Limits...
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.

Show MeSH

Related in: MedlinePlus

Accurate detection of individual bacterial species using the BactoChip.Bar plot indicates the Areas Under the receiver operating characteristic Curves (AUCs) for detection of 37 individual target species. Heatmap shows intensities of individual probes (red) and postprocessed aggregate species quantification (green). Almost all tested species (94.6%) were identified unequivocally by the specifically designed oligonucleotide probes, both based on raw data (see diagonal) and by comparing against the microbial gold standard (all AUC values >0.96).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3569451&req=5

pone-0055764-g002: Accurate detection of individual bacterial species using the BactoChip.Bar plot indicates the Areas Under the receiver operating characteristic Curves (AUCs) for detection of 37 individual target species. Heatmap shows intensities of individual probes (red) and postprocessed aggregate species quantification (green). Almost all tested species (94.6%) were identified unequivocally by the specifically designed oligonucleotide probes, both based on raw data (see diagonal) and by comparing against the microbial gold standard (all AUC values >0.96).

Mentions: As an initial validation, microbial samples each containing DNA from a single bacterial isolate were prepared for 37 randomly chosen species with available strains, from among the 54 bacterial species included during array design. Genomic DNA was either purchased from the german collection of microorganisms and cell cultures (DSMZ, from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) (Leibniz Institute DSMZ, Braunschweig, Germany) or extracted from microbial isolates; these were obtained from LGC Standards American Type Culture Collection (ATCC) (Manassas, VA, USA) and DSMZ catalog strains as vacuum dried cultures or from the Hospital Santa Chiara (Trento, Italy) as actively growing cells. The identity of hospital isolates from this study was confirmed by 16S rRNA gene sequencing (Table S1). Both types of microbial samples were prepared for the BactoChip by chemical labeling with the Universal Linkage System (ULS) linked to the Cy3 fluorophore (Agilent Technologies, Santa Clara, CA, USA). As shown in Figure 2, most individual BactoChip probes (red) and nearly all postprocessed aggregate species abundances (green) were determined to provide accurate assessments of microbial species.


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 detection of individual bacterial species using the BactoChip.Bar plot indicates the Areas Under the receiver operating characteristic Curves (AUCs) for detection of 37 individual target species. Heatmap shows intensities of individual probes (red) and postprocessed aggregate species quantification (green). Almost all tested species (94.6%) were identified unequivocally by the specifically designed oligonucleotide probes, both based on raw data (see diagonal) and by comparing against the microbial gold standard (all AUC values >0.96).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0055764-g002: Accurate detection of individual bacterial species using the BactoChip.Bar plot indicates the Areas Under the receiver operating characteristic Curves (AUCs) for detection of 37 individual target species. Heatmap shows intensities of individual probes (red) and postprocessed aggregate species quantification (green). Almost all tested species (94.6%) were identified unequivocally by the specifically designed oligonucleotide probes, both based on raw data (see diagonal) and by comparing against the microbial gold standard (all AUC values >0.96).
Mentions: As an initial validation, microbial samples each containing DNA from a single bacterial isolate were prepared for 37 randomly chosen species with available strains, from among the 54 bacterial species included during array design. Genomic DNA was either purchased from the german collection of microorganisms and cell cultures (DSMZ, from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) (Leibniz Institute DSMZ, Braunschweig, Germany) or extracted from microbial isolates; these were obtained from LGC Standards American Type Culture Collection (ATCC) (Manassas, VA, USA) and DSMZ catalog strains as vacuum dried cultures or from the Hospital Santa Chiara (Trento, Italy) as actively growing cells. The identity of hospital isolates from this study was confirmed by 16S rRNA gene sequencing (Table S1). Both types of microbial samples were prepared for the BactoChip by chemical labeling with the Universal Linkage System (ULS) linked to the Cy3 fluorophore (Agilent Technologies, Santa Clara, CA, USA). As shown in Figure 2, most individual BactoChip probes (red) and nearly all postprocessed aggregate species abundances (green) were determined to provide accurate assessments of microbial species.

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