Study cohort

We recruited a cohort of 20 subjects and sampled their saliva and feces over a 6-month period. Of the 20 subjects enrolled, there were eight separate households consisting of two individuals and four separate controls not enrolled with a housemate (Additional file 1: Table S1). In each household, one individual received treatment with an antibiotic (azithromycin or amoxicillin) for 7 days and the other individual received a placebo (vitamin C). The four separate controls did not receive any therapy. Each subject was sampled on day 0 (day prior to antibiotics), day 3 (on the third day of antibiotics), day 7, week 8, and at 6 months.

Individual-specific patterns of viruses in the gut and mouth

We isolated viruses from all 97 fecal and 95 saliva samples according to our previously described protocols [2], which involved sequential filtering to remove cellular debris, CsCl density gradient ultracentrifugation, and DNA extraction from intact virions. Resulting DNA was sequenced using Semiconductor Sequencing [14] for a total of 118,527,761 reads with a mean length of 215 nucleotides. There were 63,092,083 fecal reads with a mean GC content of 39.9% and 55,435,678 salivary reads with a mean GC content of 43.0%. We sequenced an average of 5,926,388 reads per subject and an average of 1,234,664 reads per each time point.

We characterized the ecology of the viruses in each of the eight households to decipher whether the individuals in each household could be distinguished based on their fecal or oral viruses and whether the use of antibiotics might disrupt individual-specific patterns of viruses within each household. Using principal coordinates analysis (PCOA) to visualize the beta diversity between household members, each individual could be distinguished based on their gut (Fig. 1) and oral viromes (Additional file 2: Figure S1). These individual-specific patterns were not disrupted by the use of either amoxicillin (Panels A–D) or azithromycin (Panels E–H). The individual-specific patterns were significant in the feces of 13/16 subjects and in the saliva of 7/16 subjects when using a permutation test to compare the viruses within an individual over time with that of their housemates (Table 1).

Fig. 1 Principal coordinates analysis of beta diversity present in the feces of all households. Panels a–h represent households 1–8, respectively. Subjects who received placebo are represented by squares, amoxicillin are represented by circles, and azithromycin are represented by triangles. Specimens collected from day 0 are represented in red, day 3 in blue, day 7 in green, week 8 in yellow, and month 6 in orange Full size image

Table 1 Viral homologues within and between individuals Full size table

Household-specific patterns of viruses in the gut and mouth

We also tested whether household-specific patterns of viral ecology may exist in the cohort in addition to individual-specific patterns of viruses we observed (Fig. 1). We utilized PCOA to compare all subjects in households that received amoxicillin or azithromycin and observed patterns that suggested shared fecal viral ecology within the households for each household evaluated (Fig. 2). These patterns observed were statistically significant in the feces for five out of eight households as was observed through permutation analysis comparing the shared viral ecology within a household with the shared viral ecology between different households (Additional file 1: Table S2). Similar patterns were observed in the mouths of each of the household members, but were only significant in three out of eight households (Additional file 1: Table S2 and Additional file 2: Figure S2).

Fig. 2 Principal coordinates analysis of beta diversity present in the feces of all subjects. Panel a represents subjects who received amoxicillin, their housemates who received a placebo and control subjects who received no therapy. Panel b represents subjects who received azithromycin, their housemates who received a placebo and control subjects who received no therapy. Controls who received placebo or no therapy are demonstrated by black outer circles and subjects who received antibiotics are represented by gray outer circles. Outlines are drawn around all time points representing individual households Full size image

Household viruses are persistent and shared

A prior study indicated that oral bacteriophages are highly persistent and that the gut viruses of a single individual were persistent over time [13]. We evaluated the persistence of viruses in our cohort over time to determine whether their persistence may be altered by the use of antibiotics. We found that fecal viruses were highly persistent with greater than 70% found at least 4 days apart, and 30% identifiable at 5 to 6 months (Additional file 2: Figure S3). There was no identifiable effect the antibiotics had on the persistence of phages, as similar patterns were observed in amoxicillin and azithromycin households that were observed in the controls who took no therapy. The levels of persistence in the gut far exceeded those observed in the mouth (Additional file 2: Figure S4).

Because we found patterns that suggested shared household viral ecology (Fig. 2), we quantified the proportions of shared viruses between the members of each household. We found similar patterns of shared viruses in the gut (Additional file 2: Figure S5, Panel A), and in the mouth (Panel B). There were generally more shared viruses within each household in the gut than were found in saliva, but the difference did not meet statistical significance (Additional file 2: Figure S6). The majority of the viruses that were not shared in the guts of housemates were found in those individuals who took a placebo (Panel A), while roughly equal numbers of unique oral viruses in each household were found in subjects who received either an antibiotic or placebo (Panel B).

We also measured Sorensen distances between the individuals in each household and compared them with individuals from different households to decipher whether there were significant similarities between the individuals in each household that indicated that viruses were shared within a household. We measured Sorensen rather than Bray Curtis distances because Sorensen distances are useful for measuring similarity between viromes, while Bray Curtis distances measure dissimilarity. In the gut, we found significantly higher levels of similarity amongst household members than were found between individuals who resided in different households (Fig. 3, Panel A). We also identified higher similarity in the saliva for household members, but these results were not statistically significant (Panel B).

Fig. 3 Bar graphs comparing Sorensen distances (± standard error) between subjects within a household and between subjects from different households for feces (panel a) and saliva (panel b). p values are represented above the bars Full size image

Putative transmissions and directionality of transmissions in each household

We found bacteriophages that were shared in the mouth and the gut of housemates from all households (Fig. 3, Additional file 2: Figure S5, Figure S6). We examined the structure of some of these phages and found that some were present in both housemates on most days examined (Additional file 2: Figures S7–S10). We identified virulence factors within the genome structures of many of these phages, including an enterotoxin, toxin-antitoxin system, and platelet binding protein (Additional file 2: Figure S7), mucin and m23 peptidase (Additional file 2: Figure S8), and a beta-lactamase that could be involved in the degradation of beta-lactam antibiotics such as amoxicillin (Additional file 2: Figure S9). We also identified a phage with significant similarity to crAssphage [15] shared in the members of household 6 (Additional file 2: Figure S10). Finding genes potentially involved in antibiotic resistance in the structures of phage genomes shared between household contacts suggests that the sharing of these phages could be a means by which antibiotic resistance spreads through close contact with others.

We identified numerous fecal phages whose presence/absences on certain days between the housemates suggested that they had been transmitted from one household member to the other. For example, we could identify portions of a phage present in all days in the fecal viromes of one subject in household 7 (includes subjects CA39 and CA40), but only after 6 months in their housemate (Additional file 2: Figure S11). We verified the structure of this virus by PCR amplification and Sanger sequencing of its genome from all days in subject CA40 (Fig. 4). We attempted the same in the housemate (subject CA39), but the phage could only be amplified and sequenced from the 6-month time point. While there were a few polymorphisms in the phage over the course of time, the phage only had one polymorphism between subjects CA39 and CA40 at the 6-month time point. These data suggest that between week 8 and month 6, the phage was transmitted from subject CA40 to subject CA39. We characterized the presence/absence of viruses in all households over the course of the study and defined any virus that was present in one subject, absent in their housemate, and later appeared in at least two time points in the housemate as a putative transmission. Using these criteria, we found that of the 23.7 ± 0.05% of fecal viruses that were shared, 9.8 ± 0.03% had a pattern consistent with a putative transmission between housemates (Fig. 5). When examining the directionality of the putative transmissions, the majority (7.4 ± 0.04%) were consistent with transmission from the subject taking the placebo to the subject taking antibiotics, while the minority (2.4 ± 0.01%) went in the other direction. Similar results were identified in terms of the number of putative viral transmissions in the mouth, but there was little difference in the directionality of these transmissions between housemates (Additional file 2: Figure S12).

Fig. 4 Diagram of contig71 assembled from Sanger sequences from all time points in subject CA40 and from the month 6 time point in subject CA39. The contig was not identified on days 0, 3, 7, or week 8 in subject CA39. Putative ORFs and their directions are indicated by the arrows at the top of the diagram. ORFs that had significant homologues (BLASTP E-score <10−5) are indicated by the text above each arrow. The location of polymorphisms (when compared to the day 0 time point in subject CA40) are indicated by orange vertical lines Full size image