The gut microbiota plays a crucial role in human health, influencing nutrient digestion and non-communicable diseases. While the gut microbiota of young children and adults has been extensively studied, data on older children are scarce. The gut microbiota composition varies significantly during the first year of life before stabilizing around ages 2-3. However, diversity continues to change throughout life, influenced by factors like antibiotic use and nutritional status, with diet being paramount. Studies on large, population-based cohorts highlight significant inter-individual microbial diversity, underscoring the need for research on diverse populations, particularly in non-Western countries, to understand the relationships between bacterial profiles and environmental or host factors. Researchers are exploring the gut microbiota's potential as a predictor and mitigator of various diseases. Identifying microbial biomarkers for nutritional status would advance understanding of bacteria's role in health and disease. This could lead to non-invasive malnutrition treatments targeting the gut microbiota. While associations between the microbiome and obesity have been reported, data on children with acute malnutrition are limited, but emphasize the microbiota's importance; severe acute malnutrition is linked to microbiota immaturity, and microbiota-targeted complementary food has proven more effective than standard supplementary foods in weight gain. In low- and middle-income countries, chronic malnutrition (stunting and micronutrient deficiencies) remains prevalent, raising significant health concerns. Cambodia, despite poverty reduction, still faces high rates of malnutrition. The 'Fortified Rice for School Children in Cambodia' (FORISCA) project assessed the impact of fortified rice on nutritional status, development, and anthropometry in 9500 schoolchildren. The trial demonstrated improved micronutrient status and a small impact on cognitive development, but also an increased hookworm infection prevalence. Different fortified rice formulations yielded varying effects on these parameters. Numerous studies have explored the gut microbiota's role in micronutrient status, with early work showing its contribution to B-vitamin availability. Associations between micronutrient status and gut microbiota composition have also been reported, such as the decrease in *Bifidobacterium* with iron supplementation. This study aimed to characterize the fecal microbiota of Cambodian schoolchildren, investigate associations between microbiota and various factors, and explore the impact of a nutritional intervention on fecal microbiota.

Existing literature highlights the crucial role of gut microbiota in human health, particularly in nutrient metabolism and the development of various diseases. While studies have extensively characterized the gut microbiome in young children and adults, data on school-aged children are relatively limited. The impact of diet, antibiotics, and micronutrient status on the composition and function of the gut microbiome has been well documented in several studies. However, inter-individual variability is substantial, particularly in diverse populations across different geographical regions. Studies examining the effect of dietary interventions on the gut microbiome in large cohorts are particularly rare. Previous research has shown associations between the gut microbiome and various forms of malnutrition, including acute malnutrition, with findings suggesting that microbiota immaturity is a contributing factor. Several studies have explored the role of targeted dietary interventions, including the use of microbiota-directed foods, in improving nutritional outcomes in malnourished children. These interventions have shown promise in promoting weight gain and improving micronutrient status. The role of specific micronutrients such as iron, zinc, and vitamin A in shaping the gut microbiome has also been investigated, but results have been inconsistent and often depend on the specific study design and population studied.

This study utilized a subset of 380 children from the larger FORISCA double-blind cluster-randomized controlled trial (ClinicalTrials.gov Identifier: NCT01706419), involving 9500 Cambodian schoolchildren aged 6-14 years. Children were randomly assigned to receive either normal rice (Placebo), UltraRice Original (UR-original, fortified with iron, zinc, vitamin B1 and B9), or UltraRice Improved (UR-improved, with additional vitamins A, B3, and B12) for six months. Anthropometric measurements (weight, height), blood samples (for micronutrient and inflammation markers), and fecal samples (for microbiota analysis and parasite detection) were collected at baseline and after the intervention. Fecal bacterial composition was analyzed using 16S rRNA amplicon sequencing. Alpha-diversity (within-sample diversity) and beta-diversity (between-sample diversity) were assessed using various metrics and statistical methods (Kruskal-Wallis test, PERMANOVA). Linear discriminant analysis Effect Size (LEfSe) was used to identify bacterial genera associated with different nutritional and inflammatory statuses. Linear mixed models, accounting for age and sex as covariates, were used to confirm the robustness of significant findings. PICRUSt2 software predicted the functional potential of the fecal microbiota based on 16S rRNA data. Statistical significance was determined using various tests (Mann-Whitney U test, Welch's t-test), with adjustments for multiple comparisons (Benjamini-Hochberg procedure).

The study revealed that the gut microbiota of Cambodian schoolchildren is characterized by a high proportion of *Lactobacillaceae*, which was unexpected based on previous studies in other populations. Alpha-diversity (Pielou's evenness and Shannon indices) was significantly lower in children with anaemia, iron-deficiency anaemia, and vitamin A deficiency compared to those without these deficiencies. Beta-diversity (Bray-Curtis, Jaccard, and Unifrac distances) showed significant differences between children with and without iron deficiency and vitamin A deficiency. Older children (10-14 years) had higher alpha-diversity than younger children (6-9 years). LEfSe analysis revealed associations between specific bacterial genera and several nutritional and inflammatory statuses. Anaemia was associated with genera like *Ruminococcus torques* group, *Anaerostipes*, and *Prevotella*, while iron-deficiency anaemia was associated mainly with *Prevotella*. Vitamin A deficiency was associated with *Bacilli* (Lactobacillaceae), *Gordonibacter*, and *Romboutsia*. Stunting was linked to *Prevotella 7* and *Holdemanella*. Systemic inflammation was associated with *Firmicutes*, and parasitic infection with genera like *Desulfovibrionia*, *Hungateiclostridiaceae*, and *Erysipelotrichaceae*. The six-month nutritional intervention modified the children's fecal bacterial composition and predicted functional characteristics, with differing effects depending on the rice formulation (UR-original and UR-improved). The intervention changed the microbiota even in the placebo group. MaAsLin2 analysis revealed significant associations between the two fortified rice treatments and numerous bacterial genera. PICRUSt2 analysis predicted differences in KEGG pathways based on nutritional status, with differences observed particularly in amino acid metabolism (for anaemia), and lipid metabolism (for the intervention).

This study provides valuable insights into the fecal microbiota of Cambodian schoolchildren and its association with nutritional status and inflammation. The unexpectedly high proportion of *Lactobacillaceae* warrants further investigation into its role in this population. The observed associations between specific microbiota profiles and micronutrient deficiencies (iron, vitamin A) highlight the complex interplay between diet, gut microbiota, and nutritional status. The lack of association with zinc deficiency is intriguing and might be due to the limited number of children with normal zinc status in this study. The impact of the nutritional intervention on the fecal microbiota composition and functional potential underscores the potential of dietary interventions to modify the gut microbiome. The differences in the effects of the two fortified rice formulations highlight the need for further research to optimize micronutrient supplementation strategies. The study's findings contribute to our understanding of the gut microbiome's role in child health and the potential for using microbiota-directed interventions to improve nutritional outcomes in children.

This large-scale study provides novel data on the fecal microbiota of Cambodian schoolchildren, showing a high proportion of *Lactobacillaceae* and associations between specific microbiota profiles and iron and vitamin A deficiencies but not zinc deficiency. A six-month nutritional intervention using fortified rice impacted the fecal microbiota composition and predicted functions, differing by the type of fortification. Future research could focus on exploring the mechanisms underlying these associations, optimizing micronutrient supplementation strategies, and investigating the long-term impacts of these interventions on child health.

The study's limitations include the use of multiple micronutrient-fortified rice formulations, making it challenging to isolate the effect of individual micronutrients. The relatively small number of children with normal zinc status might have limited the ability to detect associations with zinc deficiency. The study lacked a positive control in the sequencing process. Data on diarrheal episodes would have strengthened the analysis of the impact of iron consumption. Finally, whole metagenome shotgun sequencing would have provided more comprehensive functional insights.