1 (Clostridium coccoides subcluster XIVa) and Bacteroides fragilis subgroup band 45.9. Table 3 BLAST identifications of the excised DGGE bands DGGE amplicon Identification Sequence # Bp Band nr Primer # Bp Species Accession Nr % identity Identical Total 54.2 L1401-R (V6-V8) 397 Uncultured bacterium EF405354.1 98 385 389 Eubacterium contortum L34615.1 93 364 390 Clostridium oroticum M59109.1 94 367 389 Ruminococcus torques L76604.1 93 365 389 60.1 518R (V3) 139 Uncultured bacterium
EF403112.1 100 124 124 Ruminococcus productus selleck chemicals AY937379.1 98 122 124 Clostridium sp. Y10584.1 98 122 124 Ruminococcus hansenii M59114.1 97 121 124 45.9 Bfra 531F 241 Bacteroides fragilis DQ100447.1 99 210 211 Bacteroides finegoldii AB222700.1 98 207 211 Bacteroides thetaiotaomicron AY319392.1 97 206 211 With the universal V3 primers only 2 bands (band 60.1 and 95.0) correlated significantly with the API index (Chi square, respectively p = 0.03 and p = 0.04). In a logistic regression analysis including both bands, only band 60.1 (OR = 5,9; CI 1,1 – 7,9)
remained independently associated with the API index (band 95.0: OR = 5.7 109; CI 0 – NA). After further adjustment for confounders in a LY2874455 supplier multivariate logistic regression analysis, the V3 band 60.1 remained significantly associated with the API index (table 2). Excision and sequencing of band 60.1 revealed a DNA fragment of 139 bp [EMBL:FN611009] showing 100% similarity with an uncultured bacterial sequence isolated from a human fecal sample (table 3). The highest sequence similarity with
a known species was obtained for Ruminococcus RAD001 supplier productus or hansenii and Clostridium sp (table 3). These species also belong to the Clostridium subcluster XIVa proposed by Collins et al. [15] with Clostridium coccoides as their nearest neighbour. With the Bacteroides fragilis subgroup primers 4 bands (band 18.4; 27.3; 45.9 and 57.9) correlated significantly with the API index (Chi square, respectively p = 0.008; 0.048; 0.006 and 0.048). In a logistic regression analysis including all 4 bands only Astemizole band 45.9 (OR = 7.1; CI 1,1 – 46,1) remained independently associated with the API index (band 18.4: OR = 4,8; CI 0,3 – 80,0/band 27.3: OR = 8,6 107; CI 0 – NA/band 57.9: OR = 8,6 107; CI 0 – NA). After adjustment for confounders, the Bacteroides fragilis subgroup band 45.9 remained significantly associated with the API index (table 2). Excision and sequencing of band 45.9 revealed a DNA fragment of 241 bp [EMBL:FN611011] showing 99% similarity with Bacteroides fragilis (table 3). A similarity of 98 and 97% was found with respectively Bacteroides finegoldii and Bacteroides thetaiotaomicron (table 3). In a final logistic regression model including the 3 significant DGGE bands only V3 band 60.1 (OR = 3,4; CI 1,2 – 9,7) and the Bacteroides fragilis subgroup band 45.9 (OR = 9,8; CI 1,6 – 59,3) proved to be independent variables excluding the V6-V8 band 54.
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