Naydenova, P. | bioRxiv Science

bioRxiv2024

One pot RNA:DNA assembly for ribosomal RNA detection of pathogenic bacteria with single-molecule sensitivity

Ribosomal RNAs (rRNAs) serve as species-defining markers and undergo processing steps such as excision of intervening sequences (IVSs). Direct analysis of native rRNAs is hampered by amplification-induced biases and by the high conservation of rRNA sequences, which complicates discrimination of closely related variants. Here, we present modular RNA:DNA nanostructures that enable direct, amplification-free identification of rRNAs and their variants. The approach employs rationally designed RNA:DNA duplexes, named RNA identifiers (IDs), assembled onto native rRNAs via short complementary oligonucleotides bearing programmable coding motifs. We demonstrate that native bacterial 16S rRNAs can be directly converted into RNA IDs and detected with solid-state nanopores. Having established direct rRNA readout, we next show that biologically encoded rRNA processing states, including serovar-specific 23S rRNA fragmentation patterns arising from IVS excision, are resolved using RNA IDs. Finally, to extend discrimination beyond processing-level differences, we incorporate catalytically inactive Cas9 ribonucleoprotein complexes to enable single-nucleotide discrimination of rRNA variants. Our modular RNA ID-nanopore system facilitates studying rRNA processing and rRNA diversity.

biophysics↗

bioRxiv2018

One Health genomic surveillance of Escherichia coli demonstrates distinct lineages and mobile genetic elements in isolates from humans versus livestock

Livestock have been proposed as a reservoir for drug-resistant Escherichia coli that infect humans. We isolated and sequenced 431 E. coli (including 155 ESBL-producing isolates) from cross-sectional surveys of livestock farms and retail meat in the East of England. These were compared with the genomes of 1517 E. coli associated with bloodstream infection in the United Kingdom. Phylogenetic core genome comparisons demonstrated that livestock and patient isolates were genetically distinct, indicating that E. coli causing serious human infection do not directly originate from livestock. By contrast, we observed highly related isolates from the same animal species on different farms. Analysis of accessory (variable) genomes identified a virulence cassette associated previously with cystitis and neonatal meningitis that was only present in isolates from humans. Screening all 1948 isolates for accessory genes encoding antibiotic resistance revealed 41 different genes present in variable proportions of humans and livestock isolates. We identified a low prevalence of shared antimicrobial resistance genes between livestock and humans based on analysis of mobile genetic elements and long-read sequencing. We conclude that in this setting, there was limited evidence to support the suggestion that antimicrobial resistant pathogens that cause serious infection in humans originate from livestock.\n\nImportanceThe increasing prevalence of E. coli bloodstream infections is a serious public health problem. We used genomic epidemiology in a One Health study conducted in the East of England to examine putative sources of E. coli associated with serious human disease. E. coli from 1517 patients with bloodstream infection were compared with 431 isolates from livestock farms and meat. Livestock-associated and bloodstream isolates were genetically distinct populations based on core genome and accessory genome analyses. Identical antimicrobial resistance genes were found in livestock and human isolates, but there was little overlap in the mobile elements carrying these genes. In addition, a virulence cassette found in humans isolates was not identified in any livestock-associated isolate. Our findings do not support the idea that E. coli causing invasive disease or their resistance genes are commonly acquired from livestock.

genomics↗