VIM Beta-Lactamase Linkage Analysis

Hybrid sequencing study investigating whether 3 isolates from 2 patients that tested PCR positive for Verona integron-encoded metallo-β-lactamase (VIM) were likely to be linked via mobilisation of VIM. Each sample was sequenced via Nanopore with plasmid miniprep and general easyMAG libraries and via MiSeq before quality control with

• Quality control was performed using fastp --length_required 100 (v0.23.4) for short reads and filtlong --min_length 3000 (v0.2.1) for combined miniprep and easyMAG long reads

• Initial hybrid assemblies were generated for each isolate (unicycler v0.5.0: short-read assembly and long-read scaffolding/resolution and dragonflye v1.0.14: long-read first and short-read polishing)

• The best performing assembly for each isolate was selected on the basis of circular contigs and N50 (quast v5.2.0). See hybrid_assemblies/ hybrid_assemblies/qc_assembly_quast.sh for final assemblies and example run script.

• These were used to determine:

  • Isolate taxonomy (GTDB-TK v2.1.1 w/ GTDB release207v2): taxonomy_gtdb
  • Identify plasmids (mob-suite v3.1.7) and AMR genes (bmrfinderplus v3.11.17 w/ DB v2023-08-08.2) present: hybrid_assemblies/{amrfinderplus,mob_suite} hybrid_assemblies/plasmid_amr_annotate.sh

• Due to sparsity of annotation for Pseudomonas VIM-bearing plasmid

• VIM-bearing plasmids were extracted from assemblies (and in the case of Pseudomonas putida's large but poorly annotated plasmid - reassembled using trycycler v0.5.4 (following this protocol i.e., long-read assembly and contig clustering/reconciliation of raven v6.10.0, miniasm v0.3-r179, flye v2.9.2-b1786 assemblies followed by medaka v1.8.0 /w r941_min_hac_g507 and polypolish v0.5.0 short read polishing). See vim_plasmids (and vim_plasmids/pputida_trycycler.sh` for pputdia VIM-plasmid trycycler example run script).

• Each VIM plasmid was then:

  • Re-identified (mob-suite v3.1.7) vim_genes/{annotate.sh,mobsuite}
  • Annotated (Bakta v1.8.2 w/ Full DB v2023-02-20 including amrfinderplus v4.11.17 w/ DB v2023-08-08.2): vim_genes/{annotate.sh,mobsuite}
  • VIM genes sequences extracted and aligned (mafft v7.515): vim_genes/
  • Integron-finder (integron_finder v2.0.2 w/ HMMs from AMRFinderPlus DB v2023-08-08.2): vim_integrons/

• Data visualisation was performed as follows in plots:

  • Plasmids were virusalised with pycirclize v0.5.1: plots/pycirclize_plot.py
  • Integrons with visualised with clinker v0.0.26 plots/run.sh
  • VIM alignment via AliView v1.26 and inkscape v1.3 0e150ed6c4
  • Figures were combined and edited in inkscape v1.3 0e150ed6c4

Sample Data

Accession	Title	Data Archive	Links	BioSample	BioProject	Release Date	SRA.filename	BioSample.organism_name
SRR26458397	Genomic sequencing of CPO Pseudomonas clinical isolate	SRA	BioSample: 1, BioProject: 1	SAMN37915621	PRJNA1030769	2023-10-21	Pseudomonas_short_R1.fastq.gz, Pseudomonas_short_R2.fastq.gz	Pseudomonas putida
SRR26458396	Genomic sequencing of CPO Pseudomonas clinical isolate	SRA	BioProject: 1, BioSample: 1	SAMN37915621	PRJNA1030769	2023-10-21	Pseudomonas_combined_long.fastq.gz	Pseudomonas putida
SRR26458395	Genomic sequencing of CPO Enterobacter clinical isolate	SRA	BioSample: 1, BioProject: 1	SAMN37915572	PRJNA1030769	2023-10-21	Enterobacter_short_R1.fastq.gz, Enterobacter_short_R2.fastq.gz	Enterobacter soli
SRR26458394	Genomic sequencing of CPO Enterobacter clinical isolate	SRA	BioProject: 1, BioSample: 1	SAMN37915572	PRJNA1030769	2023-10-21	Enterobacter_combined_long.fastq.gz	Enterobacter soli
SRR26458393	Genomic sequencing of CPO Proteus clinical isolate	SRA	BioProject: 1, BioSample: 1	SAMN37915571	PRJNA1030769	2023-10-21	Proteus_short_R1.fastq.gz, Proteus_short_R2.fastq.gz	Proteus mirabilis
SRR26458392	Genomic sequencing of CPO Proteus clinical isolate	SRA	BioSample: 1, BioProject: 1	SAMN37915571	PRJNA1030769	2023-10-21	Proteus_combined_long.fastq.gz	Proteus mirabilis
SAMN37915621	Pathogen: clinically-associated Pseudomonas putida	BioSample	BioProject: 1, SRA: 2	SAMN37915621	PRJNA1030769	2023-10-21		Pseudomonas putida
SAMN37915572	Pathogen: clinically-associated Enterobacter soli	BioSample	BioProject: 1, SRA: 2	SAMN37915572	PRJNA1030769	2023-10-21		Enterobacter soli
SAMN37915571	Pathogen: clinically-associated Proteus mirabilis	BioSample	SRA: 2, BioProject: 1	SAMN37915571	PRJNA1030769	2023-10-21		Proteus mirabilis
PRJNA1030769	Clinical Carbapenemase Producing Organism Genomic Surveillance	BioProject	BioSample: 3, SRA: 6	SAMN37915621,SAMN37915571,SAMN37915572	PRJNA1030769	2023-10-21

Supplemental Methods

CPE Surveillance

Routine CPE surveillance by rectal swabs was done in accordance with institutional policy for patients admitted to high risk units (medicine, oncology, geriatric mental health), known history of antimicrobial resistant organisms, or recent admission to any hospital within the last 12 months. Surveillance cultures are screened using chromogenic mSuperCARBA agar (CHROMagarTM), followed by identification by MALDI-TOF (Bruker MALDI-Biotyper®). Clinical isolates undergo routine antimicrobial susceptibility testing and are screened for CPE by demonstrating non-susceptibility to ertapenem or meropenem. CPE confirmation was done phenotypically by a modified Carbapenemase Inactivation Method (mCIM), and genotypically by a laboratory validated real-time PCR testing for KPC, NDM, OXA-48 like, VIM, and GES.

DNA extraction

Bacterial colonies were resuspended in Tris-EDTA buffer (Millipore, Canada) for total nucleic acid and plasmid-specific extraction. The suspension was pre-treated with lysozyme (Biosearch Technologies, UK) and proteinase K (Biosearch Technologies, UK) followed by an automated extraction with NucliSens® easyMAG® system (bioMerieux, France). Plasmid-specific extraction was done using the QIAgen plasmid mini-prep kit (QIAgen, Canada).

Genomic Analysis

Illumina reads were trimmed for sequencing adapters and low quality regions using fastp (v0.23.2) and nanopore reads were filtered to those >=3000bp using filtlong (v0.2.1). Read statistics were calculated using seqkit (v2.3.0). Each hybrid set of reads were then combined and assembled using both DragonFlye (v1.0.14) and Unicycler (v0.5.0) (i.e., long-read and short-read first hybrid assembly approaches). QUAST (v5.2.0) was then used to determine the least fragmented assembly with the most closed contigs for each isolate. The resulting “best assembly” for each isolate were then analysed for AMR genes using AMRFinderPlus (v4.11.17 with database v2023-08-08.2), for plasmids using Mob-Suite (v3.1.7), for integrons using integron-finder (v2.0.2), and annotated in general via Bakta (v1.8.2 with database v2023-02-20) Taxonomy was determined using the Genome Taxonomy Database Tookit (v2.1.1) with database release 207v2 VIM alleles were aligned using mafft (v7.515) and visualised using AliView (v1.26). Detected plasmids were visualised using pycirclize (v0.5.1) and VIM-integrons compared with Clinker (v0.0.26). Figures were then arranged for presentation in inkscape (v1.3). Further analysis details, scripts, accession information, and results can be found in https://github.com/maguire-lab/vim_linkage_analysis (10.5281/zenodo.10032195).