Reuterin is a broad-spectrum antimicrobial system produced by specific strains of Lactobacillus reuteri during anaerobic metabolism of glycerol. Acrolein is the main component responsible for its antimicrobial activity. Here, the sensitivity of Campylobacter jejuni (n = 51) and Campylobacter coli (n = 20) isolates from chicken meat and human stool samples to reuterin was investigated. The minimum inhibitory concentration (MIC) of C. jejuni and C. coli strains was measured between 1.5 and 3.0 µM of acrolein, below the MIC of the sensitive indicator strain Escherichia coli K12 (16.5 µM acrolein). The interaction of C. jejuni N16-1419 and the reuterin-producing L. reuteri PTA5_F13 was studied during 24 h co-cultures with or without glycerol. A high C. jejuni growth was observed in cultures without glycerol. In contrast, C. jejuni growth decreased from 7.3 ± 0.1 log CFU/mL to below detection limit (1 log CFU/mL) during co-cultures added with 28 mM glycerol. This bactericidal effect could be attributed to in situ reuterin production. The low MIC observed and the high sensitivity towards in situ produced reuterin suggests L. reuteri combined with glycerol, as a possible intervention option to reduce Campylobacter in the food chain.

Vitamin B12 (cobalamin) is present in the human lower gastrointestinal tract either coming from the unabsorbed dietary fraction or from in situ production of the gut microbiota. However, it is unclear whether the gut microbial communities need exogenous B12 for growth and metabolism, or whether B12 in low and high levels could affect gut community composition and metabolite production. Here, we investigated in vitro B12 production of human fecal microbiota and the effects of different levels of B12 (as cyanocobalamin) on composition and activity. Eight fecal communities from healthy human adults distributed over three enterotypes, dominated by Firmicutes (n = 5), Bacteroides (n = 1) or Prevotella (n = 2) were used to perform batch fermentations in Macfarlane medium supplemented with low B12 medium (Control, 5 ng/ml, within the tested fecal range), no B12 addition (NB12), and high B12 addition (ExtraB12, 2500 ng/ml). The microbiota community composition (qPCR, 16S rRNA metabarcoding), metabolic activity (HPLC-RI), and B12 levels (UHPLC-DAD) were measured after 24 h incubation at 37^°C under strict anaerobic conditions. All fecal microbial communities produced B12 in the NB12 condition after 24 h, in the range from 152 ± 4 to 564 ± 25 ng/ml. None of the B12 treatments had an impact on total bacterial growth, community richness, diversity and total metabolite production, compared to the low B12 control. However, a significant increase of propionate was measured in ExtraB12 compared to NB12. Most taxonomic and metabolite changes compared to control incubations were donor-dependent, implying donor-microbiota-specific changes upon B12 treatments. Our in vitro data suggest that healthy human adult gut microbial communities have the capacity to produce B12 at levels fulfilling their own requirements, independently of the initial B12 content tested in the donor’s feces. Further, supplementation of exogenous dietary B12 may have limited impact on the healthy human gut microbial community composition and function.

Background: Folate (vitamin B9) occurs naturally mainly as tetrahydrofolate (THF), methyl-tetrahydrofolate (M-THF), and formyl-tetrahydrofolate (F-THF), and as dietary synthetic form (folic acid). While folate auxotrophy and prototrophy are known for several gut microbes, the specific folate forms produced by gut prototrophs and their impact on gut auxotrophs and microbiota remain unexplored. Methods: Here, we quantified by UHPLC-FL/UV folate produced by six predicted gut prototrophs (Marvinbryantia formatexigens DSM 14469, Blautia hydrogenotrophica 10507 ^T, Blautia producta DSM 14466, Bacteroides caccae DSM 19024, Bacteroides ovatus DSM 1896, and Bacteroides thetaiotaomicron DSM 2079 ^T) and investigated the impact of different folate forms and doses (50 and 200 µg/l) on the growth and metabolism of the gut auxotroph Roseburia intestinalis in pure cultures and during fecal anaerobic batch fermentations (48 h, 37 °C) of five healthy adults. Results: Our results confirmed the production of folate by all six gut strains, in the range from 15.3 ng/ml to 205.4 ng/ml. Different folate forms were detected, with THF ranging from 12.8 to 41.4 ng/ml and 5-MTHF ranging from 0.2 to 113.3 ng/ml, and being detected in all strains. Natural folate forms, in contrast to folic acid, promoted the growth and metabolism of the auxotroph R. intestinalis L1-82, with dose-dependent effects. During fecal batch fermentations, folate forms at both levels had no detectable effect on total bacteria concentration, on gut community composition and metabolic activity and on Roseburia spp. abundance, compared to the control without folate addition. Conclusions: Our study demonstrates for the first time in vitro the production of different natural folate forms by predicted gut prototrophs and the stimulation on the growth of the folate auxotrophic butyrate-producing R. intestinalis L1-82. Surprisingly, folate did not impact fecal fermentations. Our data suggest that the dietary folate forms at the tested levels may only have limited effects, if any, on the human gut microbiota in vivo.

Lactobacillus reuteri is a natural inhabitant of selected animal and human gastrointestinal tract (GIT). Certain strains have the capacity to transform glycerol to 3-hydroxypropionaldehyde (3-HPA), further excreted to form reuterin, a potent antimicrobial system. Reuterin-producing strains may be applied as a natural antimicrobial in feed to prevent pathogen colonization of animals, such as in poultry, and replace added antimicrobials. To date, only seven L. reuteri strains isolated from poultry have been characterized which limits phylogenetic studies and host-microbes interactions characterization. This study aimed to isolate L. reuteri strains from poultry GIT and to characterize their reuterin production and antimicrobial resistance (AMR) profiles using phenotypic and genetic methods. Seventy reuterin-producing strains were isolated from poultry crop, faeces and caeca and twenty-five selected for further characterization. Draft genomes were generated for the new 25 isolates and integrated in a phylogenetic tree of 40 strains from different hosts. Phylogenetic analysis based on gene content as well as on core genomes showed grouping of the selected 25 L. reuteri poultry isolates within the poultry/human lineage VI. Strains harbouring pdu-cob-cbi-hem genes (23/25) produced between 156 mM ± 11 and 330 mM ± 14 3-HPA, from 600 mM of glycerol, in the conditions of the test. All 25 poultry strains were sensitive to cefotaxime (MIC between 0.016 and 1 μg/mL) and penicillin (MIC between 0.02 and 4 μg/mL). Akin to the reference strains DSM20016 and SD2112, the novel isolates were resistant to penicillin, possibly associated with identified point mutations in ponA , pbpX , pbpF and pbpB . All strains resistant to erythromycin (4/27) carried the ermB gene, and it was only present in poultry strains. All strains resistant to tetracycline (5/27) harbored tetW gene. This study confirms the evolutionary history of poultry/human lineage VI and identifies pdu-cob-cbi-hem as a frequent trait but not always present in this lineage. L. reuteri poultry strains producing high 3-HPA yield may have potential to prevent enteropathogen colonization of poultry.