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FP5

INFABIO Report Summary

Project ID: QLK1-CT-2002-02606
Funded under: FP5-LIFE QUALITY
Country: France

FISH probe for C difficile

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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