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, 74 (6), 1876-85

Novel Microarray Design Strategy to Study Complex Bacterial Communities

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Novel Microarray Design Strategy to Study Complex Bacterial Communities

Antoine Huyghe et al. Appl Environ Microbiol.

Abstract

Assessing bacterial flora composition appears to be of increasing importance to fields as diverse as physiology, development, medicine, epidemiology, the environment, and the food industry. We report here the development and validation of an original microarray strategy that allows analysis of the phylogenic composition of complex bacterial mixtures. The microarray contains approximately 9,500 feature elements targeting 16S rRNA gene-specific regions. Probe design was performed by selecting oligonucleotide sequences specific to each node of the seven levels of the bacterial phylogenetic tree (domain, phylum, class, order, family, genus, and species). This approach, based on sequence information, allows analysis of the bacterial contents of complex bacterial mixtures to detect both known and unknown microorganisms. The presence of unknown organisms can be suspected and mapped on the phylogenetic tree, indicating where to refine analysis. Initial proof-of-concept experiments were performed on oral bacterial communities. Our results show that this hierarchical approach can reveal minor changes (<or=1%) in gingival flora content when samples collected in individuals from similar geographical origins are compared.

Figures

FIG. 1.
FIG. 1.
Schematic representation of the probe selection process on a small subset of 16S rRNA gene sequences. Candidate probes are compared to the library of 16S rRNA gene sequences and assigned to the most distal common node. For example, probe A, which is common to the Lactobacillus, Paralactobacillus, Pediococcus, and Streptococcus genera, is assigned to the Lactobacillales order level. Probe C is assigned to the family level of the Streptococcaceae since it detects both Streptococcus and Lactococcus spp. In contrast, probe B is specific to some Pediococcus species, and it is therefore assigned to the genus level.
FIG. 2.
FIG. 2.
Microarray coverage for the 32 phyla described in the RDP (see Materials and Methods). Bars represent the percentage of strains detected within each phylum. The number of selected probes for providing this phylum coverage is specified to the right of the figure.
FIG. 3.
FIG. 3.
Volcano plots display a summary of test statistics as a function of fluorescence intensity ratios (i.e., fold change). Each plot compares spiked versus nonspiked samples, using material of increasing bacterial complexity. Red dots in the upper left or upper right corners depict significant targets in the nonspiked sample or in the spiked sample, respectively. (A) Comparison of one defined bacterial mixture containing three bacterial species with or without a spike of 25% of the nucleic acid amounts (significance is defined as a fold change of ≥2 and P ≤ 0.01). (B and C) Comparisons of gingival flora from two healthy volunteers (B1 [B] and B2 [C]) with or without a spike of 1% of the nucleic acid amounts (significance is defined as a fold change of ≥2 and P ≤ 0.05).
FIG. 4.
FIG. 4.
Phylogenic tree representation of all phyla detected by statistical analysis (P < 0.05) comparing a gingival sample (B2) to its replicate spiked with 1% F. necrogenes ATCC 25556. Red dots and lines depict nodes and branches where at least 1 probe yielded a statistically significant signal. For better readability, only the Alphaproteobacteria class is fully represented among the Proteobacteria phylum.
FIG. 5.
FIG. 5.
Cumulative prevalence of phylotypes identified using microarrays (samples B1 and B2) compared to previously published analyses of gingival flora. The figure depicts all phylotypes identified by any of these studies. Lib1, Lib2, and Lib3 describe libraries generated for three healthy subjects as described by Paster et al. (; B. Paster, unpublished data).

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