The acquisition of the infant gut microbiota is key to establishing a host-microbiota symbiosis. Microbially produced metabolites tightly interact with the immune system, and the fermentation-derived short-chain fatty acid butyrate is considered an important mediator linked to chronic diseases later in life. The intestinal butyrate-forming bacterial population is taxonomically and functionally diverse and includes endospore formers with high transmission potential. Succession, and contribution of butyrate-producing taxa during infant gut microbiota development have been little investigated. We determined the abundance of major butyrate-forming groups and fermentation metabolites in faeces, isolated, cultivated and characterized the heat-resistant cell population, which included endospores, and compared butyrate formation efficiency of representative taxa in batch cultures. The endospore community contributed about 0.001% to total cells, and was mainly composed of the pioneer butyrateproducing Clostridium sensu stricto. We observed an increase in abundance of Faecalibacterium prausnitzii, butyrate-producing Lachnospiraceae and faecal butyrate levels with age that is likely explained by higher butyrate production capacity of contributing taxa compared with Clostridium sensu stricto. Our data suggest that a successional arrangement and an overall increase in abundance of butyrate forming populations occur during the first year of life, which is associated with an increase of intestinal butyrate formation capacity.

Glycerol/diol dehydratases (GDH) are enzymes that catalyse the production of propionate from 1,2-propanediol, and acrolein from glycerol. Acrolein reacts with dietary carcinogenic heterocyclic amines (HCA), reducing HCA mutagenicity, but is itself also an antimicrobial agent and toxicant. Gut microbial GDH activity has been suggested as an endogenous acrolein source; however, there is limited information on the potential of the intestinal microbiota to have GDH activity, and what impact it can have on the intestinal ecosystem and host health. We hypothesized that GDH activity of gut microbiota is determined by the abundance and distribution of GDH-active taxa and can be enhanced by supplementation of the GDH active Anaerobutyricum hallii, and tested this hypothesis combining quantitative profiling of gdh, model batch fermentations, microbiota manipulation, and kinetic modelling of acrolein formation. Our results suggest that GDH activity is a common trait of intestinal microbiota shared by a few taxa, which was dependent on overall gdh abundance. Anaerobutyricum hallii was identified as a key taxon in GDH metabolism, and its supplementation increased the rate of GDH activity and acrolein release, which enhanced the transformation of HCA and reduced fermentation activity. The findings of this first systematic study on acrolein release by intestinal microbiota indicate that dietary and microbial modulation might impact GDH activity, which may influence host health.

Besides nutritional components, breast milk contains diverse microbes, which may be involved in colonization of the infant gut. Expressed milk is often stored for few days in the refrigerator. The aim of this study was to determine the abundance, prevalence and diversity of facultative and strict anaerobic bacteria using culture-dependent and -independent methods, and to determine changes in milk microbial and chemical composition during storage. Samples of mature breast milk from 21 women were collected 3–6 months post-partum and were analyzed fresh or after anaerobic storage for 6 days at 4°C. The cultivable bacterial population was analyzed using the most probable number (MPN) method or plate counts and different media. The abundance of major bacterial groups was determined using quantitative PCR and 16S rRNA gene sequencing. Lactose, lactate, short chain fatty acids (SCFA) and human milk oligosaccharides (HMO) were analyzed using chromatography techniques. Highest mean viable cell counts were obtained in yeast casitone fatty acids (YCFA) broth supplied with mucin (log 4.2 ± 1.8 cells/ml) and lactose (log 3.9 ± 1.4 cells/ml), or Columbia broth (log 3.0 ± 0.7 cells/ml). Mean total bacterial counts estimated by qPCR was 5.3 ± 0.6 log cells/ml, with Firmicutes being the most abundant phylum. The most prevalent bacterial groups were Streptococcus spp. (15/19 samples), Enterobacteriaceae (13/19) and Lactobacillus/Lactococcus/Pediococcus group (12/19). While the average total number of bacterial cells did not change significantly during storage, the prevalence of strict anaerobic Bacteroidetes increased threefold, from 3/19 to 9/19, while in 7 samples Clostridium clusters IV or XIVa became detectable after storage. Major HMO were not degraded. Lactate was present in 18/21 samples after storage (2.3–18.0 mM). Butyrate was detected in 15/21 and 18/21 samples before and after storage, respectively, at concentrations ranging from 2.5 to 5.7 mM. We demonstrate enhanced prevalence and/or abundance of viable strict anaerobes from the Bacteroidetes and Clostridiales after 6-day anaerobic storage of human milk. Our data indicate that anaerobic cold storage did not markedly change total viable bacterial load, while HMO profiles were stable. Anaerobic cold storage of human milk for up to 6 days may be suitable for preserving milk quality for potential microbial transfer to the infant gut.

Background: Impaired microbial development and decreased levels of short-chain fatty acids, particularly butyrate, is suggested to have a role in the development of atopic dermatitis (AD).Methods: Faecal microbiota composition, abundance of selected bacterial groups and fermentation metabolites were compared at 90, 180 and 360 days of life between 27 children who developed AD by age one (AD group), and 39 controls (non-AD group) among the CARE (Childhood AlleRgy, nutrition and Environment) study cohort.Results: Diversity within the Firmicutes and Bacteroidetes phyla in the faecal microbiota was lower in the AD group compared with the non-AD group. Longitudinal analysis showed multiple amplicon sequence variants (ASV) within the same bacterial family to be differentially abundant. Namely, Ruminococcus bromii, a keystone primary starch degrader, and Akkermansia muciniphila, a mucin-utilizer, had lower abundance among the AD group. Children with AD were less likely to have high levels of faecal butyrate at 360 days compared with those without AD (11.5% vs 34.2%). At 360 days, children with high abundance of R. bromii had higher level of butyrate as well as lower proportion of children with AD compared to children with low abundance of R. bromii (11.1-12.5% vs 44.4-52.5%), which was independent of the abundance of the major butyrate producers. Conclusion:Our results suggested that R. bromii and other primary degraders might play an important role in the differences in microbial cross-feeding and metabolite formation between children with and without AD, which may influence the risk of developing the disease.

Bacteria harboring glycerol/diol dehydratase (GDH) encoded by the genes pduCDE metabolize glycerol and release acrolein during growth. Acrolein has antimicrobial activity, and exposure of human cells to acrolein gives rise to toxic and mutagenic responses. These biological responses are related to acrolein’s high reactivity as a chemical electrophile that can covalently bind to cellular nucleophiles including DNA and proteins. Various food microbes and gut commensals transform glycerol to acrolein, but there is no direct evidence available for bacterial glycerol metabolism giving rise to DNA adducts. Moreover, it is unknown whether pathogens, such as Salmonella Typhymurium, catalyze this transformation. We assessed, therefore, acrolein formation by four GDH-competent strains of S. Typhymurium grown under either aerobic or anaerobic conditions in the presence of 50 mM glycerol. On the basis of analytical derivatization with a heterocyclic amine, all wild-type strains were observed to produce acrolein, but to different extents, and acrolein production was not detected in fermentations of a pduC-deficient mutant strain. Furthermore, we found that, in the presence of calf thymus DNA, acrolein-DNA adducts were formed as a result of bacterial glycerol metabolism by two strains of Limosilactobacillus reuteri, but not a pduCDE mutant strain. The quantification of the resulting adducts with increasing levels of glycerol up to 600 mM led to the production of up to 1.5 mM acrolein and 3600 acrolein-DNA adducts per 108 nucleosides in a model system. These results suggest that GDH-competent food microbes, gut commensals, and pathogens alike have the capacity to produce acrolein from glycerol. Further, the acrolein production can lead to DNA adduct formation, but requires high glycerol concentrations that are not available in the human gut.