This method measures the phylogenetic distance among bacterial communities in a phylogenetic tree [43], and provides a measure of similarity among communities in different samples. To compare the similarity of the jejunal microbiota in all dogs at the three time points, all the pair-wise distances between the communities were computed. To visualize the clustering of the samples along the first 3 axes of maximal variance,

Principal Coordinate Analysis (PCA) was used. PCA allows visualization whether any environmental factors (i.e., tylosin treatment) would group the communities together (Figure 5). Differences in bacterial groups between time points were determined using repeated measures ANOVA or Friedman’s test where appropriate (Prism5, GraphPad Software Inc, San Diego, Calif). Fisher’s exact tests FK506 order were used to compare proportions of dogs that harbor specific bacterial taxa among time points. The data were used to calculate the FRAX597 ic50 Shannon-Weaver bacterial diversity index, which yields information about species diversity in bacterial communities. The Shannon-Weaver index (Hs) was defined as -∑p i ln(p i ), where p i is the proportion of individual bacteria found in a certain species [44]. The Shannon-Weaver index takes into account the abundance and the evenness of the species

present within a community. Microbial communities with higher species richness and an even distribution (i.e., each species is present in similar proportions) will have a higher Hs than communities with a lower Tyrosine-protein kinase BLK species richness, or communities with high species richness but where a few species predominate. To estimate the total number of OTUs present in each sample, the coverage-based nonparametric richness estimators Ace and Chao

1 were calculated. Rarefaction curves were produced using the software NCT-501 datasheet program DOTUR [45]. Rarefaction analysis is used to estimate diversity and can serve as an indicator for the completeness of sampling [46]. To predict the maximum number of OTUs present in the canine jejunum, a Richards equation [47] was fit to the rarefaction curves [20]. The Richards equation has parameters C1 and C2 with the equation C1 = A × (1+(B – 1) × EXP (-C × ((C2) – D)))(1/(1-B)), where C1 is the OTU estimated and C2 is the number of sequences sampled [20]. Acknowledgements This study and publication was supported through internal funding by the Gastrointestinal Laboratory at Texas A&M University, College Station, TX, USA. The authors thank Mr. Seppo Lasanen for his excellent technical assistance. References 1. Suau A, Bonnet R, Sutren M, Godon JJ, Gibson GR, Collins MD, Dore J: Direct analysis of genes encoding 16S rRNA from complex communities reveals many novel molecular species within the human gut. Appl Environ Microbiol 1999, 65:4799–4807.PubMed 2.