Role of Cutibacterium avidum during gut colonization and in the alleviation of Infant Colic
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Open access
Autor(in)
Datum
2019Typ
- Doctoral Thesis
ETH Bibliographie
yes
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Abstract
Infant colic (IC) affects 5-19% of infants younger than five months of age and is characterized by crying, fussing or irritability that last more than three hours per day, during three or more days a week and occur without obvious cause. Several studies have identified an altered fecal microbiota in colic infants, a status of low-grade inflammation and high breath hydrogen (H2) excretion compared to healthy infants and different mechanisms have been hypothesized, but causality remains unclear.
A newborn’s diet is based on breast milk or formula, with high content of lactose and for the former of human milk oligosaccharides (HMO), which could serve as substrate for microbial metabolism in the colon. The infant gut microbiota is characterized by high abundance of lactate-producing bacteria and consequently, to prevent accumulation and toxic effect, lactate must be concomitant metabolized by lactate-utilizing bacteria (LUB). The LUB community in infants until 4 months of age is mainly composed of propionate-producing bacteria, Bacteroides spp., Propionibacterium/Cutibacterium (former Propionibacterium spp.) and Veillonella spp., with lower numbers of butyrate-producing bacteria, Anaerostipes spp., Eubacterium hallii and Eubacterium limosum, and sulphide-reducing bacteria (SRB) like Desulfovibrio. Concomitantly with lactate utilization, there is formation of H2 and hydrogen sulphide (H2S) by some LUB members, gases associated with pain and intestinal discomfort in humans. The theoretical model sustaining this thesis states that the over-production of detrimental lactate, H2 and H2S is a contributing factor causing IC. Propionibacterium/Cutibacterium convert lactate into propionate, acetate and CO2, without H2. In doing so, they could prevent lactate accumulation and divert lactate from the production of harmful metabolites, thus potentially alleviating colic symptoms in infants. Therefore, the aim of this thesis was to investigate the role of Propionibacterium/Cutibacterium during gut colonization and in the alleviation of IC.
We first characterized the abundance, diversity and persistence of Propionibacterium/Cutibacterium in a small infant cohort at 2 (n=19), 4 (n=37), 8 (n=38) and 12 (n=32) weeks of age. For this purpose, we developed and validated a quantitative PCR method to enumerate Propionibacterium/Cutibacterium in infant feces that we used together with culture-based enumeration to investigate colonization dynamic in infants. The prevalence of Propionibacterium/Cutibacterium was high (84%) at 2 weeks of age and decreased to 41% by 12 weeks of age. Persistence of Propionibacterium/Cutibacterium was dependent on colonization levels at 2 weeks and decreased simultaneously with introduction of milk-formula in the diet and increasing abundance of butyrate-producing bacteria. The development of a selective medium with lactate as sole carbon source and supplemented with antibiotics allowed for the isolation of 87 C. avidum isolates from infant feces (n=10; age range: 1.5-26 weeks). Colonization potential and metabolic effects of non-hemolytic C. avidum isolates were tested in an in vitro continuous intestinal fermentation model mimicking infant proximal colon conditions. C. avidum spiked daily at 108 or 109 cells mL-1 colonized, decreased formate, and persisted during the washout period. We identified correlations between Propionibacterium/Cutibacterium and lactate-producers in two reactors as well as in infant feces.
To gain further insights on the evolutionary adaption of C. avidum, we used novel phylogenetic markers to explore the relationship of infant C. avidum to C. avidum strains from other body sites. We described a subpopulation C. avidum depicting specific phenotypic characteristics (negative hemolysis, α-galactosidase and β-glucuronidase activity), which suggest adaptation to the infant gut. We further observed ability to utilize in vitro an array of energy sources (D- and L-lactate, glycerol, glucose, galactose, N-acetyl-D-glucosamine and maltodextrins) normally available in the infant gut, which could support their colonization. In addition, we observed increased growth and propionate production by C. avidum P279 in co-culture with infant Bifidobacterium strains further indicating their role as lactate-utilizers.
For the last step, we developed a gnotobiotic animal model of germ-free rats inoculated with fecal slurries from two- to three-month-old infants, either healthy (n=2) or colicky (n=2). Quantitative PCR and 16S rRNA gene amplicon sequencing revealed engraftment of dominant fecal microbiota in infant-microbiota associated rats (IMA) independently of the dominant taxa in donors. We identified an altered functionality, with high H2 excretion compared to previous gnotobiotic experiments using fecal microbiota from healthy adults and irritably bowel syndrome patients in IMA rats. We used this new model to investigate the effect of C. avidum P279, selected for its colonization ability observed in in vitro colonic fermentations, on metabolism, especially on levels of H2 production and on microbial composition in IMA rats. In line with our hypothesis, colic IMA rats supplemented with C. avidum P279 excreted significantly lower levels of H2 after intervention compared to animals assigned to placebo treatment.
Overall, our findings provide novel scientific data regarding infant gut colonization by C. avidum and their role as lactate-degraders and propionate-producers in the infant gut. Our data showed increased microbial production of H2 by the infant GM and potential ability of C. avidum P279 to reduce it. Based on our observations, we propose a new mechanistic model with intestinal production of H2 as key factor for IC development. Future research focusing on the relationship between H2 metabolism and the inflammatory and nociceptive response of the host may help to further elucidate the mechanism behind IC. Mehr anzeigen
Persistenter Link
https://doi.org/10.3929/ethz-b-000381110Publikationsstatus
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Beteiligte
Referent: Lacroix, Christophe![cc](/themes/Mirage2//images/orcid_icon.png)
Referent: Braegger, Christian
Referent: Schwab, Clarissa
Referent: Bernalier-Donadille, Annick
Referent: Chassard, Christophe
Verlag
ETH ZurichThema
Gut microbiota; infant colic; Propionibacterium; Cutibacterium; GnotobioticOrganisationseinheit
03626 - Lacroix, Christophe (emeritus) / Lacroix, Christophe (emeritus)
ETH Bibliographie
yes
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