Rapid screening of the human microbiome by bacterial profiling
Abstract number: O179
Budding A., Akol H., van Bodegraven A., Savelkoul P.
Objectives: It is well known that the human microbiota plays an important role in health and disease. However, the exact role of the commensal bacteria and the ways in which they influence our wellbeing remains elusive. One of the major obstacles in solving these and related questions is the analysis of the microbiome. New insights have recently been gained with high throughput sequence analysis but, although valuable, these methods are not suited for large scale screening. Here we present a rapid and straightforward assay for the profiling of the human microbiota.
Methods: The molecular method involves two features: the length of the interspace (IS) region between the 16s and the 23s rDNA and specific primer sequences discriminating Firmicuta and Bacteroidetes, the two major phyla in the human colon. The IS region is conserved within a species, but varies between species. Thus bacteria can in principle be identified by the length of their IS region. Unknown species can be recognised using the specific primer sequences. The primers were combined with non-specific reverse primers in a double label multiplex PCR. Size and colour sorting of fragments was performed in an ABI 3130XL sequencer.
For validation purposes an in silico profile of 5 bacteria was constructed based on known sequence information and tested with the cultured bacteria. Then, colonic mucosal biopsies from 20 patients were analysed using this method. Intra- and interpatient composition of colonic microbiota was analysed in 5 biopsies per patient, from coecum, flexura hepatica, flexura lienalis, sigmoid and rectum.
Results: All cultured bacteria showed a profile identical to the in silico profile. Subsequently, excellent bacterial profiles were obtained for the clinical samples. Each patient had a unique bacterial profile, with only a few fragments being identical between patients. These fragments corresponded to well known colonic bacteria such as Faecalibacterium prausnitzii and Bacteroides thetaiotaomicron. Patterns obtained from the different sites of the colon of a single patient were almost identical.
Conclusions: The bacterial profiling method proved to be identical to expected profiles based on sequence data. In addition, the method was suited for characterisation of the complex human microbiome. With this method, profiles of the human colonic microbiota can be obtained in a straightforward fashion. The method is universal, fast, reproducible and ideally suited for analysis of large sample sets.
|Session name:||18th European Congress of Clinical Microbiology and Infectious Diseases|
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