Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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

ABSTRACT: We have designed and experimentally validated the BactoChip, a 60-mer oligonucleotide microarray for simultaneous detection and quantification of multiple bacterial species of clinical interest. The Bactochip microarray targets a novel set of high-resolution marker genes, those genes that most unequivocally characterized each bacterial species. The accuracy of the BactoChip microarray was evaluated using the labeled total DNA of single bacterial species at different concentrations (from 65ng to more than 250ng). The specificity of the developed array was further validated using mixed cultures containing up to 15 different bacterial species in even or staggered amount. We employed the Agilent 'Custom HD-CGH 8x15k Array" (catalogue number: G4427A) and the Agilent'Genomic DNA ULS labeling Kit" (catalogue number: 5190-0419). The microarray successfully distinguished among bacterial species from 21 different genera. The BactoChip additionally proved accurate in determining species-level relative abundances over a 10-fold dynamic range in complex bacterial communities. 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.

ORGANISM(S): Corynebacterium jeikeium

SUBMITTER: Nicola Segata

PROVIDER: E-MEXP-3420 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Similar Datasets

Project description:MicroRNAs have well-established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type-dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly up-regulate NF-κB associated microRNAs; strikingly, these regulations which are induced by the bacterial lipopolysaccharides (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti-bacterial defence. However, a suppression of the global immune regulator miR-155 in endotoxintolerant macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let-7 family as the common denominator of Salmonella regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let-7 relieves cytokine IL-6 and IL-10 mRNAs from negative post-transcriptional control. Our results establish a paradigm of microRNA-mediated feed-forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens. The murine macrophage cell-line RAW 264.7 was exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. We compared mouse mRNA abundances between cells infected with wild-type ("24WT") or SPI1/2 knockout Salmonella ("24SPI1/2") and non-infected, mock-treated controls ("24C"). Both strains mounted a similar inflammatory response as judged from the abundance of pro-inflammatory genes compared to the controls. These data indicate, that macrophages can mount a full inflammatory response to extracellular bacteria, that is not further enhanced or attenuated by the sensing of intracellular bacteria. Murine RAW 264.7 cells were rendered endotoxin-tolerant by pre-exposure to heat-killed Salmonella typhimurium SL1344 ("HKS") at an MOI of 10 for 20 h. 5 days upon removal of the HKS stimulus cells were exposed to wild-type Salmonella typhimurium SL1344 or to the corresponding knockout strain lacking both major genomic loci associated with Salmonella pathogenicity ("SPI1" and "SPI2"). To induce infectivity overnight cultures of Salmonella were dilluted 1:100 and grown to OD2. Host cells at a density of approximately 10e6 per well of a 6-well plate were infected at an MOI of 1. Upon initial incubation for 30 min cells were further incubated for 24 h in RPMI supplemented with gentamicin (10 µg / ml). As determined by a gentamicin protection and plating assay the SPI1/2 double knockout strain is deficient in both invasion and intracellular replication. The wild-type Salmonella strain invades and replicates in endotoxin-tolerant RAW 264.7 cells, though the intracellular replication rate is significantly reduced (by roughly 40 %) compared to naive RAW 264.7 cells. We compared mouse mRNA abundances between cells infected with wild-type ("wt") and non-infected, mock-treated controls ("Mock") or between wild-type ("wt") and SPI1/2-knockout ("SPI1/2") infected cells. As apparent from the Microarray data the wild-type Salmonella strain induces a typical inflamatory response in endotoxin-tolerant RAW 264.7 cells (as judged from the fold-induction of NFkB-inducible genes). As expected however, mRNAs corresponding to known inflammatory genes are significantly less abundant in response to the non-invasive SPI1/2 knockout strain, compared to wild-type infection. These data indicate, that endotoxin-tolerance renders macrophages largely insensitive to extracellular, but not to intracellular bacteria. ***This submission represents the mRNA component of the study

Project description:We created a multi-species microarray platform, containing probes to the whole genomes of seven different Saccharomyces species, with very dense coverage (one probe every ~500 bp) of the S. cerevisiae genome, including non-S288c regions, mitochondrial and 2 micron circle genomes, plus probes at fairly dense coverage (one probe every ~2,100 bp) for each of the genomes of six other Saccharomyces species: S. paradoxus, S. mikatae, S. kudriavzevii, S. bayanus, S. kluyveri and S. castellii. We performed array-Comparative Genomic Hybridization (aCGH) using this platform, examining 83 different Saccharomyces strains collected across a wide range of habitats; of these, 69 were widely used commercial S. cerevisiae wine strains, while the remaining 14 were from a wide range of other industrial and natural habitats. Thus, we were able to sample much of the pan-genome space of the Saccharomyces genus. We observed interspecific hybridization events, introgression events, and pervasive copy number variation (CNV) in all but a few of the strains. These CNVs were distributed throughout the strains such that they did not produce any clear phylogeny, suggesting extensive mating in both industrial and wild strains. To validate our results and to determine whether apparently similar introgressions and CNVs were identical by descent or recurrent, we also performed whole genome sequencing on nine of these strains. These data may help pinpoint genomic regions involved in adaptation to different industrial milieus, as well as shed light on the course of domestication of S. cerevisiae. Control arrays of the following types are included in the dataset: (1) "Self-self" hybridizations (called "Self cntrl" in the dataset), where the reference DNA mix was labeled with either Cy3 or Cy5 (in separate reactions) and then mixed and hybridized. (2) Reference sub-pool hybridizations: the mix of 6 non-cerevisiae strains' DNA was labeled with Cy5, mixed with the final Reference pool (labeled in Cy3) and hybridized (called "OtherSppCompletePool" control) or the mix of 42 S. cerevisiae strains' DNA was labeled with Cy5, mixed with the final Reference pool (labeled in Cy3) and hybridized (called "Scer only ref pool") (3) Single Saccharomyces species hybridizations, where each of the 6 non-cerevisiae species, and also the S288c S. cerevisiae lab strain, was labeled with Cy5, mixed with the final Reference pool (labeled in Cy3) and hybridized (these arrays are called by the strain number followed by the species nickname). Four separate replicates of the self-self hybridizations were performed, and duplicate arrays (done on different days using different DNA preparations) were performed for three of the wine strains: GSY2175, GSY2176 and GSY2196.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets