Uses of Staphylococcus aureus GeneChips in Genotyping and Genetic Composition Analysis

Understanding the relatedness of strains within a bacterial species is essential for monitoring reservoirs of antimicrobial resistance and for epidemiological studies. Pulsed-field gel electrophoresis (PFGE), ribotyping, and multilocus sequence typing are commonly used for this purpose. However, these techniques are either nonquantitative or provide only a limited estimation of strain relatedness. Moreover, they cannot extensively define the genes that constitute an organism. In the present study, 21 oxacillin-resistant Staphylococcus aureus (ORSA) isolates, representing eight major ORSA lineages, and each of the seven strains for which the complete genomic sequence is publicly available were genotyped using a novel GeneChip-based approach. Strains were also subjected to PFGE and ribotyping analysis. GeneChip results provided a higher level of discrimination among isolates than either ribotyping or PFGE, although strain clustering was similar among the three techniques. In addition, GeneChip signal intensity cutoff values were empirically determined to provide extensive data on the genetic composition of each isolate analyzed. Using this technology it was shown that strains could be examined for each element represented on the GeneChip, including virulence factors, antimicrobial resistance determinants, and agr type. These results were validated by PCR, growth on selective media, and detailed in silico analysis of each of the sequenced genomes. Collectively, this work demonstrates that GeneChips provide extensive genotyping information for S. aureus strains and may play a major role in epidemiological studies in the future where correlating genes with particular disease phenotypes is critical

Transcriptional adaptation of Mycobacterium ulcerans in an original mouse model: New insights into the regulation of mycolactone

Mycobacterium ulcerans is the causal agent of Buruli ulcer, a chronic infectious disease and the third most common mycobacterial disease worldwide. Without early treatment, M. ulcerans provokes massive skin ulcers, caused by the mycolactone toxin, its main virulence factor. However, spontaneous healing may occur in Buruli ulcer patients several months or years after the disease onset. We have shown, in an original mouse model, that bacterial load remains high and viable in spontaneously healed tissues, with a switch of M. ulcerans to low levels of mycolactone production, adapting its strategy to survive in such a hostile environment. This original model offers the possibility to investigate the regulation of mycolactone production, by using an RNA-seq strategy to study bacterial adaptation during mouse infection. Pathway analysis and characterization of the tissue environment showed that the bacillus adapted to its new environment by modifying its metabolic activity and switching nutrient sources. Thus, M. ulcerans ensures its survival in healing tissues by reducing its secondary metabolism, leading to an inhibition of mycolactone synthesis. These findings shed new light on mycolactone regulation and pave the way for new therapeutic strategies

Molecular Classification of Crohn’s Disease and Ulcerative Colitis Patients Using Transcriptional Profiles in Peripheral Blood Mononuclear Cells

Ulcerative colitis (UC) and Crohn’s disease (CD) are common inflammatory bowel diseases producing intestinal inflammation and tissue damage. Although emerging evidence suggests these diseases are distinct, ∼10% of patients remain classified as indeterminate inflammatory bowel disease even after invasive colonoscopy intended for diagnosis. A molecular diagnostic assay using a clinically accessible tissue would greatly assist in the classification of these diseases. In the present study we assessed transcriptional profiles in peripheral blood mononuclear cells from 42 healthy individuals, 59 CD patients, and 26 UC patients by hybridization to microarrays interrogating more than 22,000 sequences. Supervised analysis identified a set of 12 genes that distinguished UC and CD patient samples with high accuracy. The alterations in transcript levels observed by microarray were verified by real-time polymerase chain reaction. The results suggest that a peripheral blood mononuclear cell-based gene expression signature can provide a molecular biomarker that can complement the standard dia-gnosis of UC and CD

Pathways activated during human asthma exacerbation as revealed by gene expression patterns in blood

This study has shown that analysis of PBMCs reveals systemic changes accompanying asthma exacerbation and has laid the foundation for future comparative studies using PBMCs