Risultati finali
Preweaning period:
There are many differences in diet and lifestyle characteristics across Europe. These may influence the development of the infant gut microbiota and its metabolism. The aim of this work was to determine the faecal microbiota composition of 606 young infants coming from 5 European countries with different lifestyle characteristics, and to correlate this composition to different socio-demographic parameters, as well as to way of delivery, feeding method and antibiotic treatment.
Faecal samples obtained from 6-weeks old infants were analysed by fluorescent in situ hybridisation combined with flow cytometry (FISH-FC) with a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes for assessment of the microbiota composition. The Bifidobacterium genus was predominately detected (40% average proportion of total bacteria), followed by Bacteroides (11.4%) and enterobacteria (7.5%).
Large differences were observed between countries in the composition of the infant gut, as well as a north-south gradient, opposing high bifidobacteria and low diversity faecal microbiota in the North and early diversification and high Bacteroides proportions in the South. Spain was the most different, while Germany was the closest to the average microbiota composition observed. Bifidobacteria largely dominated among breast-fed infants, while formula-fed babies presented significantly higher proportions of Bacteroides, and members of the Clostridium coccoides and Lactobacillus groups.
Vaginally delivered newborns presented higher proportions of Bacteroides and members of the Atopobium cluster compared to those born by Caesarean section, having higher proportion of members of the Clostridium coccoides and Streptococcus groups. Infants receiving antibiotic treatment presented significantly higher proportions of enterobacteria, while when mothers received antibiotics during pregnancy, newborns presented lower proportions of Bacteroides and members of the Atopobium cluster.
Weaning and post-weaning period:
With the introduction of the first solid foods in the infant diet, the faecal microbiota changes and weaning is a critical stage in gut development. The aim of this work was to determine the faecal microbiota composition of infants from 5 European countries with different lifestyle characteristics (Sweden, Scotland, Germany, Italy and Spain) one month after weaning, and to comparatively assess the impact of geographic origin as well as way of delivery, feeding method, and perinatal antibiotic treatment.
Faecal samples obtained from 605 infants 4 weeks after the introduction of first solid foods were analysed by fluorescent in situ hybridisation combined with flow cytometry using a panel of 10 rRNA targeted group- and species-specific oligonucleotide probes. The Bifidobacterium genus (36.5% average proportion of total detectable bacteria), Clostridium coccoides group (14%) and Bacteroides (13.6%) were predominant after weaning.
Northern-European countries were associated with higher proportions of bifidobacteria in infant gut microbiota while higher Bacteroides and lactobacilli characterized southern countries. Beyond the country of birth that led to a north-south gradient, several factors that influenced the microbiota at 6 weeks (formerly assessed for the same infants) had a persistent effect after weaning.
The initial feeding method influenced the C. leptum group and C. difficile + C. perfringens species, and bifidobacteria still dominated after weaning among initially breast-fed infants, while formula-fed babies presented significantly higher proportions of Bacteroides and members of the Clostridium coccoides group. The way of delivery influenced changes in proportions of Bacteroides and Atopobium. No impact of perinatal antibiotic treatment was still observed after weaning.
It is postulated that a lack of certain bacteria or colonisation with inappropriate strains could delay the development of tolerance in infancy. Clostridium perfringens and Clostridium difficile are pathogenic clostridia potentially associated with gastrointestinal infections and allergy in infants.
Due to their importance, the aim of this work was to develop two species-specific oligonucleotide probes applicable to epidemiological investigations using FISH combined with flow cytometry (FISH-FC). To enable the molecular detection and quantification of these species in the infant gut, two 16S rRNA oligonucleotide probes were developed: Cdif198 for C. difficile and Cperf191 for C. perfringens. We defined the probes in silico using the RDP sequence database.
The probes were then validated using FISH combined with flow cytometry and a collection of target and non-target strains, and faecal samples inoculated with dilutions of C. difficile and C. perfringens strains. The probes Cdif198 and Cperf191 resulted as a class I and a class II probes, respectively, as defined by Fuchs and colleagues (Fuchs et al., 1998). These new probes were used, in association with a panel of 8 probes targeting the predominant faecal bacterial groups of humans, to assess the composition of the intestinal microbiota of 33 infants of 1.5 to 18.5 months of age.
The probes designed allowed detection and quantification of the relative proportions of C. difficile (0.5% ± 1.0%) and C. perfringens (2.1% ± 2.3%) in the microbiota of infants. The Bifidobacterium genus was predominant with an average proportion of 32% of cells detected. When the proportions of the bacterial cells detected were added together, a mean of 78.5% was obtained with the panel of 10 non-overlapping phylogenetic probes.
Reference: Clostridium difficile and Clostridium perfringens species detected in infant faecal microbiota using 16S rRNA targeted probes. Journal of Microbiological Methods, 67 (1) 150-161, 2006
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