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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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Quantification of the relative abundances of multiple species from the same genus contained within a single sample.True versus detected relative abundances for each of 6 Staphylococcus species are shown in blue (gold standard) and red (inferred), respectively. Even DNA relative abundances were targeted, with true experimental abundances of cell copy numbers varying due to differences in genome size. Mean Squared Error (MSE) of relative abundance over all predictions was below 0.0019.
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pone-0055764-g003: Quantification of the relative abundances of multiple species from the same genus contained within a single sample.True versus detected relative abundances for each of 6 Staphylococcus species are shown in blue (gold standard) and red (inferred), respectively. Even DNA relative abundances were targeted, with true experimental abundances of cell copy numbers varying due to differences in genome size. Mean Squared Error (MSE) of relative abundance over all predictions was below 0.0019.

Mentions: We next assessed the BactoChip’s ability to additionally discriminate multiple mixed bacterial species within the same genus. This is of particular interest for genera such as Staphylococcus in which some species may be pathogenic and others commensal; these are often poorly resolved by single 16S genes [36], [37]. This genus contains extremely diverse species found throughout the human microbiome [38] and includes organisms of clinical interest such as S. aureus. We thus tested the BactoChip on 6 species from the clinically relevant staphylococci comprising S. aureus, S. carnosus, S. epidermidis, S. haemolyticus, S. lugdunensis, and S. saprophyticus. Hybridization of this mixed sample on the BactoChip resulted in all six species being correctly identified (Figure 3).


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)

Quantification of the relative abundances of multiple species from the same genus contained within a single sample.True versus detected relative abundances for each of 6 Staphylococcus species are shown in blue (gold standard) and red (inferred), respectively. Even DNA relative abundances were targeted, with true experimental abundances of cell copy numbers varying due to differences in genome size. Mean Squared Error (MSE) of relative abundance over all predictions was below 0.0019.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3569451&req=5

pone-0055764-g003: Quantification of the relative abundances of multiple species from the same genus contained within a single sample.True versus detected relative abundances for each of 6 Staphylococcus species are shown in blue (gold standard) and red (inferred), respectively. Even DNA relative abundances were targeted, with true experimental abundances of cell copy numbers varying due to differences in genome size. Mean Squared Error (MSE) of relative abundance over all predictions was below 0.0019.
Mentions: We next assessed the BactoChip’s ability to additionally discriminate multiple mixed bacterial species within the same genus. This is of particular interest for genera such as Staphylococcus in which some species may be pathogenic and others commensal; these are often poorly resolved by single 16S genes [36], [37]. This genus contains extremely diverse species found throughout the human microbiome [38] and includes organisms of clinical interest such as S. aureus. We thus tested the BactoChip on 6 species from the clinically relevant staphylococci comprising S. aureus, S. carnosus, S. epidermidis, S. haemolyticus, S. lugdunensis, and S. saprophyticus. Hybridization of this mixed sample on the BactoChip resulted in all six species being correctly identified (Figure 3).

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