the strains were found to be oxidase positive and arginine dihydrolase negative and to have lateral flagella as well as a single polar flagellum. Additionally the strains produced acid oxidatively from some carbohydrates. Other phenotypic characteristics, including cellular fatty acids, agreed with the original description. Based on our emended description, M. timonae and M. timonae-like strains can be differentiated from other aerobic nonfermentative gram-negative rods by conventional biochemical tests combined with cellular fatty acid analysis.Massilia timonae was described in 1998 based on a single isolate from the blood of an immunocompromised patient. It was classified as a novel bacterium based on its unique phenotypic and genotypic characteristics (11). Subsequently, the use of 16S rRNA sequence analysis led to the identification of a second isolate of M. timonae from a surgical wound infection in an immunocompetent 36-year-old male who had undergone orthopedic surgery (14). The phenotypic characteristics described in both these reports were based largely on commercial test kit results.As the reference laboratory for the state of California, the Microbial Diseases Laboratory receives unusual bacteria for identification. Two isolates (99A9205 and 97A4424) that could not be identified by phenotypic testing by conventional algorithms (18) were subsequently identified by 16S rRNA analysis as M. timonae, a species we had not knowingly encountered before. In order to compare these strains with a known isolate of M. timonae, we characterized the type strain (CIP 105350) by the same test protocols. A search of our culture collection turned up two additional isolates (85A2206 and 96A14209) that appeared phenotypically similar to 99A9205 and 97A4424 as well as to the type strain of M. timonae. Herein we present our results, including 16S rRNA sequence analysis, conventional biochemical test results, morphological and flagellar characteristics, and cellular fatty acid (CFA) analysis, from examining our four strains and the type strain. Based on these results, we provide an emended description of this new species. MATERIALS AND METHODSBacterial strains. The strains studied are summarized in ...