6 Type 2 Diabetes,Metformin,and Gut Microbiota Diabetes Care A ND T2D-met OTUO2).Baresiellaceae(Bacteroidetes Enterococcus casselifiavus(OTU048 Prevotella (OTU236) OTU080)were enriched in T2D-met compared with T2D-met*participants 3 2 0 1 2 3 and F ND T2D-met' obe we ound that A. (3.4 and 44 times.respectively)in T2D. met*than in T2D-met participants tridium celatum (OTU14 Bulleidia p-1630-c5(OTUO7 Mollicutes RF39(OTUO6 00o,0si0eoh0ofbunotur Mollicutes RF39 (OTU284 2 Bifidobacterium bifidum (OTU176 tdiffer Megasphaera(OTU069 otbutyraiteproducesbeteenRC Prevotella (OTU028) n and nonme Butyrivibrio (OTU062) =0001.p -3 -2 0 3 C T2D-met T2D-met+ any of these groups of bacteria betweer Clostridia tes and ND p nd q values>0.2 Me CONCLUSIONS Prevotel a(0u028 mmnity-ba ple of Colom .3 .1 0 3 tent with previous literature (6.11-13.17 LDA score(log10) that the a association between gut micro of -(C)par higher relative abundance of purportedly netcidlmucin.degradingandSCfA 3 out of 273 statistically significant if two OTUs of Molli metndND participants matchedon age,sex,and BMI. hen co that OTand ND eve es hav o Clostridiac 02d06 (Firmicutes T2D-met'than i ND participants.In tMCobiaacompcsion,buiimd lostridiaceae OTU074)and Prev contras fou OTUs idiales in ngs on the taxa involved have been in e cipants,whereas Enterococcus c 556 SMB53(Firmicutes|Clostridiaceael plained by confounding factors,includ- Ent 0TU026 body weight,and ints (Figs.2A and 3A and B).W After matching on age sex and articipa ts t Ruminoc aelOTU025)were more s on we s(E OTU062).a different OTU of Pre Prevotel a(Bacteroide t microbiota composition.One OTU 02 8)and M was higher among OTU)bifidum (Acti in T2D-met*compared with T2D- other otu related to f casseliflayus was nobacterial BifidobacteriaceaelOTU176). participants,whereas OTUs from lower among participants with diabetes. Fig. 3 out of 273 statistically significant if not taking LDA scores into account). When comparing T2D-met2 and ND par￾ticipants, we found that OTUs belonging to Clostridiaceae 02d06 (Firmicutes| Clostridiaceae|OTU074) and Prevotella (Bacteroidetes|Prevotellaceae|OTU236) were overrepresented in T2D-met2 par￾ticipants, whereas Enterococcus casse￾liflavus (Firmicutes|Enterococcaceae| OTU048) was more abundant in ND par￾ticipants (Figs. 2A and 3A and B). When comparing T2D-met+ participants to ND participants, we found that OTUs of Butyrivibrio (Firmicutes|Lachnospiraceae| OTU062), a different OTU of Prevotella (Bacteroidetes|Prevotellaceae|OTU028), Megasphaera (Firmicutes|Veillonellaceae| OTU069), Bifidobacterium bifidum (Acti￾nobacteria|Bifidobacteriaceae|OTU176), two OTUs ofMollicutes RF39 (Tenericutes| OTU284 and OTU067), and Bulleidia p-1630-c5 (Firmicutes|Erysipelotricha￾ceae|OTU077) were more abundant in T2D-met+ than in ND participants. In contrast, four OTUs of Clostridiales in￾cluding Clostridium celatum (Firmicutes| Clostridiaceae|OTU014), Clostridiaceae SMB53 (Firmicutes|Clostridiaceae| OTU026), Oscillospira (Firmicutes| Ruminococcaceae|OTU024), and Cellu￾losibacter alkalithermophilus (Firmicutes| Ruminococcaceae|OTU025) were more abundant in ND than in T2D-met+ partic￾ipants (Figs. 2B and 3C and D). OTUs from Prevotella (Bacteroidetes|Prevotellaceae| OTU028) and Megasphaera (Firmicutes| Veillonellaceae|OTU069) were enriched in T2D-met+ compared with T2D-met2 participants, whereas OTUs from Oscillospira (Firmicutes|Ruminococcaceae| OTU024), Barnesiellaceae (Bacteroidetes| OTU102), and a different OTU of Clostri￾diaceae 02d06 (Firmicutes|Clostridiaceae| OTU080) were enriched in T2D-met2 compared with T2D-met+ participants (Figs. 2C and 3E and F). Finally, when we pooled mucin￾degrading and butyrate-producing mi￾crobes, we found that A. muciniphila and Butyrivibrio were more abundant (3.4 and 4.4 times, respectively) in T2D￾met+ than in T2D-met2 participants; differences were statistically significant for A. muciniphila (F1, 109 = 9.46, P = 0.003, q value = 0.01) but not for Butyr￾ivibrio (F1, 109 = 3.03, P = 0.08, q value = 0.21) (Fig. 3G and H). There were no sig￾nificant differences in the other groups of butyrate producers between metfor￾min and nonmetformin users (Roseburia: F1, 109 = 1.44, P = 0.23, q value = 0.39; Subdoligranulum: F1, 109 = 0.001, P = 0.97, q value = 0.97; Faecalibacterium: F1, 109 = 0.53, P = 0.47, q value = 0.59). There were no significant differences in any of these groups of bacteria between participants with diabetes and ND partic￾ipants (all P . 0.1 and q values .0.2). CONCLUSIONS In our community-based sample of Colom￾bian adults, we provide evidence consis￾tent with previous literature (6,11–13,17) that the association between gut micro￾biota and type 2 diabetes is modified by metformin use. T2D-met+ participants had higher relative abundance of purportedly beneficial mucin-degrading and SCFA￾producing bacteria compared with T2D￾met2 and ND participants matched on age, sex, and BMI. Several studies have demonstrated that type 2 diabetes is associated with gut microbiota composition, but find￾ings on the taxa involved have been in￾consistent (2–5). Some of the variance in previous study findings may be ex￾plained by confounding factors, includ￾ing demographics, body weight, and treatment with drugs, such as metfor￾min. After matching on age, sex, and BMI and stratifying comparisons on metformin, we found only modest asso￾ciations between type 2 diabetes and gut microbiota composition. One OTU related to Prevotella was higher among participants with diabetes, whereas an￾other OTU related to E. casseliflavus was lower among participants with diabetes. -4 -3 -2 -1 0 1 2 3 4 Butyrivibrio (OTU062) Prevotella (OTU028) Megasphaera (OTU069) Bifidobacterium bifidum (OTU176) Mollicutes RF39 (OTU284) Mollicutes RF39 (OTU067) Bulleidia p-1630-c5 (OTU077) Clostridium celatum (OTU014) Clostridiaceae SMB53 (OTU026) Oscillospira (OTU024) Cellulosibacter alkalithermophilus (OTU025) ND T2D-met+ -4 -3 -2 -1 0 1 2 3 4 Clostridiaceae 02d06 (OTU074) Prevotella (OTU236) Enterococcus casseliflavus (OTU048) ND T2D-met- -4 -3 -2 -1 0 1 2 3 4 Prevotella (OTU028) Megasphaera (OTU069) Oscillospira (OTU024) Barnesiellaceae (OTU102) Clostridiaceae 02d06 (OTU080) T2D-met- T2D-met+ LDA score (log10) B A C Figure 2—LDA scores (log10) of the OTUs displaying differences between pairs of groups of participants. ND vs. T2D-met2 (A), ND vs. T2D-met+ (B), T2D-met+ vs. T2D-met2 (C) participants. 6 Type 2 Diabetes, Metformin, and Gut Microbiota Diabetes Care