Project description:<p>Traveler's diarrhea (TD) is caused by enterotoxigenic Escherichia coli (ETEC), other pathogenic gram-negative pathogens, norovirus and some parasites. Nevertheless, standard diagnostic methods fail to identify pathogens in more than 30% of TD patients, so it is predicted that new pathogens or groups of pathogens may be causative agents of disease. A comprehensive metagenomic study of the fecal microbiomes from 23 TD patients and seven healthy travelers was performed, all of which tested negative for the known etiologic agents of TD in standard tests. Metagenomic reads were assembled and the resulting contigs were subjected to semi-manual binning to assemble independent genomes from metagenomic pools. Taxonomic and functional annotations were conducted to assist identification of putative pathogens. We extracted 560 draft genomes, 320 of which were complete enough to be enough characterized as cellular genomes and 160 of which were bacteriophage genomes. We made predictions of the etiology of disease in individual subjects based on the properties and features of the recovered cellular genomes. Three subtypes of samples were observed. First were four patients with low diversity metagenomes that were predominated by one or more pathogenic E. coli strains. Annotation allowed prediction of pathogenic type in most cases. Second, five patients were co-infected with E. coli and other members of the Enterobacteriaceae, including antibiotic resistant Enterobacter, Klebsiella, and Citrobacter. Finally, several samples contained genomes that represented dark matter. In one of these samples we identified a TM7 genome that phylogenetically clustered with a strain isolated from wastewater and carries genes encoding potential virulence factors. We also observed a very high proportion of bacteriophage reads in some samples. The relative abundance of phage was significantly higher in healthy travelers when compared to TD patients. Our results highlight that assembly-based analysis revealed that diarrhea is often polymicrobial and includes members of the Enterobacteriaceae not normally associated with TD and have implicated a new member of the TM7 phylum as a potential player in diarrheal disease. </p>
Project description:This study aims to determine the epidemiology of Enterobacteriaceae resistant to antibiotics of last resort in pregnant women in labour at a tertiary hospital, Pretoria, South Africa. Rectal swabs shall be used to screen for colonisation with CRE and colistin-resistant Enterobacteriales in pregnant women during labour. Carbapenem and colistin-resistant Enterobacterales can cause the following infections: bacteraemia; nosocomial pneumonia; urinary tract infections, and intra-abdominal infections. Due to limited treatment options, infections caused by these multidrug-resistant organisms are associated with a mortality rate of 40-50%. Screening for colonisation of carbapenem-resistant Enterobacteriaceae (CRE) and colistin-resistant Enterobacteriaceae will help implement infection and prevention measures to limit the spread of these multidrug-resistant organisms.
Project description:The Enterobacteriaceae are a scientifically and medically important clade of bacteria, containing the gut commensal and model organism Escherichia coli, as well as several major human pathogens including Salmonella enterica and Klebsiella pneumoniae. Essential gene sets have been determined for several members of the Enterobacteriaceae, and the E. coli Keio single-gene deletion library is often regarded as a gold standard for gene essentiality studies. However, it remains unclear how much essential genes vary between strains and species. To investigate this, we have assembled a collection of thirteen sequenced high-density transposon mutant libraries from five genera of the Enterobacteriaceae. We first benchmark a number of gene essentiality prediction approaches, investigate the effects of transposon density on essentiality prediction, and identify biases in transposon insertion sequencing data. Based on these investigations we develop a new classifier for gene essentiality. Using gene essentiality defined by this new classifier, we define a core essential genome in the Enterobacteriaceae of 201 universally essential genes, and reconstruct an ancestral essential gene set of 296 genes. Despite the presence of a large cohort of variably essential genes, we find an absence of evidence for genus-specific essential genes. A clear example of this sporadic essentiality is given by the set of genes regulating the σE extracytoplasmic stress response, which appears to have independently become essential multiple times in the Enterobacteriaceae. Finally, we compare our essential gene sets to the natural experiment of gene loss in obligate insect endosymbionts closely related to the Enterobacteriaceae. This isolates a remarkably small set of genes absolutely required for survival, and uncovers several instances of essential stress responses masked by redundancy in free-living bacteria.
Project description:Antibiotic resistance associated with the expression of the clinically significant carbapenemases, IMP, KPC, and NDM and OXA-48 in Enterobacteriaceae is emerging as a worldwide calamity to health care. In Australia, IMP-producing Enterobacteriaceae is the most prevalent carbapenemase-producing Enterobacteriaceae (CPE). Genomic characteristics of such carbapenemase-producing Enterobacteriaceae (CPE) are well described, but the corresponding proteome is poorly characterised. We have thus developed a method to analyse dynamic changes in the proteome of CPE under antibiotic pressure. Specifically, we have investigated the effect of meropenem at sub-lethal concentrations to develop a better understanding of how antibiotic pressure leads to resistance. Escherichia coli, producing either NDM, IMP or KPC type carbapenemase were included in this study, and their proteomes were analysed in growth conditions with or without meropenem.
Project description:Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of inactive double hexamers of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic origins are not irrevocably defined by selection of the loading site for the replicative helicase, but can shift in position after helicase loading. Using purified proteins, we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. In yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a shift in origin positions that correlates with the direction of transcription. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict. 4 samples: one replicate for WT at 37C, two replicates for rat1-1 at 37C, and one replicate for rat1-1 at 24C. All are single-end sequenced via Ion Torrent PGM methodology. rat1 = Nuclear 5' to 3' single-stranded RNA exonuclease; involved in RNA metabolism (http://www.yeastgenome.org/locus/S000005574/overview). 4 ChIP seq samples and their duplicates are submitted. rat1-1 ORC ChIP at 24C and 37C; rat1-1 MCM ChIP at 24C and 37C.
Project description:Chimaerism and mixoploidy define the presence of cell lineages with different parental genomes or different ploidy states in a single individual. Our knowledge on their mechanistic origin results from indirect observations, often when the cell lineages have been subject to rigorous selective pressure during development. Here, we applied haplarithmisis to infer the haplotypes and the copy number of parental genomes in 116 single blastomeres comprising entire preimplantation stage bovine embryos (n=23) following in vitro fertilization. Not only abnormal fertilizations leading to triploid zygotes, but also normally fertilized zygotes can spontaneously segregate entire parental genomes into different cell lineages during cleavage of the zygote. We coin the term “heterogoneic division” to indicate the events leading to non-canonical zygotic cytokinesis segregating the parental genomes into distinct lineages. Persistence of those cell lines during development is the likely cause of chimaerism and mixoploidy in mammals.