Biology subjects

Harmsen, D.

Publications and source records attributed to Harmsen, D..

3 recordsLinked to original sources

bioRxiv2024

Evaluation of a Real-time Plasmid Transmission Detection Pipeline

The spread of antimicrobial resistance among bacteria by horizontal plasmid transmissions poses a major challenge for clinical microbiology. Here, we evaluate a new real-time plasmid transmission detection pipeline implemented in the SeqSphere+ (Ridom GmbH, Munster, Germany) software. Within the pipeline, a local Mash plasmid database is created and Mash searches with a distance threshold of 0.001 are used to trigger plasmid transmission early warning alerts (EWA). Clonal transmissions are detected using cgMLST allelic differences. The integrated tools MOB-suite, NCBI AMRFinderPlus, CGE MobileElementFinder, pyGenomeViz, and MUMmer are used to characterize plasmids and for visual pairwise plasmid comparisons, respectively. We evaluated the pipeline using published hybrid assemblies (Oxford Nanopore Technology/Illumina) of a surveillance and outbreak dataset with plasmid transmissions. To emulate prospective usage, samples were imported in chronological order of sampling date. Different combinations of the user-adjustable parameters sketch size (1,000 vs 10,000) and plasmid size correction were tested and discrepancies between resulting clusters were analyzed with Quast. When using a sketch size of 1,000 with size correction turned on, the SeqSphere+ pipeline agreed with the published data and produced the same clonal and carbapenemase-carrying plasmid clusters. EWAs were in the correct chronological order. In summary, the developed pipeline presented here is suitable for integration into clinical microbiology settings with limited bioinformatics knowledge due to its automated analyses and alert system, which are combined with the GUI-based SeqSphere+ platform. Thus, with its integrated sample database, (near) real-time plasmid transmission detection is within reach in bacterial routine-diagnostic settings when long-read sequencing is employed. ImportancePlasmid-mediated spread of antimicrobial resistance (AMR) is a major challenge for clinical microbiology and monitoring of potential plasmid transmissions is essential to combat further dissemination. Whole-genome sequencing (WGS) is often used to surveil nosocomial transmissions but usually limited to the detection of clonal transmissions (based on chromosomal markers). Recent advances in long-read sequencing technologies enable full reconstruction of plasmids and the detection of very similar plasmids but so far easy-to-use bioinformatic tools for this purpose were missing. Here we present an evaluation of an innovative real-time plasmid transmission detection pipeline. It is integrated into the GUI-based SeqSphere+ software, which already offers cgMLST based pathogen outbreak detection. It requires very limited bioinformatics knowledge, and its database, automated analyses, and alert system make it well suited for prospective clinical application.

bioinformatics↗

bioRxiv2021

Establishment of a publicly available core genome multilocus sequence typing scheme for Clostridium perfringens

Clostridium perfringens is a spore-forming anaerobic pathogen responsible for a variety of histotoxic and intestinal infections in humans and animals. High-resolution genotyping aiming to identify bacteria at strain level has become increasingly important in modern microbiology to understand pathogen transmission pathways and to tackle infection sources. This study aimed at establishing a publicly available genome-wide multilocus sequence-typing scheme for C. perfringens. 1,431 highly conserved core genes (1.34 megabases; 50% of the reference genome genes) were indexed for a core-genome-based MLST scheme for C. perfringens. As an example, we applied the scheme to 87 poultry and 73 non-poultry strains (total=160). The genotyping results of the 160 genomes were congruent in terms of resolution and tree topology between allele-based and single-nucleotide-polymorphism-based core-genome typing. For the analysis of poultry strains of C. perfringens concerning the country of isolation, NetB-toxin gene carriage and clinical disease, we used 60 allelic differences as a clustering threshold. The results showed that poultry strains from a single country formed a cluster (n=17 clusters including 46 strains). Two clusters included six strains from four different countries. These strains were netB-positive, as were seven strains from Denmark and two strains from Finland, possibly indicating common sources of netB-positive strains. In terms of clinical presentation, different clusters of strains were associated with cases of suspected necrotic enteritis. Strains from sick birds grouped with strains from healthy birds or meat samples showing that potentially virulent strains are widespread and that host-related factors contribute significantly to NE. In summary, a publicly available scheme and an allele nomenclature database for genomic typing of C. perfringens has been established and can be used for broad-based and standardised epidemiological studies.

microbiology↗

bioRxiv2018

Core Genome Multi Locus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections

The use of whole genome sequencing (WGS) using next generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through Single Nucleotide Polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome Multilocus Sequence Typing (cgMLST) has not been explored so far. The current study developed an allele-based method cgMLST and compared its performance to the genome-wide SNP approach and the traditional MLVA on a defined sample collection. The dataset comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 epidemiologically unrelated human and animal isolates collected in Italy. The cgMLST scheme generated in this study contained 2,687 targets of the B. melitensis 16M reference genome (75.4% of the complete genome). We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be [≤]4 loci in the cgMLST and [≤]10 in WGS SNP analysis. CgMLST and SNP analysis provided much higher phylogenetic distance resolution than MLVA, particularly for strains belonging to the same lineage thus allowing diverse and unrelated genotypes to be identified with greater confidence. The application of this cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

microbiology↗