The objective of this study was to examine the impact of the amoxicillin-clavulanic acid intervention and probiotic supplementation on the gut bacteria microbiota and its resistome on a subset of 70 healthy adult participants, in order to explain the positive outcome of probiotic supplement at reducing diarrhea-like defecation events12. Studies investigating the impact of antibiotics on the gut microbiota and resistome have reported inter-individual variations in microbiota before antibiotic treatment to prompt enrichment of ABR genes and shifts in microbiota composition and diversity during antibiotic treatment6,9,18. Other studies have reported that microbiota recovery after antibiotic treatment was in some cases transient and in other cases incomplete16,19,20. Work by Pérez-Cobas et al. evaluated the impact on the gut microbiota of four antimicrobials, including amoxicillin, with different modes of action on four human healthy participants before, during and after treatment with antimicrobials4. They reported that there were marked differences in the microbiota composition due to the specific antimicrobial, as well as notable differences in relative abundance of resistome genes by database search of metagenomes against the Antibiotic Resistance Genes Database4,21. It was also found that the composition between gut microbiota and resistome correlated to the surviving microbiota community4.

These studies provided important insights, however, one limitation of these investigations is the fact that there were very few participants in each of these studies (between 3–8 participants)4,6,16,19,20, thus making deductions on a population level incomplete and uncertain. A recent study by Raymond et al. using shotgun metagenomics sequencing of a larger cohort of 18 healthy human participants evaluated the impact of antibiotic treatment on both gut resistome and microbiota6. They concluded that antibiotic treatment can alter gut resistome and microbiome composition in a specific, reproducible and predictable manner and that enrichment of ABR genes and shifts in microbiome composition returned to baseline levels 90 days after antibiotic treatment cessation6. Interestingly, they also reported that a subgroup had enrichment in E. cloacae that was linked to lower microbiota diversity of the Bacteroides enterotype, suggesting that the initial composition of the microbiota ultimately determines the reshaping by antibiotics6.

Our results are also in accordance with the observation of inter-individual and transient variation in both gut microbiota composition (Figs 2 and 4) and resistome (Fig. 6), not only in the baseline of all the participants, but in all the visits of both probiotic and placebo groups. Our data shows that each participant harbors a unique core microbiota that reacts differently to the amoxicillin-clavulanic acid intake (Supplementary Fig. S3). In that regard, our study highlights the importance of having a large cohort of participants in order to infer statistically significant relevant conclusions, which to the best of our knowledge has not been previously reported on microbiota and its resistome. Furthermore, our results were also consistent with observations reported by Raymond et al. that in both the probiotic and placebo groups there were shifts in both microbiota composition and resistome incurred by antibiotic treatment that were specific, reproducible and predictable for a number of bacterial families and ABR classes/genes for both groups6. For example, in the microbiota results in Fig. 3 there were major relative shifts in such families as Lachnospiraceae (increase), Bacteroidaceae (decrease), unidentified Clostridiales and Enterobacteriaceae (increase). These trends were also observed in the resistome, especially with regards to specific ABR genes of the classes of aminoglycosides, beta-lactams and tetracycline (Figs 7 and 8), thus indicating and confirming that this antimicrobial treatment can induce specific, reproducible and predictable changes6. It must be noted that our ABR microarray does not comprise the full list of currently known and clinically relevant ABR genes to date and it is most likely that we do not have the complete picture of the gut resistome. Nevertheless, the ABR genes that we do have on our microarray clearly showed enrichment of ABR genes upon antimicrobial treatment and reversion back to baseline levels after antimicrobial cessation. It was also shown that there were no discernible shifts that were impacted by probiotic supplement on the administration of the antimicrobial combination with these genomic analyses, as the gut microbiota and resistome profiles were very similar in both groups. However, there are a few notable observations that may have been impacted by the probiotic supplement (visit 3) during antimicrobial treatment compared to placebo that warrant further investigation such as, 1) an increase in the family of Porphyromonadaceae (genus Parabacteroides) and 2) a relative decrease in the diversity of Enterobacteriaceae from the shotgun sequencing results (Fig. 5) in the probiotic group. Interestingly, Zackular and co-workers reported and hypothesized that Porphyromonadaceae may have an important protective role as anti-inflammatory mediators of gut health in a murine model of inflammation-driven colorectal cancer22. In another study, a randomized clinical study of 28 participants supplemented with the probiotic Lactobacillus casei Shirota to evaluate the impact on metabolic syndrome, reported the enrichment of the genus Parabacteroides of the Porphyromonadaceae family23, supporting the possibility that probiotic supplementation during antibiotic treatment may have increased the relative amount of the family Porphyromonadaceae in our study.

Although not a direct causality of probiotic supplement, the results obtained here, when taken together, provide new insights to investigate further the effect of this probiotic combination such as the impact of the family Porphyromonadaceae and inhibition of the growth of opportunistic pathogens. Furthermore, the major impact on gut microbiota and resistome composition was due to the combination of amoxicillin-clavulanic acid. However, according to Raymond et al. we must not exclude the possibility that the shifts that were observed may not only be due to the antibiotic treatment, but possibly other unknown fitness factors6.

More importantly, the major finding in our study was the prompt recovery or dynamic reversion to similar pre-treatment baseline levels of gut microbiota and resistome one week after amoxicillin-clavulanic acid cessation for both probiotic and placebo groups (with the exception of minor taxon differences). This rapid reversion has not been previously reported to our knowledge. Our results contradict other reports, where recovery of gut bacterial microbiota and resistome composition was either incomplete or was achieved in 28 to 90 days after antibiotic treatment4,6,16,19,20. It must be noted that other antibacterial agents, in addition to amoxicillin, were used in these studies such as cefprozil, clarithromycin, metronidazole, ciprofloxacin that are not directly comparable to amoxicillin-clavulanic acid in terms of the impact on gut microbiota. Thus, the difference may be explained by the type of antibacterial agent, the duration of the treatment or the health status of the study participants. Our study was done in healthy participants with a short-term antimicrobial intervention so a possible explanation for the prompt reversion to baseline levels may be explained by the hypothesis of microbiota community resilience19,24. This seems to be the case for short-term exposure to antimicrobials, but studies of long-term antibiotic use have reported that perturbations in gut microbiota and resistome can have either long-lasting irreversible effects or can persist for longer periods16,18,25. Moreover, according to Jakobsson et al. this highlights the proposal of restrictive antibiotic usage, in order to minimize the unknown consequences of long-term antibiotic use16.

It is interesting to note that Raymond et al. also reported inter-individual specific changes in resistance gene abundance, explaining that enrichment of important ABR genes may be below the level of detection and present at low abundance levels before beta-lactam treatment6. For example, they reported that beta-lactam resistance genes such as bla OXA-1 and bla TEM-1 were not detected before antibiotic treatment, but increased during antibiotic treatment and subsequently were not detected after antibiotic treatment6. These findings are also in agreement with our findings where specific ABR genes of beta-lactams such as bla CMY-1 , bla CTX-M-1 , bla CTX-M-12 , bla DHA-1 , bla SHV-1 , bla SHV-37 , bla TEM-1B , bla TEM-1A , and bla OXA-1 (Figs 8 and 9) were not detected in baseline, but were enriched after amoxicillin-clavulanic acid + supplement and subsequently decreased after supplement and washout in both groups. The explanation from Raymond et al. for the enrichment of certain ABR genes that are not detected in the baseline levels does not imply they are not present, but are, in fact, present at very low abundance levels that are simply below the level of detection6.

It has been reported in a number of studies that specific ABR genes are present in the gut microbiota of many individuals from different countries15,16. For example, the tetracycline genes of tet32, tet40, tetO, tetQ and tetW have been reported to be ubiquitously present in the gut microbiota of many individuals and to be the most abundant family of resistance genes18,26. These described results are also in agreement with our results for the genes tet32, tetO, tetQ, tetW, as well as tetX, which were found in all the visits of both arms. Moreover, the results in Fig. 7 for the class of tetracycline showed that there were 36 distinct ABR probes on our microarray, but there were many more tetracycline genes detected in all the visits of both placebo and probiotic arms. This is explained by the fact that there are many participants that carry the same tetracycline genes and, thus the frequency of occurrence of these same tetracycline genes is found throughout many participants in all the visits of both arms of the study.

We also observed specific and potentially important ABR genes such as the class of aminoglycosides and beta-lactams that were enriched after the antimicrobial + supplement, which were subsequently decreased, not detected or detected at very low levels after the supplement and washout. The enrichment of selected ABR genes in the aminoglycosides and beta-lactams was analyzed by the CARD database to deduce a possible link to the relative enrichment of microbiota families, thus connecting the two datasets. Interestingly, our in silico database search of enriched ABR genes had hits to the Gram-negative bacteria family of Enterobacteriaceae and in some cases specific genera of Salmonella, Escherichia, Klebsiella, Shigella, Enterobacter and Citrobacter. This not only validates the gut resistome and microbiota results for the enrichment of the family of Enterobacteriaceae, but implies that many of the members of this family harbor multiple ABR genes, potentially on plasmids, and thereby impart multidrug resistance.

Investigators have also reported on the enrichment of the Enterobacteriaceae family and have concluded that such enrichment may be due to a rise in pathogenic and opportunistic pathogens such as Escherichia, Salmonella and Enterobacter4,6,27. Raymond et al. also showed by shotgun sequencing increases in a subgroup of participants that were enriched with E. cloacae after exposure to antibiotic, as well as enrichment of beta-lactam resistance genes6. Although enrichment of the Enterobacteriaceae family may be due, in part, to the increase of pathogenic and opportunistic pathogens, such assumption may not be entirely accurate, or justified, as there are many commensal Gram-negative bacteria that belong to the Enterobacteriaceae family (e.g., strains of E. coli, and E. cloacae) that are normal constituents of the gut microbiota. To probe deeper into the Enterobacteriaceae family, shotgun metagenomics sequencing was also used to confirm the link of the enrichment of specific ABR genes to the possible enrichment of opportunistic pathogens. To this end, the shotgun results agreed with the in silico analysis (CARD and KEGG databases) that the enrichment of specific ABR genes may be linked to species within the Enterobacteriaceae family, namely E. coli, E. cloacae, and K. pneumoniae; three of the most abundant species found in the shotgun metagenomics data (Figs 5, 9 and Supplementary Fig. 4).

Overall, the results obtained in this study clearly showed that treatment with amoxicillin-clavulanic acid induced many community-wide shifts in gut bacterial microbiota and resistome composition. In both probiotic and placebo groups, enrichment of specific ABR genes of aminoglycosides and beta-lactams appeared to be associated with the enrichment of members of the Enterobacteriaceae family. Typically members of this family are normal intestinal residents, but some members may potentially be opportunistic pathogens. Moreover, perturbations in gut microbiota and resistome composition occurred in a specific, reproducible and predictable manner for both arms and the subsequent return to pre-treatment baseline-like levels one week after short-term antimicrobial treatment can be explained by the hypothesis of community resilience19,24. Lastly, further investigation into the impact of the probiotics on Porphyromonadaceae relative abundance may help explain the positive outcome of reducing the duration of diarrhea-like defecation events.