Characterization of the fecal microbiota of rural Malawian children, associations with biomarkers of environmental enteric dysfunction and the impact of a mass drug administration program

Abstract

Environmental Enteric Dysfunction (EED) is a subclinical condition of the gut characterized by changes in gut morphology and function with underlying chronic inflammatory responses. EED affects more than two-thirds of young children living in low-income settings. EED has been linked with growth faltering in young children living in low-income settings, however, the mechanisms by which EED impacts growth are not well defined. A proposed pathway suggests that ingestion of enteric pathogens and toxins, through contaminated food and water, changes gut microbiota composition and function. This results in intestinal inflammation and changes in the intestinal epithelium structure. There is limited data examining the association between the gut microbiota and EED. The present work was nested in a cluster-randomized trial of mass drug administration of azithromycin and was carried out to characterize composition and diversity of the gut microbiota in rural Malawian children with and without signs of EED. The work also tested for associations between the gut microbiota and stunting. Proteins were extracted from fecal samples and used for the quantification, by ELISA, of neopterin, myeloperoxidase and alpha-1 antitrypsin, which are biomarkers of intestinal inflammation and permeability that have been proposed as potential proxies of EED. Concentrations of the three biomarkers were combined to form a proxy EED score using principal component analysis. Total genomic DNA was also extracted from the fecal samples for V4-16SrRNA sequencing and for species-specific PCR to determine intestinal carriage of bacteria that have previously been shown to be associated with growth in a mouse model. All three biomarkers decreased with age. Faecalibacterium prausnitzii and Dorea formicigenerans were prevalent in over 70% of children while Bifidobacterium longum was the least prevalent of the bacteria assayed. No associations were found between fecal biomarkers of EED or intestinal bacteria carriage and stunting, however, intestinal carriage of D. formicigenerans was associated with normal biomarker concentration. Increased fecal microbiota diversity was associated with a reduction in neopterin concentration. Increased abundance of Succinivibrio was associated with reduced EED scores. Mass treatment with azithromycin appeared to have no long-term impact on alpha diversity of fecal samples but was weakly associated with increased abundance of Prevotella at 24-months follow-up. A negative correlation between age and the three biomarkers of EED was found and confirms trends shown in other children living in similar low-income settings. The association between D. formicigenerans and normal biomarker concentration suggests a potential beneficial role of the bacterium in gut health. An increase in microbiota diversity is potentially associated with reduced intestinal inflammation, but larger studies are needed to confirm this. The association between Succinivibrio and biomarkers of EED shown here is consistent with recent data from another study conducted in Malawi. Succinivibrio plays a role in fiber-degradation and metabolites of fiber-degradation have been shown to inhibit intestinal colonization by pathogenic bacteria, therefore, further studies are needed to investigate the significance of this bacterium in EED. Mass treatment with azithromycin may have long-lasting effects on the abundance of defined bacterial taxa but not overall microbiota diversity.