**8. Discussion**

The Anna Karenina principle, based on Leo Tolstoy's grea<sup>t</sup> book and cited in 1878 (*All happy families are alike: each unhappy family is unhappy in its own way*), has been recently translated by Zaneveld et al. [53] as the response to stress against the stability of animal microbiomes. These authors discussed how healthy microbiomes may be quite similar between individuals, but each dysbiotic microbiota is dysbiotic in its own way. The associations between microbiome instability/variability and many confounding factors as well as with diseases, sugges<sup>t</sup> that microbiome may have many and simultaneous multiple faces.

This "stochastic" drift, occurring at any stage of life under stress conditions, can create several phenotypes that need to be known and harmonized when planning a study on microbiota.

Early childhood possesses distinct microbiota tracts compared with later ones, where di fferent clusters and phyla may be di fferently represented. One common characteristic during this early stage of life is that bacterial richness and diversity increase during growth. Therefore, pediatricians should know that there are several age-related microbiota profiles, and should also be aware of the need to categorize each individual in a defined, monthly range by carefully considering the above-mentioned interference factors.

Several specialties need to be involved in this aim as well as the combination of di fferent knowledge. The "Clinical Microbiota Expert" is not only a new job, but represents a step forward to create competence in this field where clinical microbiologists, clinicians, and bioinformaticians are merged into one. This new job-role will have to create awareness on the study of the "dynamic body" such as the gu<sup>t</sup> microbiota during early age by creating novel models and approaches as well as solutions to solve and interpret the clinical microbiology results. Therefore, translational methodologies to approach a new way of designing clinical trials need to use feasibility and e fficacy tools, and a deeper preparation in the field to avoid uncontrolled errors, unsubstantiated results, and excessive costs.

#### **9. Conclusions and Future Perspectives**

Next-generation sequencing methodologies still remain expensive and the diagnostic market is offering di fferent solutions, thus a proper, and especially judicious, use of these methods is definitively mandatory. The clinical microbiota expert and pediatricians involved in the field will also have to guide through this jungle by trying to avoid false myths and promises that could be di fficult to realize. In the near future, all of these studies and experiences will necessarily lead to a better understanding of the real key phases of microbiome progression from birth throughout childhood.

A final consideration to underline is that the metagenomics community still needs to fully converge toward standardized methods and procedures, leading to an investigation of the sources of variability and bias at each step of the workflow, and to an improved reproducibility and comparability between studies. This is a necessary premise for moving from correlation studies to causation investigations and to answer complex questions in a translational setting.

**Author Contributions:** L.D. designed and conceived the study; S.P. revised the paper and the technical aspects and checked the literature; C.B. and G.Z. revised the manuscript; M.P. conceived the figures and revised the manuscript; E.D. revised the clinical aspects and the manuscript.

**Acknowledgments:** The authors wish to thank the Fondazione "Romeo ed Enrica Invernizzi".

**Conflicts of Interest:** The authors declare no conflicts of interest.
