Project description: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.
Project description:During an intracellular bacterial infection, the host cell and the infecting pathogen interact through a progressive series of events that may result in many distinct outcomes. To understand the specific strategies our immune system employs to manage attack by diverse pathogens, we sought to identify the unique and the core host and pathogen interactions that occur during infection: We compared in molecular detail the pathways induced across infection by seven diverse bacterial species that constitute many of the main human pathogens: Staphylococcus aureus, Listeria monocytogenes, Enterococcus faecalis, Group B Streptococcus, Yersinia pseudotuberculosis, Shigella flexneri and Salmonella enterica. We infected primary human macrophages with each species and used scRNA-Seq to generate a comprehensive dataset of gene expression profiles during bacterial infection. Examining the expression profiles of the infected macrophages across the pathogens, we discovered different modules of infection representing different states through which the infection progresses. The early module captures intra-cellular activity such as lysosome and degranulation, followed by type I IFN signaling, from which results in a cell death module, with a last mode of inflammatory response through response to IL-1. Comparing these modules across the pathogens, we found that their dynamics differ, with some modules active in all species and others which are present in some, but not all pathogens. Our work defines the hallmarks of host-pathogen interactions by identifying recurring properties of infection that can provide insight into diagnostics and therapeutic timing.
Project description:A prototype oligonucleotide microarray was designed to detect and identify viable bacterial species with the potential to grow of common beer spoilage microorganisms from the genera Lactobacillus, Megasphaera, Pediococcus and Pectinatus. Probes targeted the intergenic spacer regions (ISR) between 16S and 23S rRNA, which were amplified in a combination of reverse transcriptase (RT) and polymerase chain reaction (PCR) prior to hybridization. This method allows the detection and discrimination of single bacterial species in a complex sample. Furthermore, microarrays using oligonucleotide probes targeting the ISR allow the distinction between viable bacteria with the potential to grow and non-growing bacteria. The results demonstrate the feasibility of oligonucleotide microarrays as a contamination control in food industry for the detection and identification of spoilage microorganisms within mixed population. Keywords: microarray, oligonucleotide, species-specific, detection, beer spoilage bacteria