Project description:The model prokaryote Escherichia coli can exist as a either a commensal or a pathogen in the gut of diverse mammalian hosts. These associations, coupled with its ease of cultivation and genetic variability, have made E. coli a popular indicator organism for tracking the origin of fecal water contamination. Source tracking accuracy is predicated on the assumption that E. coli isolates recovered from contaminated water present a genetic signature characteristic of the host from which they originated. In this study, we compared the accuracy with which E. coli isolated from humans, bear, cattle and deer could be identified by standard fingerprinting methods used for library-based microbial source tracking (repetitive element PCR and pulsed-field gel electrophoresis) in relation to microarray-based analysis of genome content. Our results show that patterns of gene presence or absence were more useful for distinguishing E. coli isolates from different sources than traditional fingerprinting methods, particularly in the case of human strains. Host-associated differences in genome composition included the presence or absence of mobile IS1 elements as well as genes encoding the ferric dicitrate iron transporter (fec), E. coli common pilus (ECP), type 1 fimbriae and the CRISPR associated cas proteins. Many of these differences occurred in regions of the E. coli chromosome previously shown to be M-bM-^@M-^\hot spotsM-bM-^@M-^] for the integration of horizontally-acquired DNA. PCR primers designed to amplify the IS1 and fec loci confirmed array results and demonstrated the ease with which gene presence/absence data can be converted into a diagnostic assay. The data presented here suggest that, despite the high level of genetic diversity observed among isolates by PFGE, human-derived strains may constitute a distinct ecotype distinguished by multiple potential library-independent source tracking markers. Twelve isolates of E. coli ( 3 from bear, 3 from cattle, 3 from deer and 3 from humans) were isolated from feces and/or raw sewage. Genome content for each strain was assessed in duplicate using comparative genome hybridization with E. coli K12 MG1655 as the reference for a total of 24 arrays.
Project description:Borreliella burgdorferi is the causative agent of Lyme disease, which is the most common vector-borne disease in the United States. While cases of Lyme disease are geographically limited to the Northeast, mid-Atlantic, and Midwest states, there are still estimates of almost 500,000 cases annually in the United States alone. In this study, we sought to assess transcriptional changes that occur in response to B. burgdorferi infection in the ankle joints of C57BL/6 mice. To this end, we performed single cell RNA sequencing (scRNA-seq) to transcriptionally profile cells, identify population heterogeneity, and analyze how cellular environments change over time. Sequencing of immune and non-immune cells in the mouse ankle joint was performed over five distinct time points, starting with uninfected animals and progressing to eight weeks post-infection in two-week increments.
Project description:The model prokaryote Escherichia coli can exist as a either a commensal or a pathogen in the gut of diverse mammalian hosts. These associations, coupled with its ease of cultivation and genetic variability, have made E. coli a popular indicator organism for tracking the origin of fecal water contamination. Source tracking accuracy is predicated on the assumption that E. coli isolates recovered from contaminated water present a genetic signature characteristic of the host from which they originated. In this study, we compared the accuracy with which E. coli isolated from humans, bear, cattle and deer could be identified by standard fingerprinting methods used for library-based microbial source tracking (repetitive element PCR and pulsed-field gel electrophoresis) in relation to microarray-based analysis of genome content. Our results show that patterns of gene presence or absence were more useful for distinguishing E. coli isolates from different sources than traditional fingerprinting methods, particularly in the case of human strains. Host-associated differences in genome composition included the presence or absence of mobile IS1 elements as well as genes encoding the ferric dicitrate iron transporter (fec), E. coli common pilus (ECP), type 1 fimbriae and the CRISPR associated cas proteins. Many of these differences occurred in regions of the E. coli chromosome previously shown to be “hot spots” for the integration of horizontally-acquired DNA. PCR primers designed to amplify the IS1 and fec loci confirmed array results and demonstrated the ease with which gene presence/absence data can be converted into a diagnostic assay. The data presented here suggest that, despite the high level of genetic diversity observed among isolates by PFGE, human-derived strains may constitute a distinct ecotype distinguished by multiple potential library-independent source tracking markers.
2013-03-01 | GSE21115 | GEO
Project description:Fecal source tracking for Bong stream
Project description:For expression analysis of wild-type V. cholerae, hapR, and rpoS deletion mutants in mid-exponential or stationary phase, the strains were grown to either OD600 of 0.3 or for 11 h in LB media at 37 0C, and bacteria from 2-ml culture were quickly pelleted, resuspended in Trizol reagent (GIBCO/BRL, San Diego, California, United States), and frozen on dry ice. RNA was isolated from the Trizol agent, treated with DNaseI (Ambion, Austin, Texas, United States), and cleaned by using the RNeasy kit (Qiagen, Valencia, California, United States). Labeling of cDNA and microarray hybridizations were performed as described [Yiliz et al. 2001, Mol. Micro. 53: 497-515]. Microarrays were scanned with a GenePix 400A instrument (Axon Instruments), using the GENEPIX 5.0 software. At least four microarray experiments were performed for each of two biological replicates for the tested strains. Gene expression of V. cholerae, rpoS, and hapR deletion mutants in stationary phase LB cultures was analyzed and compared to the wild-type parent under identical conditions. Gene expression of the wild-type parent during stationary phase after 11 h growth in LB was analyzed using RNA from an exponentially growing culture as a reference.
Project description:For expression analysis of wild-type V. cholerae, hapR, and rpoS deletion mutants in mid-exponential or stationary phase, the strains were grown to either OD600 of 0.3 or for 11 h in LB media at 37 0C, and bacteria from 2-ml culture were quickly pelleted, resuspended in Trizol reagent (GIBCO/BRL, San Diego, California, United States), and frozen on dry ice. RNA was isolated from the Trizol agent, treated with DNaseI (Ambion, Austin, Texas, United States), and cleaned by using the RNeasy kit (Qiagen, Valencia, California, United States). Labeling of cDNA and microarray hybridizations were performed as described [Yiliz et al. 2001, Mol. Micro. 53: 497-515]. Microarrays were scanned with a GenePix 400A instrument (Axon Instruments), using the GENEPIX 5.0 software. At least four microarray experiments were performed for each of two biological replicates for the tested strains. Gene expression of V. cholerae, rpoS, and hapR deletion mutants in stationary phase LB cultures was analyzed and compared to the wild-type parent under identical conditions. Gene expression of the wild-type parent during stationary phase after 11 h growth in LB was analyzed using RNA from an exponentially growing culture as a reference. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set