The glyphosate formulation Roundup® LB plus influences the global metabolome of pig gut microbiota in vitro.

Glyphosate is the world's most widely used herbicide, and its potential side effects on the intestinal microbiota of various animals, from honeybees to livestock and humans, are currently under discussion. Pigs are among the most abundant livestock animals worldwide and an impact of glyphosate on their intestinal microbiota function can have serious consequences on their health, not to mention the economic effects. Recent studies that addressed microbiota-disrupting effects focused on microbial taxonomy but lacked functional information. Therefore, we chose an experimental design with a short incubation time in which effects on the community structure are not expected, but functional effects can be detected. We cultivated intestinal microbiota derived from pig colon in chemostats and investigated the acute effect of 228 mg/d glyphosate acid equivalents from Roundup® LB plus, a frequently applied glyphosate formulation. The applied glyphosate concentration resembles a worst-case scenario for an 8-9 week-old pig and relates to the maximum residue levels of glyphosate on animal fodder. The effects were determined on the functional level by metaproteomics, targeted and untargeted meta-metabolomics, while variations in community structure were analyzed by 16S rRNA gene profiling and on the single cell level by microbiota flow cytometry. Roundup® LB plus did not affect the community taxonomy or the enzymatic repertoire of the cultivated microbiota in general or on the expression of the glyphosate target enzyme 5-enolpyruvylshikimate-3-phosphate synthase in detail. On the functional level, targeted metabolite analysis of short chain fatty acids (SCFAs), free amino acids and bile acids did not reveal significant changes, whereas untargeted meta-metabolomics did identify some effects on the functional level. This multi-omics approach provides evidence for subtle metabolic effects of Roundup® LB plus under the conditions applied.