**4. Modulation of the gut microbiome by probiotics**

Probiotic mechanisms resulting in human gut microbiome alteration include effects on the microbial composition and function of these native organisms. More recent studies have utilized culture-dependent methods and metagenomic sequencing techniques to evaluate probiotic effects on changes in microbiome composition, diversity, and function. Certain strains of probiotics have been shown to release antimicrobial proteins or metabolic waste products that suppress the growth of other bacteria in the local vicinity. Others have been shown to compete with local bacterial populations for receptors and binding sites on the intestinal epithelial cells [34–36]. *Lactobacillus reuteri* is an anaerobic probiotic that converts glycerol into reuterin, a

potent antimicrobial compound that inhibits the growth of pathogenic gram-negative and gram-positive bacteria. Agar spot testing has demonstrated these inhibitory effects on enterohemorrhagic *E*. *coli* (EHEC), enterotoxigenic *E*. *coli* (ETEC), *Salmonella enterica*, *Shigella sonnei*, and *Vibrio cholerae* [34]. Gut microbiota growth and metabolism are heavily dependent on the supply of dietary carbohydrates. The probiotic *Bifidobacterium* has been observed to contribute to interspecies crossfeeding resulting in an increase in beneficial microorganisms, including *Firmicutes* bacteria. This occurs as *Bifidobacterium* can utilize starch and fructo-oligosaccharides for energy and release lactate as a metabolic byproduct. The lactate is then used by local *Firmicutes* bacteria for energy. This relationship is important for the host as *Firmicutes* bacteria produce butyrate, a beneficial short-chain fatty acid [37, 38]. Interestingly, cross-feeding between different *Bifidobacterial* strains has been shown to upregulate the transcription and expression of various genes resulting in metabolic profile changes, primarily genes that play a role in carbohydrate metabolism [38]. Shifts in metabolic gene expression have also been observed in murine models when supplemented with fermented milk products that harbored a variety of probiotic bacteria. Results of metatranscriptomic analysis on fecal samples revealed a significant change in carbohydrate enzyme gene expression, further strengthening the proposed relationship between probiotic bacteria supplementation and shifts in the metabolic function of the gut microbiome [39].

A study analyzing the fecal microbiota of 6-month-old infants explored the changes in intestinal microbiota communities when supplemented with *L*. *rhamnosus*. Their results showed an abundance of *L*. *rhamnosus* and an increased microbial species evenness index suggesting ecological stability and diversity [40]. In murine models, supplementation of *L*. *reuteri* resulted in an increase in microbial community evenness and diversity when compared to vehicle-treated mice [41]. These findings are notable as maintaining diversity in microbial communities is associated with ecological stability [42]. Interestingly, insults such as infections or antibiotic therapy that result in a decline in microbial diversity have been associated with autoimmune diseases such as Crohn's disease and eczema [43, 44]. These findings suggest that probiotics may induce local changes in the gut microbiota and directly contribute to healthy diversity and stabilization of microbial communities.
