Technique for Identifying Flavobacterium columnare Using Whole-Cell Fatty Acid Profiles

Abstract

Isolates of Flavobacterium columnare (29 from diseased fish and three American Type Culture Collection cultures ATCC 23463, 49512, 43622]) were identified by use of biochemical characteristics prior to generating whole-cell fatty acid profiles. The microbial identification system (MIS; Microbial ID, Newark, Delaware), a gas chromatography system, was used to generate the fatty acid profiles of F. columnare. The MIS contains databases of clinically and environmentally important bacteria that are represented by over 100 genera, including Flavobacterium spp. (F. aquatile ATCC 11947] and F. mizutaii). Flavobacterium columnare is not included in the databases because it does not grow on standard media. Fatty acid profiles of F. columnare were generated with the CLIN40 protocol established by MIS after growth of the bacteria in modified Shieh broth. The fatty acid composition of F. columnare isolates determined by the CLIN40 method consisted of 10 major fatty acids (those present at levels > 1%): 11-methyl-dodecanoic acid (13:0 iso the term “iso” designates the methyl group at the penultimate carbon atom]), 13-methyl tetradecenoic acid isomer G (15:1 iso G), 13-methyl tetradecanoic acid (15:0 iso), 12-methyl tetradecanoic acid (15:0 anteiso the term “anteiso” designates the methyl group at the third carbon atom from the end]), pentadecanoic acid (15:0), 14-methyl pentadecanoic acid (16:0 iso), 3-hydroxy-13-methyl tetradecanoic acid (15:0 iso 3OH), 15-methyl cis-9-hexadecenoic acid (iso 17:1 ω9c), 3-hydroxy-14-methyl pentadecanoic acid (16:0 iso 3OH), and 3-hydroxy-15-methyl hexadecanoic acid (17:0 iso 3OH) (94.8% of profile). Five fatty acids found in the highest percentages from all isolates (CLIN40 method) included 15:1 iso G (16.12%), 15:0 iso (46.54%), 15:0 iso 3OH (6.81%), iso 17:1 ω9c (7.32%), and 17:0 iso 3OH (9.42%). Fatty acid profiles were also established by means of the MIS rapid protocol (RCLN50) in which identifications can be completed in 7 min instead of 20 min. Fatty acids found in the highest percentages for the RCLN50 protocol included 15:1 iso G (15.36%), 15:0 iso (43.03%), 15:0 iso 3OH (7.43%), iso 17:1 ω9c (6.83%), and 17:0 iso 3OH (10.17%). Both methods will allow reliable identification of F. columnare.