Overall, this systematic review demonstrates that there is no convincing evidence for consistent effects of probiotics on fecal microbiota composition in healthy adults.

No effects were observed on the fecal microbiota composition in terms of α-diversity, richness, or evenness in any of the included studies when compared to placebo. Only in the paper by Ferrario et al. [27] was it reported that probiotic supplementation significantly modified the overall structure of the fecal bacterial community in terms of compositional dissimilarity (β-diversity) when compared to placebo.

Study design and reporting of results

Overall, the reporting of the analyses and results was non-transparent and difficult to assess with very few effect sizes, confidence intervals, and P values reported. This is possibly due to the fact that fecal microbiomics is a relatively new research area that currently relies heavily on non-parametric statistics and lacks an internationally accepted standard approach of reporting results. Unfortunately, this impedes the comparison of results in the present review. As the only study, Ferrario et al. [27] used a cross-over design, which may not be the ideal design to assess the effects of a probiotic intervention due to the risk of carry-over effects [35]. In the study the probiotic cell count was decreased after the 4-week wash-out period compared with baseline count, suggesting that wash-out was effective. However, a carry-over effect at the outcome level cannot be excluded. Only two studies provided a priori sample size calculations [20, 24], of which two calculated statistical power based on alterations in fecal microbiota composition [20, 22]. Thus, several of the studies may well have been underpowered, with an inherent risk of unequal distribution of confounding factors. A potential confounder in the studies reviewed is habitual diet. Human studies have revealed that short-term and long-term changes in diet (such as plant-based vs. animal-based, amount of dietary fibers and fat) influence the fecal microbiota composition and function [36–38]. Hence, the enormous inter-individual variation in the dietary intake and its effect on the fecal microbiota may mask the true picture of the impact of a probiotic treatment. Only one of the included studies monitored habitual diet with the aim of accounting for differences in dietary habits, specifically considering prebiotic fibers, during the intervention period [27]. A major limitation of most included studies is an unclear, inexplicit statement of the pre-specified primary outcome and delimitation of secondary outcomes. Only one study [24] is explicitly labeled as a pilot trial, reporting a multitude of outcomes, only in part addressing multiple testing. Two studies do not address the issue of multiple testing [23, 27], while others report multiple primary outcomes or make no distinction between primary and secondary outcomes [19–22]. Reporting of the results is generally unclear, with between-group comparisons on primary outcomes intermixed with results on secondary outcomes and within-group comparisons of differences between baseline and post-intervention measures.

Heterogeneity

Although study participants in the included trials were all healthy adults, the demographic makeup varied widely among studies. Rampelli et al. [23] included only elderly individuals, who may respond differently to probiotics than young individuals and Brahe et al. [20] included postmenopausal women only.

Considering that the impact on the fecal microbiota may differ among strains of the same bacterial species [39], despite close phylogenetic relationships, a potential source of heterogeneity is the use of various probiotic agents. Six studies used single-strain interventions with probiotic products belonging to the genera Lactobacillus [19, 20, 22, 24, 27] or Bacillus [21]. One study used a double-strain probiotic mixture of bacteria belonging to the genera Lactobacillus and Bifidobacterium [23]. Whereas the use of different probiotic agents makes it difficult to draw any meta-analytical conclusions, the choice between single-strain and multi-strain intervention is probably of less importance. In most cases, inert bacteria are administered and within a few hours are entering a diverse ecosystem where they are numerically a minority. So while additive or synergistic effects might be observed in vitro, the opportunity for metabolically active strains delivered in combination to result in similar effects in vivo may not present itself.

None of the studies included in the present review comment on the rationale behind their choice of dosage. The International Scientific Association for Probiotics and Prebiotics provides a list of dosages ranging from 1 × 108 to 1.8 × 1012 CFU twice daily depending on strain and disease, based on at least one well designed clinical trial showing a beneficial effect for a health promoting or therapeutic outcome [40]. However, the list covers gastrointestinal disorders only and does not address fecal microbiota in healthy participants. In general, different dosages should be assessed to facilitate an interpretation of the dose-response relationship of probiotic consumption on relevant outcomes, rather than on safety and viability alone. The information provided by such studies would enable the identification of the dosage needed to observe an impact on the relevant outcome and add to the likelihood that an observed association is causal [41]. Hanifi et al. [21] examined and detected oral dose-response relationships, but for tolerance and gastrointestinal viability only. As of now, it is impossible to draw any conclusions on the ideal dosage regarding effects on the fecal microbiota composition. Likewise, the optimal duration of intervention remains elusive.

Mode of administration may also contribute to the observed lack of impact on fecal microbiota. Ingested probiotics must survive hostile environments including acidic, protease and bile-salt rich conditions during their passage through the gastrointestinal tract [42, 43]. Currently, screening feces is the only way to assess whether the probiotics have survived through the gastrointestinal tract. Yet, the site of action may be proximal to the colon and it is not necessarily possible to conclude on the degree of colonization or even the amount of bacteria that produce the effect [44]. In contrast to the findings in five of the included studies, the study by Rampelli et al. [23] showed only a trend towards enrichment of the probiotic strain, perhaps due to the use of biscuits as the mode of administration, yet, another reason may be low compliance. This may add to the explanation why little effect of probiotics was found in Rampelli et al. [23]. Compliance was evaluated as sufficient in the remaining studies [19–22, 24, 27].

Further adding to the heterogeneity between studies is the application of different methods for assessing fecal microbiota. Even though all studies applied high-throughput metagenomics approaches, the resolution and specificity levels varied tremendously and no studies used the same methodological approach. Only one study examined the fecal microbiota by an untargeted metagenomic approach using shotgun sequencing and thereby providing information on microbial gene and derived functional levels, free of bias introduced by amplification of a specific genomic region as is the case in targeted 16S rRNA sequencing and array-based analyses [45]. Compositional information can be achieved by mapping the reads to a microbial gene reference catalogue [34], as was done by Brahe et al. [20]. Still, only a fraction of sequencing reads can be mapped to the existing reference catalogues. The targeted 16S rRNA approach provides information at the taxonomic level in the form of abundance and phylogenetic relationship, but the method has pitfalls in PCR amplification steps [46] and cross-platform comparison is not straightforward. Of the included studies, two use 454 pyrosequencing [19, 21], one uses Illumina MiSeq [24], another uses Ion Torrent sequencing [27], and two use phylogenetic microarrays [22, 23]. The sequencing platforms differ in costs, coverage, and length of reads with the Illumina platform becoming more widely used [45]. Community profiles from HITChip correlate well with pyrosequencing-based compositions (Pearson correlations at phylum (average r = 0.94), class (0.93), order (0.94) and family levels (0.77)) and the HTF microbe.array has demonstrated good reproducibility by cluster analysis of the phylogenetic fingerprint in samples from the same participant [47, 48]. In general, using phylogenic microarray approaches have the advantages of being cost-efficient for compositional characterization; however cross-hybridization may occur and only taxa that are covered by the reference sequences can be detected [47]. Another well-known source of bias in 16S rRNA-based studies is the targeted hypervariable region of the 16S rRNA gene. The region used for analysis in the included studies applying 16S rRNA-based methods varies with one study using V1 and V6 [22], one study using V3 [27], one study using V3 and V4 [19], one study using V5 and V6 [24], and one study does not specify [23]. Several studies have examined the effects of region choice when evaluating fecal microbiota composition with no current international consensus [47, 49–51].

Probiotics in health and disease

In a recent systematic review including 29 trials studying healthy adults with undisturbed microbiota (using non-high-throughput molecular techniques) only ~20 % showed an effect of probiotic treatment on fecal microbiota. It is concluded that there is little, if any, evidence of an effect of probiotic treatment in circumstances where the microbiota is unperturbed by pathophysiological processes or pharmaceutical treatment (antibiotics or chemotherapy), either concurrent with or prior to intervention. However, where dysbiosis is present or where the microbiota is perturbed, there is some evidence for a restorative or protective effect of certain strains of probiotics, both on the fecal microbial community itself, but more importantly, also on host physiology, e.g. alleviation of gastrointestinal symptoms [18].

In the case of dysbiotic or perturbed microbial communities, any restorative or protective effect on the microbiota alone, without any measurable beneficial effect for the host, would predominantly be of academic interest by improving our understanding of the intestinal ecosystem. In the case of undisturbed microbiota, any inference of health benefit from changes to the microbiota alone would be highly speculative without a direct linkage to relevant host phenotypes. Ideally, hard endpoint data would determine the effects of probiotics in healthy individuals, but considering the time perspective of generating such data this may be long in coming. Until such studies are available, any statement on health benefits of probiotic supplementation in healthy participants would rely on observed effects on biomarkers or other intermediate outcomes.

Limitations

Limitations of this review include the search terms used to identify relevant papers. In addition to probio*, we specifically searched on bifido* and lacto*, but other search terms such as Bacillus and Saccharomyces could have been relevant. Publication bias is a well-known challenge within the field of systematic reviews and meta-analyses; however, the majority of the studies included in the present review provide null findings, indicating that this concern may be settled to some extent. Language bias cannot be ruled out since our search was exclusively based on English language dominated sources.