Influence of bacteriocins and the supernatant of a co-culture (OMNi BiOTiC® AAD10) in vitro and on the fecal microbiome in mice

Abstract

Introduction: Postbiotics as alternatives to probiotics for the supportive treatment of dysbiosis may be of interest for certain high-risk patients (as neonates and immunocompromised or elderly patients). The aim of this study was to gain insights into the production, composition, antimicrobial activity and effect on the fecal microbiome of 3 different postbiotics. Methods: Culture supernatants from OMNi BiOTiC® AAD10, the reuterin system as supernatant from Lentilactobacillus diolivorans and commercially available (S)-Reutericyclin were chosen as postbiotics. The supernatants were characterized by HPLC (Reuterin) or shotgun analysis, liquid chromatography-mass spectroscopy and gas chromatography-mass spectroscopy (OMNi BiOTiC® AAD10). The antimicrobial effect was examined in cultures of Gram-positive and Gram-negative bacteria and Candida albicans. To determine the effect on the fecal microbiome a murine model with male BALBc mice was chosen. Mice were gavage fed with either postbiotic substance or control medium for 4-8 weeks (depending on the postbiotic). At the end stool was harvested and mice were euthanized. Stool samples were used for microbiome analysis and determination of volatile organic compounds (GC-MS). Results: Characterization of the 3 different supernatants: The 10 probiotic bacteria of the AAD10 showed different growth patterns during the observation period of 196h. The relative abundance (RA) of L. salivarius, L. paracasei, E. faecium and B. longum/lactis decreased over time. The RA of L. plantarum increased while L. rhamnosus showed an initial increase to 96h of culture time followed by a decrease towards the end of the observation period. Gene expression analysis revealed a dominance of gene activity in the group’s nucleotide and amino-acid biosynthesis in accordance with cell growth. The peaks of the gene expressions of the different probiotic strains coincided with their peak RA. The metabolomics analysis confirmed these findings with a dominance of amino acids, peptides and nucleotides in the cell free AAD10 supernatant. Fumaric, pantothenic, malic, 9,3-methyl-2-oxovaleric and aspartic acid, cytidine monophosphate, creatine, tryptophan, orotidine and phosphoserine correlated with culture time. For susceptibility testing and murine application cell free supernatant was harvested after 48h and 196h. The fermenter culture of L. diolivorans allowed production of a supernatant containing 13,4g/l 3-HPA (Reuterin) and 6.9g/l glycerol. Due to a potential commercial interest storage stability was tested for this supernatant and revealed a good stability over 35 days at -20°C. As (S)-Reutericyclin was obtained as pure substance no further characterizations were performed for this postbiotic. In susceptibility testing the AAD10 supernatant showed activity against S. epidermidis, L. monocytogenes, P. aeruginosae, E. faecium and C. albicans (48h), or against S. agalactiae and S. epidermidis (196h). The Reuterin system showed activity against S. aureus, S. agalactiae and S. epidermidis and (S)-Reutericyclin against S. epidermidis only. The murine application revealed a significant decrease of beta diversity in the fecal microbiome of mice receiving (S)–Reutericyclin. Neither AAD10 Supernatant nor the Reuterin system had effects on alpha or beta diversity. At species level, AAD10 supernatant increased Faecalibacterium prausnitzii and Anaeroplasma. Reuterin caused an increase in Ruminococcae and Eubacterium xylanophilum and a decrease in Lachnospiraceae and Ruminoclostridium. (S)-Reutericyclin caused an increase in Muribaculum and Streptococcus and a decrease in butyrate-producing Roseburia, Ruminoclostridium and Eubacterium xylanophilum. The VOC testing in the stool was only performed for Reuterin and (S)-Reutericyclin groups. A significant decrease in heptane and an increase in 3-methylbutanal could be demonstrated for Reuterin. Significant increases in heptane and pentane and decreases in 2-heptanone and 2,3-butanedione were found for (S) - Reutericyclin. Conclusions: The 3 different postbiotic culture supernatants showed different antimicrobial profiles against aerobic bacteria. These effects could be used as “selective” antibiotic in the future – for instance in the therapy of atopic dermatitis (effect of Reuterin against staphylococci and streptococci), food stabilizer (effect of AAD10 supernatant against L. monocytogenes) or targeted antibiotic (effect of AAD10 supernatant against L. monocytogenes, P. aeruginosae and E. faecium). While (S) – Reutericyclin with reduced beta diversity in the microbiome analysis and a decrease in the relative abundance of butyrate producers had rather negative effects on the murine fecal microbiome, the effects of Reuterin and AAD10 supernatant were rather unspecific. The importance of the observed changes of volatile stool gases as a marker of bacterial metabolism remains to be addressed in future research projects.

Datum der Bewilligung	2023
Originalsprache	Englisch
Gradverleihende Hochschule	Medizinische Universität Graz
Betreuer/-in	Christoph Castellani (Betreuer*in), Georg Singer (Betreuer*in) & Daniela Ingrid Sperl (Betreuer*in)