A patient being treated with amoxicillin-clavulanic acid for acute sinusitis was monitored for the development of AAD. The patient developed loose stool within 24 h after the first dose of antibiotics. DNA was purified from stool samples collected (i) from the first voided stool after the initiation of antibiotics (day 0), (ii) 4 days after the initiation of antibiotics (day 4), and (iii) 2 weeks after the last dose of antibiotics (day 24). A 16S rDNA library was constructed from each of three DNA samples following PCR amplification using primers that target bacterial 16S rRNA genes. Ninety-six randomly selected clones from each library were subjected to DNA sequence analysis using a sequencing primer (519R) that yielded an average of 500 bp of readable sequence. Ultimately, 84, 74, and 84 sequences were utilized from each library, respectively, for phylogenetic analysis. Phylogenetic trees were constructed using the ARB suite of programs (Fig. ).
In the day 0 sample, the majority of the sequences clustered within four bacterial groups (Fig. ): (i) Bacteroides spp., (ii) Clostridium sp. rRNA cluster IV, (iii) Clostridium sp. rRNA cluster XIVa, and (iv) Bifidobacterium spp. Four days after the initiation of antibiotics, there was a marked shift in the representation of the major bacterial groups. Bacteroides spp. were still a major component of the microbiota, but whereas members of the Bacteroides fragilis cluster were predominant in the day 0 sample, Bacteroides distasonis was the predominant group at day 4. Strikingly, no sequences corresponding to Clostridium rRNA cluster XIVa or to Bifidobacterium spp. were detected at day 4, whereas these two groups represented a third of the sequences detected on day 0. Conversely, 34% of the sequences detected on day 4 were members of the Enterobacteriaceae, which represented only 2% of the day 0 sequences.
Two weeks after the cessation of antibiotics (day 24), there was partial reversal of the changes seen on day 4. B. fragilis was once again the predominant Bacteroides species, and there was reappearance of members of Clostridium rRNA cluster XIVa. No sequences of members of the Enterobacteriaceae were detected on day 24. Interestingly, Bifidobacterium spp., which was one of the major groups seen on day 0 and not present on day 4, did not return by day 24.
LIBSHUFF analysis (17
) of each pairwise comparison between the libraries indicated that the libraries were significantly different from one another: P
values were <0.001 with the exception of the day 24 library compared to the day 1 library, where the P value was 0.004. Visual inspection of the phylogenetic analysis of cloned sequences (Fig. ) suggests that the day 24 library is a subset of the day 0 library, with all well-represented phylogenetic groups present at day 0 also present at day 24 (with the exception of the bifidobacteria).