Empirical gonorrhea treatment at initial diagnosis reduces onward transmission. However, increasing resistance to multiple antibiotics may necessitate waiting for culture-based diagnostics to select ...an effective treatment. There is a need for same-day culture-free diagnostics that identify infection and detect antimicrobial resistance. We investigated if Nanopore sequencing can detect sufficient
DNA to reconstruct whole genomes directly from urine samples. We used
-spiked urine samples and samples from gonorrhea infections to determine optimal DNA extraction methods that maximize the amount of
DNA sequenced while minimizing contaminating host DNA. In simulated infections, the Qiagen UCP pathogen mini kit provided the highest ratio of
to human DNA and the most consistent results. Depletion of human DNA with saponin increased
yields in simulated infections but decreased yields in clinical samples. In 10 urine samples from men with symptomatic urethral gonorrhea, ≥92.8% coverage of an
reference genome was achieved in all samples, with ≥93.8% coverage breath at ≥10-fold depth in 7 (70%) samples. In simulated infections, if ≥10
CFU/ml of
was present, sequencing of the large majority of the genome was frequently achieved.
could also be detected from urine in cobas PCR medium tubes and from urethral swabs and in the presence of simulated
coinfection. Using Nanopore sequencing of urine samples from men with urethral gonorrhea, sufficient data can be obtained to reconstruct whole genomes in the majority of samples without the need for culture.
F-type plasmids are diverse and of great clinical significance, often carrying genes conferring antimicrobial resistance (AMR) such as extended-spectrum β-lactamases, particularly in ...Enterobacterales. Organising this plasmid diversity is challenging, and current knowledge is largely based on plasmids from clinical settings. Here, we present a network community analysis of a large survey of F-type plasmids from environmental (influent, effluent and upstream/downstream waterways surrounding wastewater treatment works) and livestock settings. We use a tractable and scalable methodology to examine the relationship between plasmid metadata and network communities. This reveals how niche (sampling compartment and host genera) partition and shape plasmid diversity. We also perform pangenome-style analyses on network communities. We show that such communities define unique combinations of core genes, with limited overlap. Building plasmid phylogenies based on alignments of these core genes, we demonstrate that plasmid accessory function is closely linked to core gene content. Taken together, our results suggest that stable F-type plasmid backbone structures can persist in environmental settings while allowing dramatic variation in accessory gene content that may be linked to niche adaptation. The association of F-type plasmids with AMR may reflect their suitability for rapid niche adaptation.
and other Enterobacteriaceae are diverse species with "open" pangenomes, where genes move intra- and interspecies via horizontal gene transfer. However, most analyses focus on clinical isolates. The ...pangenome dynamics of natural populations remain understudied, despite their suggested role as reservoirs for antimicrobial resistance (AMR) genes. Here, we analyze near-complete genomes for 827 Enterobacteriaceae (553
and 274 non-
spp.) with 2292 circularized plasmids in total, collected from 19 locations (livestock farms and wastewater treatment works in the United Kingdom) within a 30-km radius at three time points over a year. We find different dynamics for chromosomal and plasmid-borne genes. Plasmids have a higher burden of AMR genes and insertion sequences, and AMR-gene-carrying plasmids show evidence of being under stronger selective pressure. Environmental niche and local geography both play a role in shaping plasmid dynamics. Our results highlight the importance of local strategies for controlling the spread of AMR.
Multi-drug-resistant
infection is a significant public health risk. Rapidly detecting
and antimicrobial-resistant (AMR) determinants by metagenomic sequencing of urine is possible, although high ...levels of host DNA and overgrowth of contaminating species hamper sequencing and limit
genome coverage. We performed Nanopore sequencing of nucleic acid amplification test-positive urine samples and culture-positive urethral swabs with and without probe-based target enrichment, using a custom SureSelect panel, to investigate whether selective enrichment of
DNA improves detection of both species and AMR determinants. Probes were designed to cover the entire
genome, with tenfold enrichment of probes covering selected AMR determinants. Multiplexing was tested in a subset of samples. The proportion of sequence bases classified as
increased in all samples after enrichment, from a median (IQR) of 0.05 % (0.01-0.1 %) to 76 % (42-82 %), giving a corresponding median improvement in fold genome coverage of 365 times (112-720). Over 20-fold coverage, required for robust AMR determinant detection, was achieved in 13/15(87 %) samples, compared to 2/15(13 %) without enrichment. The four samples multiplexed together also achieved >20-fold genome coverage. Coverage of AMR determinants was sufficient to predict resistance conferred by changes in chromosomal genes, where present, and genome coverage also enabled phylogenetic relationships to be reconstructed. Probe-based target enrichment can improve
genome coverage when sequencing DNA extracts directly from urine or urethral swabs, allowing for detection of AMR determinants. Additionally, multiplexing prior to enrichment provided enough genome coverage for AMR detection and reduces the costs associated with this method.
Deserts represent an extreme challenge for photosynthetic life. Despite their aridity, they are often inhabited by diverse microscopic communities of cyanobacteria. These organisms are commonly found ...in lithic habitats, where they are partially sheltered from extremes of temperature and UV radiation. However, living under the rock surface imposes additional constraints, such as limited light availability, and enrichment of longer wavelengths than are typically usable for oxygenic photosynthesis. Some cyanobacteria from the genus Chroococcidiopsis can use this light to photosynthesize, in a process known as far-red light photoacclimation, or FaRLiP. This genus has commonly been reported from both hot and cold deserts. However, not all Chroococcidiopsis strains carry FaRLiP genes, thus motivating our study into the interplay between FaRLiP and extreme lithic environments. The abundance of sequence data and strains provided the necessary material for an in-depth phylogenetic study, involving spectroscopy, microscopy, and determination of pigment composition, as well as gene and genome analyses. Pigment analyses revealed the presence of red-shifted chlorophylls d and f in all FaRLiP strains tested. In addition, eight genus-level taxa were defined within the encompassing Chroococcidiopsidales, clarifying the phylogeny of this long-standing polyphyletic order. FaRLiP is near universally present in a generalist genus identified in a wide variety of environments, Chroococcidiopsis sensu stricto, while it is rare or absent in closely related, extremophile taxa, including those preferentially inhabiting deserts. This likely reflects the evolutionary process of gene loss in specialist lineages.
Hybrid assemblies are highly valuable for studies of
due to their ability to fully resolve the structure of mobile genetic elements, such as plasmids, which are involved in the carriage of clinically ...important genes (e.g. those involved in antimicrobial resistance/virulence). The widespread application of this technique is currently primarily limited by cost. Recent data have suggested that non-inferior, and even superior, hybrid assemblies can be produced using a fraction of the total output from a multiplexed nanopore Oxford Nanopore Technologies (ONT) flowcell run. In this study we sought to determine the optimal minimal running time for flowcells when acquiring reads for hybrid assembly. We then evaluated whether the ONT wash kit might allow users to exploit shorter running times by sequencing multiple libraries per flowcell. After 24 h of sequencing, most chromosomes and plasmids had circularized and there was no benefit associated with longer running times. Quality was similar at 12 h, suggesting that shorter running times are likely to be acceptable for certain applications (e.g. plasmid genomics). The ONT wash kit was highly effective in removing DNA between libraries. Contamination between libraries did not appear to affect subsequent hybrid assemblies, even when the same barcodes were used successively on a single flowcell. Utilizing shorter run times in combination with between-library nuclease washes allows at least 36
isolates to be sequenced per flowcell, significantly reducing the per-isolate sequencing cost. Ultimately this will facilitate large-scale studies utilizing hybrid assembly, advancing our understanding of the genomics of key human pathogens.
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