Plasmid Sequence Analysis from Long Reads v2

This protocol demonstrates how to assemble reads from plasmid DNA, and generate a circularised and non-repetitive consensus sequence At the moment, this protocol uses Canu to de-novo assemble high-quality single-cut reads. Input(s): demultiplexed fastq files (see protocol Demultiplexing Nanopore reads with LAST). I've noticed that the default demultiplexing carried out by Guppy (at least up to v4.2.2, as used in the first version of this protocol) has issues with chimeric reads, which can affect assembly. Output(s): Consensus sequence per barcode as a fasta file

Complete genome and plasmid sequence data of three Xanthomonas arboricola pv. corylina strains, the bacterium responsible for bacterial blight of hazelnut

Xanthomonas arboricola pv. corylina is the causal agent of bacterial blight of hazelnut. The bacterium is listed as A2 quarantine pathogen in Europe since 1978 and on the Regulated Non-Quarantine Pest (RNQP) list since 2020. Three strains from various geographic regions and isolated at different times were sequenced using a hybrid approach with short- and long-read technologies to generate closed genome and plasmid sequences in order to better understand the biology of this pathogen.

Plasmid Sequence Analysis from Long Reads v2

This protocol demonstrates how to assemble reads from plasmid DNA, and generate a circularised and non-repetitive consensus sequence At the moment, this protocol uses Canu to de-novo assemble high-quality single-cut reads. Input(s): demultiplexed fastq files (see protocol Demultiplexing Nanopore reads with LAST). I've noticed that the default demultiplexing carried out by Guppy (at least up to v4.2.2, as used in the first version of this protocol) has issues with chimeric reads, which can affect assembly. Output(s): Consensus sequence per barcode as a fasta file

Laboratory Stock Variants of the Archetype Silver Resistance Plasmid pMG101 Demonstrate Plasmid Fusion, Loss of Transmissibility, and Transposition of Tn7/pco/sil Into the Host Chromosome

Salmonella Typhimurium carrying the multidrug resistance (MDR) plasmid pMG101 was isolated from three burns patients in Boston United States in 1973. pMG101 was transferrable into other Salmonella spp. and Escherichia coli hosts and carried what was a novel and unusual combination of AMR genes and silver resistance. Previously published short-read DNA sequence of pMG101 showed that it was a 183.5Kb IncHI plasmid, where a Tn7-mediated transposition of pco/sil resistance genes into the chromosome of the E. coli K-12 J53 host strain had occurred. We noticed differences in streptomycin resistance and plasmid size between two stocks of E. coli K-12 J53 pMG101 we possessed, which had been obtained from two different laboratories (pMG101-A and pMG101-B). Long-read sequencing (PacBio) of the two strains unexpectedly revealed plasmid and chromosomal rearrangements in both. pMG101-A is a non-transmissible 383Kb closed-circular plasmid consisting of an IncHI2 plasmid sequence fused to an IncFI/FIIA plasmid. pMG101-B is a mobile closed-circular 154 Kb IncFI/FIIA plasmid. Sequence identity of pMG101-B with the fused IncFI/IncFIIA region of pMG101-A was >99%. Assembled host sequence reads of pMG101-B showed Tn7-mediated transposition of pco/sil into the E. coli J53 chromosome between yhiM and yhiN. Long read sequence data in combination with laboratory experiments have demonstrated large scale changes in pMG101. Loss of conjugation function and movement of resistance genes into the chromosome suggest that even under long-term laboratory storage, mobile genetic elements such as transposons and insertion sequences can drive the evolution of plasmids and host. This study emphasises the importance of utilising long read sequencing technologies of plasmids and host strains at the earliest opportunity.

The Genomic Epidemiology of Carbapenemase-Producing Enterobacterales (CPE) at a Hospital System in Toronto, Ontario, Canada, 2007-2018

Background: At a hospital system (H1) in Ontario, Canada, we investigated whether whole-genome sequencing (WGS) altered initial epidemiological interpretation of carbapenemase-producing Enterobacterales (CPE) transmission. Methods: We included patients with CPE colonization/infection identified by population-based surveillance from October 2007 to August 2018 who received healthcare at H1 in the year before/after CPE detection. H1 reported epidemiological transmission clusters. We combined single nucleotide variant (SNV) analysis, plasmid characterization, and epidemiological data. Results: Eighty-five patients were included. H1 identified 7 epidemiological transmission clusters A-G involving 24/85 (28%) patients. SNV analysis confirmed transmission clusters C, D, and G, and identified two additional cases belonging to cluster A. One was a travel-related case that was the likely index case (0-6 SNVs from other isolates); this case stayed on the same unit as the initially presumed index case 4 months prior to detection of the initially presumed index case on another unit. The second additional case occupied a room previously occupied by 5 cluster A cases. Plasmid sequence analysis excluded a case from cluster A and identified clusters E and F as possibly two parts of a single cluster. SNV analysis also identified a case without direct epidemiologic links that was 18-21 SNVs away from cluster B, suggesting possible undetected inter-hospital transmission. Conclusions: SNV and plasmid sequence analysis identified cases belonging to transmission clusters that conventional epidemiology missed and excluded other cases. Implementation of routine WGS to complement epidemiological transmission investigations has the potential to improve prevention and control of CPE in hospitals.

Genetic Characterization of Plasmid-mediated qepA Gene Among ESBL-producing Escherichia Coli Isolates in Mexico

A molecular characterization of a plasmid-born qepA gene in (ESBL)-producing E. coli clinical isolates were performed. An 2.63% (11/418) were qepA positive isolates, of which a 90.0% carried CTX-M-15 (9/11) and SHV-12 (1/11). All isolates showed chromosomal mutations in the gyrA and parC genes. The clonal groups A, B and C were identified and belonged to, respectively, phylogroups A, B1 and D, as well as the sequence types 205, 405 and 617. Several plasmid profiles were determined with incompatibility groups FIA, FIB and FII. The genetic environment of the qepA in plasmid pEC8020 was different from those reported previously. The plasmid sequence included genes conferring resistance to β-lactams (blaCTX-M-15), macrolides (mphA), fluoroquinolones (qepA1), trimethoprim (dfrB4) and sulphonamides (sul1). Likewise, the IncF-pEC8020 plasmid carried several insertion sequences including ISCR3, IS6100 and multiple copies of IS26. This work contributes to the epidemiology and genetics of plasmid-born qepA genes of ESBL-producing E. coli.

Draft Genome Sequence of a Salmonella enterica subsp. enterica Serotype Choleraesuis Strain Isolated from the Pulp of Muskmelons

ABSTRACT Salmonella enterica subsp. enterica serotype Choleraesuis is a foodborne pathogen with zoonotic potential. We report the draft genome sequence and a closed plasmid sequence from a plant-internalized S. Choleraesuis strain that was isolated from the pulp of a Spanish Galia melon purchased from a German supermarket in 2015.

Draft Genome Sequence of Weissella paramesenteroides STCH-BD1, Isolated from Ensiled Sorghum bicolor

ABSTRACT Weissella paramesenteroides has potential as an industrial biocatalyst due to its ability to produce lactic acid. A novel strain of W. paramesenteroides was isolated from ensiled sorghum. The genome was sequenced using a hybrid assembly of Oxford Nanopore and Illumina data to produce a 2-Mbp genome and 22-kbp plasmid sequence.