Gut Microbiota Profiling of PediatricNonalcoholic Fatty Liver Diseaseand Obese Patients Unveiled by anIntegrated Meta-omics-Based Approach

There is evidence that nonalcoholic fatty liver disease (NAFLD) is affected by gut microbiota. Therefore, we investigated its modifications in pediatric NAFLD patients using targeted metagenomics and metabolomics. Stools were collectedfrom 61 consecutive patients diagnosed with nonalcoholic fatty liver (NAFL), nonalcoholic steatohepatitis (NASH), orobesity and 54 healthy controls (CTRLs), matched in a case-control fashion. Operational taxonomic units were pyrose-quenced targeting 16S ribosomal RNA and volatile organic compounds determined by solid-phase microextraction gaschromatography-mass spectrometry. The a-diversity was highest in CTRLs, followed by obese, NASH, and NAFLpatients; and b-diversity distinguished between patients and CTRLs but not NAFL and NASH. Compared to CTRLs,in NAFLD patients Actinobacteria were significantly increased and Bacteroidetes reduced. There were no significant differences among the NAFL, NASH, and obese groups. Overall NAFLD patients had increased levels of Bradyrhizobium,Anaerococcus, Peptoniphilus, Propionibacterium acnes, Dorea,andRuminococcus and reduced proportions of Oscillospira andRikenellaceae compared to CTRLs. After reducing metagenomics and metabolomics data dimensionality, multivariateanalyses indicated a decrease of Oscillospira in NAFL and NASH groups and increases of Ruminococcus, Blautia, andDorea in NASH patients compared to CTRLs. Of the 292 volatile organic compounds, 26 were up-regulated and 2down-regulated in NAFLD patients. Multivariate analyses found that combination of Oscillospira, Rickenellaceae, Para-bacteroides, Bacteroides fragilis, Sutterella, Lachnospiraceae, 4-methyl-2-pentanone, 1-butanol, and 2-butanone could dis-criminate NAFLD patients from CTRLs. Univariate analyses found significantly lower levels of Oscillospira and higherlevels of 1-pentanol and 2-butanone in NAFL patients compared to CTRLs. In NASH, lower levels of Oscillospira wereassociated with higher abundance of Dorea and Ruminococcus and higher levels of 2-butanone and 4-methyl-2-pentanonecompared to CTRLs. Conclusion: An Oscillospira decrease coupled to a 2-butanone up-regulation and increases in Rumi-nococcus and Dorea were identified as gut microbiota signatures of NAFL onset and NAFL-NASH progression, respectively.