4. Discussion
diarrheal, well-nourished
MALNOURISHED, ZINC DEFICIENCY, WASTED
In this pilot study, we investigated the nutritional status and composition, relative abundance, and diversity of the gut microbiota of children in urban and rural areas of Pakistan from 3 to 24 months of age. The most significant findings were age-related changes with a decrease in HAZ score, increase in alpha and beta diversity, and changes in relative abundance of the major taxa at the level of phylum and genus with increasing age from 3 to 24 months of age. Differentially abundant taxa were identified between all of the children in the first and second years of life, between those residing in rural and urban areas, and those who received different interventions at different ages from 3 to 24 months.Several studies, many of them longitudinal, have investigated the gut microbiota using 16S rRNA sequencing in children under the age of five years in low- and middle-income countries [In the present study, we found that the most abundant phyla were Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes, in descending order. This pattern of relative abundance of the major phyla differs by country. For example, in a similar longitudinal study from South India, Firmicutes and Proteobacteria were most abundant, followed by Actinobacteria and Bacteroidetes [Increasing age also had an impact on the relative abundance of the major Phyla, with significant increases in Firmicutes and Bacteroidetes and significant decreases in Actinobacteria and Proteobacteria with increasing age. Similarly, there were changes in the relative abundance of the four most abundant genera, with significant increases in In the present study, we did not investigate eukaryotic components of the gut microbiota. Recently, Popovic et al., analyzed stool samples and data from a subset of 80 children at 12 and 24 months of age from the same clinical trial (ClinicalTrials.gov Identifier: NCT00705445) [The age-related changes in the gut microbiota in the current study were similar to those from a longitudinal pilot study comparing low birth weight stunted children to normal birth weight non-stunted children from birth to 2 years of age in South India [Specific taxa associated with linear growth stunting of children in longitudinal studies vary with geographic location, age, and analytic techniques. In the longitudinal study from India referred to above [Zinc deficiency has been shown to be associated with alteration in the gut microbiota in school-age children [There are a few limitations to our pilot study. The numbers of well-nourished and malnourished children at each age, in each location, and intervention arm were small. We did not account for antibiotic use, diarrheal episodes, or diet, all of which are known to impact the gut microbiota. We used the Roche 454 platform for 16S rRNA sequencing, which was what was available at the time. In future studies, we will use the Illumina MiSeq platform for 16S sequencing and will also perform whole genome shotgun sequencing to characterize the gut microbiome of a percentage of subjects. The study design of the parent study did not include a cohort of healthy children. In future studies, we will also use the “relative microbiota maturity index” and “microbiota-for-age Z score” developed by Subramanian et al. [This study was nested within a larger cluster-randomized trial on the effect of zinc and micronutrients on growth and morbidity. Consonant with the findings of the larger main trial [Further studies of larger numbers of malnourished (including underweight, wasted, and stunted) and well-nourished children using additional analytic tools such as “relative microbiota maturity index” and “microbiota-for-age Z score”, as well as metagenomics and metabolomics, are needed to identify differences in the gut microbiota between them and to design microbiota-directed interventions.