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Simultaneous quantification of multiple bacteria by the BactoChip microarray designed to target species-specific marker genes.

Ballarini A, Segata N, Huttenhower C, Jousson O - PLoS ONE (2013)

Bottom Line: The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes.The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%).In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Biology, University of Trento, Trento, Italy. ballarini@science.unitn.it

ABSTRACT
Bacteria are ubiquitous throughout the environment, the most abundant inhabitants of the healthy human microbiome, and causal pathogens in a variety of diseases. Their identification in disease is often an essential step in rapid diagnosis and targeted intervention, particularly in clinical settings. At present, clinical bacterial detection and discrimination is primarily culture-based, requiring both time and microbiological expertise, especially for bacteria that are not easily cultivated. Higher-throughput molecular methods based on PCR amplification or, recently, microarrays are reaching the clinic as well. However, these methods are currently restricted to a small set of microbes or based on conserved phylogenetic markers such as the 16S rRNA gene, which are difficult to resolve at the species or strain levels. Here, we designed and experimentally validated the BactoChip, an oligonucleotide microarray for bacterial detection and quantification. The chip allows the culture-independent identification of bacterial species, also determining their relative abundances in complex communities as occur in the commensal microbiota or in clinical settings. The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes. The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%). In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

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Accurate determination of microbial community composition of up to 15 species at even and staggered abundances.(A) Bar plots show true (blue) versus predicted (red and green) relative abundances for a 9-species bacterial community with evenly distributed abundances and high or low DNA concentrations. MSE of relative abundance over all predictions was below 0.0006. (B) True (blue) versus predicted (red) abundances for a 15-species evenly distributed community, total MSE <0.0013. (C) Evaluation of the BactoChip’s overall quantitation of relative microbial abundances in all four staggered communities. Each point represents the predicted vs. true relative abundance for one species in one experiment, with total R2>0.75.
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pone-0055764-g004: Accurate determination of microbial community composition of up to 15 species at even and staggered abundances.(A) Bar plots show true (blue) versus predicted (red and green) relative abundances for a 9-species bacterial community with evenly distributed abundances and high or low DNA concentrations. MSE of relative abundance over all predictions was below 0.0006. (B) True (blue) versus predicted (red) abundances for a 15-species evenly distributed community, total MSE <0.0013. (C) Evaluation of the BactoChip’s overall quantitation of relative microbial abundances in all four staggered communities. Each point represents the predicted vs. true relative abundance for one species in one experiment, with total R2>0.75.

Mentions: The BactoChip was able to correctly identify bacterial presence and absence within these two communities with species-level specificity (Figure 4, Figure S2). Quantitatively, the estimated relative abundances closely approximated the expected community compositions. In the smaller of the two communities, evenly distributed abundance estimations deviated from reference values by less than 15% in half of the cases, the worst result being an estimation of 3.9% of Acinetobacter baumanii against an expected value of 7.7%. The staggered abundance setting achieved similar results, with an overall correlation of 0.97 (p<10−10) of predicted values with reference. Even in the larger community, microbial composition was correctly identified in each case, with only two outliers (Stenotrophomonas maltophilia and Streptococcus pyogenes) in a formulation of the community with staggered abundances; these were likely due to a swapped sample label and were not considered in subsequent experiments, as available resources precluded the possibility of proving or disproving a mislabeling. For the evenly distributed formulation of this community, all relative abundance estimations were within at most two-fold of the expected value.


Simultaneous quantification of multiple bacteria by the BactoChip microarray designed to target species-specific marker genes.

Ballarini A, Segata N, Huttenhower C, Jousson O - PLoS ONE (2013)

Accurate determination of microbial community composition of up to 15 species at even and staggered abundances.(A) Bar plots show true (blue) versus predicted (red and green) relative abundances for a 9-species bacterial community with evenly distributed abundances and high or low DNA concentrations. MSE of relative abundance over all predictions was below 0.0006. (B) True (blue) versus predicted (red) abundances for a 15-species evenly distributed community, total MSE <0.0013. (C) Evaluation of the BactoChip’s overall quantitation of relative microbial abundances in all four staggered communities. Each point represents the predicted vs. true relative abundance for one species in one experiment, with total R2>0.75.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3569451&req=5

pone-0055764-g004: Accurate determination of microbial community composition of up to 15 species at even and staggered abundances.(A) Bar plots show true (blue) versus predicted (red and green) relative abundances for a 9-species bacterial community with evenly distributed abundances and high or low DNA concentrations. MSE of relative abundance over all predictions was below 0.0006. (B) True (blue) versus predicted (red) abundances for a 15-species evenly distributed community, total MSE <0.0013. (C) Evaluation of the BactoChip’s overall quantitation of relative microbial abundances in all four staggered communities. Each point represents the predicted vs. true relative abundance for one species in one experiment, with total R2>0.75.
Mentions: The BactoChip was able to correctly identify bacterial presence and absence within these two communities with species-level specificity (Figure 4, Figure S2). Quantitatively, the estimated relative abundances closely approximated the expected community compositions. In the smaller of the two communities, evenly distributed abundance estimations deviated from reference values by less than 15% in half of the cases, the worst result being an estimation of 3.9% of Acinetobacter baumanii against an expected value of 7.7%. The staggered abundance setting achieved similar results, with an overall correlation of 0.97 (p<10−10) of predicted values with reference. Even in the larger community, microbial composition was correctly identified in each case, with only two outliers (Stenotrophomonas maltophilia and Streptococcus pyogenes) in a formulation of the community with staggered abundances; these were likely due to a swapped sample label and were not considered in subsequent experiments, as available resources precluded the possibility of proving or disproving a mislabeling. For the evenly distributed formulation of this community, all relative abundance estimations were within at most two-fold of the expected value.

Bottom Line: The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes.The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%).In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

View Article: PubMed Central - PubMed

Affiliation: Centre for Integrative Biology, University of Trento, Trento, Italy. ballarini@science.unitn.it

ABSTRACT
Bacteria are ubiquitous throughout the environment, the most abundant inhabitants of the healthy human microbiome, and causal pathogens in a variety of diseases. Their identification in disease is often an essential step in rapid diagnosis and targeted intervention, particularly in clinical settings. At present, clinical bacterial detection and discrimination is primarily culture-based, requiring both time and microbiological expertise, especially for bacteria that are not easily cultivated. Higher-throughput molecular methods based on PCR amplification or, recently, microarrays are reaching the clinic as well. However, these methods are currently restricted to a small set of microbes or based on conserved phylogenetic markers such as the 16S rRNA gene, which are difficult to resolve at the species or strain levels. Here, we designed and experimentally validated the BactoChip, an oligonucleotide microarray for bacterial detection and quantification. The chip allows the culture-independent identification of bacterial species, also determining their relative abundances in complex communities as occur in the commensal microbiota or in clinical settings. The microarray successfully distinguished among bacterial species from 21 different genera using 60-mer probes targeting a novel set of in silico identified high-resolution marker genes. The BactoChip additionally proved accurate in determining species-level relative abundances over a 100-fold dynamic range in complex bacterial communities and with a low limit of detection (0.1%). In combination with the continually increasing number of sequenced bacterial genomes, future iterations of the technology could enable to highly accurate clinically-oriented tools for rapid assessment of bacterial community composition and relative abundances.

Show MeSH
Related in: MedlinePlus