Study cohort

We recruited and sampled the feces, saliva, and skin from a cohort of 56 subjects over a 6-month period from the University of California, San Diego, campus. Of those 56 individuals, there were 24 separate households consisting of 2 individuals and 8 separate controls not enrolled with a housemate (Fig. 1). In each household, 1 individual received treatment with an antibiotic (amoxicillin or azithromycin), and the other individual received treatment with a placebo (vitamin C). Twelve households received amoxicillin, with 6 households receiving 3 days and another 6 receiving 7 days of therapy. In these households, amoxicillin was given twice daily and the placebo also was given twice daily. Another 12 households received azithromycin, with 6 receiving 3 days and the other 6 receiving 7 days of therapy. The azithromycin was given once daily in these households, and the placebo was given once daily as well. The additional 8 subjects enrolled in the study were not enrolled with a housemate and did not receive antibiotic or placebo. Households were randomized into the separate arms of the study; however, the study subjects were not blinded because we had to account for existing drug allergies and medication interactions in our decisions to give them antibiotics. Study subjects were sampled on day 0 (day prior to antibiotics), day 3 (on the third day of antibiotics), day 7, week 8, and at 6 months.

Fig. 1 Flowchart of study design Full size image

There were no significant differences identified in the demographics of the subjects enrolled in the amoxicillin arm, azithromycin arm, or control arm of the study (Table 1). The mean age of subjects was 24 in the amoxicillin arm, 29 in the azithromycin arm, and 25 in the control arm. Approximately 50 % of the study participants were female, and none of the study subjects had taken systemic antibiotics for a year prior to the initiation of the study. The subjects lived together ranging from 1 to 54 months prior to the initiation of this study, and 58 % in the amoxicillin arm were romantic couples compared to 50 % in the azithromycin arm. The majority of study participants were Caucasian (54 %), with roughly equal numbers of Latino (25 %) and Asian subjects (21 %) (Additional file 1: Table S1). Five of the 24 households (2 in amoxicillin arm and 3 in azithromycin arm) were lost to follow-up after the 8-week time point and did not complete the 6-month time point.

Table 1 Demographics of study participants Full size table

Household-specific patterns in the mouth, gut, and skin

We sequenced the V1–V2 hypervariable segment of the 16S rRNA gene from the feces, saliva, and skin of all subjects at all time points in the study to characterize the bacterial biota present on each body surface with a total of 806 specimens (269 feces, 270 saliva, and 267 skin). Because previous studies that demonstrate household-specific patterns of bacterial biota are confounded by genetically related individuals living in those households [28, 29], we tested whether there were household-specific patterns in the bacterial biota among the genetically unrelated individuals in this cohort. By measuring weighted UniFrac distances [33] between household pairs longitudinally and comparing with individuals from separate households, we found smaller distances among the household pairs, which was statistically significant (p < 0.05) in the gut, saliva, and skin for all households (Additional file 2: Figure S1). When comparing household pairs with control subjects who were not enrolled with housemates, the distances were also significantly smaller in the gut and saliva, but not on the skin. The smaller distances indicate a higher degree of similarity among taxa and their relative abundances. The similarity observed in the bacterial biota was not significantly affected by the use of antibiotics, as the same patterns were observed in households that received azithromycin and those that received amoxicillin (Fig. 2). Additionally, the distances between subjects in each household was not significantly altered on any body surface over the course of the study (Additional file 2: Figure S2), suggesting that the use of antibiotics did not substantially change the collective microbiota of the household.

Fig. 2 Bar graph representing mean weighted UniFrac distances (±standard error) within a household (white bars) and between different households (gray bars) based on the antibiotic received in each household. The y-axis represents mean weighted UniFrac distances, while the body site sampled and antibiotic received within each household is represented on the x-axis. p values were determined using the Mann Whitney U test Full size image

We measured weighted UniFrac distances among the households based on whether the individuals in the households were romantic couples or roommates to discern whether personal relationships significantly shape the shared microbiota within the household. We found that in all households, there was a statistically significant pattern of shared microbiota (p < 0.05 in each case; data not shown); however, there was no significant relationship identified in the feces, saliva, or skin based on whether the individuals were couples or roommates. There was a trend toward more shared taxa in couples, but the trend was not statistically significant on any body surface (Additional file 2: Figure S3 and S4).

Effects of time on microbiota compositions

We evaluated whether microbiota compositions within subjects were becoming more dissimilar over time, and whether the use of antibiotics may accelerate this process. We measured weighted UniFrac distances between day 0 (day prior to antibiotics or placebo) and each subsequent time point to identify whether the degree of dissimilarity increased over time. In the gut, the microbiota of subjects taking amoxicillin and azithromycin generally grew more dissimilar over each time point measured (Fig. 3). While these data were not statistically significant, a clear trend could be observed for most time points. For those subjects taking placebo, the exact same trend was identified. This trend was not a direct result of the placebo or antibiotic therapy, as those control subjects who received no treatment also demonstrated the same trend. The magnitude of the dissimilarity among the different antibiotic, placebo, and control groups did not differ significantly. These data suggest that while the gut microbiota grow more dissimilar within a subject over time, the use of short courses of common antibiotics does not significantly accelerate the process. No similar trends were identified in the mouth (Additional file 2: Figure S5) or on the skin (Additional file 2: Figure S6), suggesting that the gut is unique among these body surfaces in the evolution of its microbial communities.

Fig. 3 Bar graph (±standard error) representing the mean weighted UniFrac distances from day 0 in the feces of all subjects over time. The y-axis represents mean weighted UniFrac distances, and the x-axis represents the different subject groups over time based on the therapy they received. D3 represents day 3, D7 represents day 7, W8 represents week 8, and M6 represents month 6. p values were determined using the Kruskal Wallis test Full size image

Taxonomic responses to antibiotics

We examined the taxonomic compositions of all subjects across the different body surfaces over time to decipher whether there were differences attributable to the antibiotics. We first measured beta diversity within and between all subjects and visualized the output using principal coordinates analysis (Fig. 4). Most of the samples reflected the body site from which they were derived and could not be distinguished based on whether or not the subject received an antibiotic or placebo.

Fig. 4 Principal coordinates analysis of beta diversity present in all subjects, time points, and sample types based on whether they received antibiotics (amoxicillin or azithromycin) or placebo Full size image

In the gut, the most abundant taxa identified belonged to families Bacteroidaceae, Lachnospiraceae, and Ruminococcaceae. In both the azithromycin and amoxicillin arms of the study, the abundance of Lachnospiraceae was significantly diminished after antibiotic therapy and remained diminished at 6 months (Additional file 2: Figure S7). We compared the taxa from each individual taking antibiotics with their housemate taking a placebo to decipher whether there were taxa in each household whose relative abundance was altered as a response to the antibiotics. Lachnospiraceae were significantly diminished (p ≤ 0.01) in response to amoxicillin on days 3 and 7 in most households, but their relative abundances increased significantly in comparison to their housemates by week 8 (Fig. 5). Veillonellaceae, Bacteroidales, and Porphyromonadaceae (anaerobic bacteria) were significantly decreased in response to amoxicillin, while Fusobacteriaceae (also anaerobes) were increased. Bifidobacteriales and Erysipelotrichaceae were initially decreased, and subsequently increased in comparison to their housemates taking placebo. In response to azithromycin therapy, Erysipelotrichaceae, Veillonellaceae, and Clostriales were significantly diminished, while Alcaligenaceae were increased compared to their housemates.

Fig. 5 Heatmaps representing the relative abundances of taxa in individuals taking antibiotics that were significantly different when compared to their housemates receiving placebo. Each individual taking an antibiotic is shown next to their housemate taking a placebo. Each household consisting of two subjects is separated by gray vertical boxes. a Feces, b saliva, and c skin. The family or order for each OTU shown on the heatmaps is shown to the right of each heatmap, and the antibiotic received is shown to the left of each heatmap. The index color scale is shown below Full size image

In the saliva, the most abundant taxa identified were Prevotellaceae, Streptococcaceae, Veillonellaceae, and Neisseriaceae (Additional file 2: Figure S7). In response to amoxicillin, Veillonellaceae, Actinomycetaceae, Neisseriaceae, Prevotellaceae, and Porphyromonadaceae were all significantly increased in comparison to their housemates, while Streptococcaceae and Gemellaceae were diminished (Fig. 5). In response to azithromycin, Bifidobacteriales and Veillonellaceae were increased, while Clostridiales, Neisseriaceae, and Erysipelotrichaceae were diminished in comparison to their housemates.

On the skin, the most abundant taxa identified were Corynebacteriaceae, Propionibacteriaceae, Staphylococcaceae, and Streptococcaceae (Additional file 2: Figure S7). In response to both amoxicillin and azithromycin, the relative abundances of Streptococcaceae, Staphylococcaceae, Actinomycetales, Corynebacteriaceae, and Propionibacteriaceae were all altered in comparison to their housemates (Fig. 5).

Reduction in microbial diversity

We characterized the diversity of the microbial communities on each body surface in response to antibiotics to decipher whether diversity was substantially impacted longitudinally by the use of typical antibiotic courses. We first examined changes in microbiota diversity in each subject compared to their housemate taking a placebo. In the gut, we found that there was a significant reduction in diversity compared to their housemates for subjects taking amoxicillin (Fig. 6a). While there was a trend toward a sustained reduction in microbiota diversity compared to their housemates at 8 weeks and 6 months, the data were not statistically significant. Reductions in diversity were observed in subjects taking only 3 days of amoxicillin, but they were generally less than those observed in subjects who took 7 days of amoxicillin. Similar results were identified when comparing subjects who took azithromycin with their housemates. At 3 and 7 days, significant reductions in gut microbial diversity was observed, but this reduction was not sustained throughout the study (Fig. 6b). There were no significant differences observed between subjects who took 3 or 7 days of azithromycin. We also analyzed reductions in gut diversity in subjects who took amoxicillin and azithromycin and compared to control subjects rather than their housemates (Fig. 7a). We found that there was a substantial reduction in microbiota diversity in subjects who took either amoxicillin or azithromycin and that those reductions were sustained throughout the 6-month study. Interestingly, we also identified reductions in the diversity of their housemates, which was not observed in control subjects. When examining changes in diversity time point by time point, the majority of the diversity reductions occurred within the first 3 days of antibiotic therapy (Additional file 2: Figure S8).

Fig. 6 Bar graphs (±standard error) representing the normalized difference in Shannon diversity in the gut between individuals taking antibiotics and their housemates taking placebo at each time point studied. a Households that took amoxicillin and placebo and b households that took azithromycin and placebo. All households collectively are represented by the blue bars, households that took 3 days of an antibiotic are represented by red bars, households that took 7 days of an antibiotic are represented by green bars, and control subjects who were not enrolled with housemates and are represented by purple bars. The x-axis represents the time point and the y-axis represents the change in normalized change in Shannon diversity since the prior time point. Negative results indicate lower diversity in the subjects taking antibiotics compared to their housemates taking placebo, and positive results indicate greater diversity in the housemates taking placebo compared to the individuals taking antibiotics. For the control subjects, the bars represent the mean change in diversity among all control subjects. p values were determined using the Mann Whitney U test, and *p values ≤0.05 Full size image

Fig. 7 Bar graphs (±standard error) representing the change in Shannon diversity from day 0 across time in each subject group by body site tested. a Feces, b saliva, and c skin. Groups that received antibiotics, placebo, or no therapy (controls) are labeled across the x-axis, and the y-axis represents the change in Shannon diversity. *p values <0.05 using the Mann Whitney U test comparing subject groups at specified time points with the controls Full size image

We also compared changes in oral microbiota diversity between the subjects within a household. We identified reductions in microbiota diversity in subjects taking amoxicillin compared to their housemates, but most reductions were not statistically significant (Fig. 8a). As was found in the gut, there were generally greater reductions in microbiota diversity in response to 7 days of amoxicillin than were observed after just 3 days. In response to azithromycin, we found significant reductions in microbiota diversity after both 3 and 7 days of therapy, but those reductions were not sustained by week 8 (Fig. 8b). We also examined reductions in microbiota diversity independent of their housemates, and found that there were sustained reductions in microbiota diversity in response to amoxicillin over the entire 6 months of the study, but that reductions in diversity in response to azithromycin were not sustained (Fig. 7b). Unlike the reductions in microbiota diversity in the guts of housemates who took a placebo, there were no significant diversity reductions in the oral microbiota of housemates. The majority of the diversity reductions that took place in response to antibiotics occurred within the first 3–7 days of therapy (Additional file 2: Figure S8).

Fig. 8 Bar graphs (±standard error) representing the normalized difference in Shannon diversity in the saliva between individuals taking antibiotics and their housemates taking placebo at each time point studied. a Households that took amoxicillin and placebo and b households that took azithromycin and placebo. All households collectively are represented by the blue bars, households that took 3 days of an antibiotic are represented by red bars, households that took 7 days of an antibiotic are represented by green bars, and control subjects who were not enrolled with housemates and are represented by purple bars. The x-axis represents the time point and the y-axis represents the change in normalized change in Shannon diversity since the prior time point. Negative results indicate lower diversity in the subjects taking antibiotics compared to their housemates taking placebo, and positive results indicate greater diversity in the housemates taking placebo compared to the individuals taking antibiotics. For the control subjects, the bars represent the mean change in diversity among all control subjects. p values were determined using the Mann Whitney U test, and *p values ≤0.05 Full size image

When compared to housemates, subjects who received amoxicillin saw a reduction in their cutaneous microbiota diversity after 3 days, but not in response to azithromycin (Additional file 2: Figure S9). Independent of their housemates, this reduction in microbiota diversity in response to amoxicillin was sustained over the 6 months of the study (Fig. 7c). We also observed a reduction in diversity of the subjects who received the placebo, which was greater than that observed in response to antibiotics. The reduction in microbiota diversity observed in response to amoxicillin occurred within the first 3 days of therapy (Additional file 2: Figure S8).