care.diabetesjo nals.org de la Cuesta-Zuluaga and Associates 5 T2D R2=0019 had higher fasting glucose,HbAand in- p=0.34 rent T20 16S rRNA Gene Sequencing e ND 009 g (mean Good's cov e±5D=09901 PCo1(10.34% 0.001);9%of OTUs were detected at ast bych biota.We next tested for ween participants with 20 diabetes and ND participants(A OSIM 2519 ipants (ANOSIM statistic R=-0.018,P= 0.557 The numbe of obse ved O met"and ND than bet n T2D-met (Supplementary Fig ce- PCo1(10.34% ANOSIM statistic R=0.018,P=0.348; :ANOSIM statistic R- We c s (PE 2D-me A) with dia nd ND partic =0.009,P=0.416)(Fg18) ever,the com significance (R2=0.013,P=0.036)(Fig. C).the PCo1(10.34%) De0036 0.01 T2D-met ND Participants (R 0.008,P=0.9 43).These results suggested and partici s not takir D the -me (T t-and VA.(A high-quality color represe e in the onine issue.) We next use to examine microbiota at the OTU level.Note that (represented by OTUs with [og10]LDA relevant.OTUs (19 displayed in Fig.2 andT2D-met2 participants. Compared with ND participants, T2D-met+ participants had higher fasting glucose, HbA1c, and in￾sulin resistance than ND participants and lower levels of the insulin-sensitizing hor￾mone adiponectin (P , 0.05). No other demographic, anthropometric, or clinical parameters were statistically different. 16S rRNA Gene Sequencing Gut microbiota communities were spe￾cific to each participant with marked in￾tersubject differences (Fig. 1A) (overall interindividual generalized UniFrac dis￾tance = 0.720 6 0.009). We found high coverage across all groups of participants (mean Good’s coverage 6 SD = 0.990 6 0.001); 99% of OTUs were detected at least by two DNA reads, demonstrating thorough sampling of the gut micro￾biota. We next tested for differences in the number of observed OTUs across the groups of participants. We found no differences between participants with diabetes and ND participants (ANOSIM statistic R = 0.005, P value = 0.425) or between T2D-met+ and T2D-met2 partic￾ipants (ANOSIM statistic R = 20.018, P = 0.557). The number of observed OTUs tended to be more similar between T2D￾met+ and ND than between T2D-met2 and ND participants (Supplementary Fig. 1); however, these differences were not statistically significant (T2D-met+ vs. ND: ANOSIM statistic R = 0.018, P = 0.348; T2D-met2 vs. ND: ANOSIM statistic R = 0.009, P = 0.409). We observed no significant differences in b diversity estimates among the three groups of participants (PERMANOVA: R2 = 0.019, P = 0.335) (Fig. 1A) or between participants with diabetes and ND partic￾ipants (R2 = 0.009, P = 0.416) (Fig. 1B). However, the comparison between met￾formin and nonmetformin users reached significance (R2 = 0.013, P = 0.036) (Fig. 1C), demonstrating differences in the bacterial community structure associated with metformin use. The difference was also significant when comparing T2D￾met+ and ND participants (R2 = 0.015, P = 0.036) but not when comparing T2D-met2 and ND participants (R2 = 0.008, P = 0.943). These results suggested the microbial communities of T2D-met+ versus T2D-met2 were modestly phylo￾genetically dissimilar. We next used LEfSe to examine differ￾ences in the relative abundance of gut microbiota at the OTU level. Note that we were only interested in OTUs dis￾playing strong associations in the LDA (represented by OTUs with [log10] LDA scores $3); such stringency resulted in fewer retained, but more likely biologically relevant, OTUs (19 displayed in Fig. 2 and PCo2 (6.03%) PCo1 (10.34%) ND R2 = 0.019 P = 0.34 T2D-met+ T2D-met￾PCo2 (6.03%) R2 = 0.009 P = 0.42 ND T2D PCo1 (10.34%) PCo2 (6.03%) met￾R2 = 0.013 P = 0.036 T2D-met+ PCo1 (10.34%) A B C Figure 1—Principal coordinates analysis based on generalized UniFrac. A: Comparison among the three groups of participants. B: Comparison between participants with diabetes and ND partici￾pants. C: Comparison between T2D-met+ and participants not taking metformin (T2D-met2 and ND). Ellipses encompass 75% of data distribution in each group of participants. R2 and P values from PERMANOVA. (A high-quality color representation of this figure is available in the online issue.) care.diabetesjournals.org de la Cuesta-Zuluaga and Associates 5