Seqsphere software

MLST was performed in silico by uploading the FASTA sequence of each genome in the Oxford PubMLST database for Campylobacterhttps://pubmlst.org/bigsdb?db=pubmlst_campylobacter_seqdef&page=sequenceQuery, or by using the MLST typing scheme for C. jejuni, of the Ridom SeqSphere+software (Ridom GmbH, Münster, Germany). Minimal spanning trees were constructed using BioNumerics software (v 7.6, Applied Maths NV, Sint-Martens-Latem) and Pearson correlations.

The de novo reconstructed sequences were used to extract sequence types (ST) by using the SeqSphere⁺ software (v. 4.1.9 Ridom GmbH, Münster, Germany), which accesses the public MLST scheme for E. faecium, which is available on the PubMLST website (www.pubmlst.org/efaecium/) [49 (link), 50 (link)]. For further high-resolution genotyping, the cgMLST E. faecium scheme of the SeqSphere⁺ software was applied [33 (link)]. Discrete complex types (CT) were deduced for each sequenced isolate. On basis of the cgMLST analyses, a neighbor-joining phylogenetic tree and, further, a minimum spanning tree were calculated in SeqSphere⁺.

Core genome MLST (cgMLST) of reads was performed with Ridom SeqSphere + Software (Ridom, Munster, Germany [27 (link)]) with default settings for trimming and velvet assembly. For the assignment of cgMLST alleles, two different schemes were used: (i) a publicly-available S. enterica (species level) cgMLST scheme designed by Enterobase and (ii) an in-house serovar-specific cgMLST scheme.

To describe the clonal relationship for the samples, we performed whole genome sequencing (WGS). As previously described [24 (link)], WGS was performed using a MiSeq Illumina platform (accredited with ISO 17025 norm at the Division of Clinical Microbiology, University Hospital Basel) with 2 × 300 nt paired-end sequencing after Nextera XT library preparation. After sequencing, the resulting reads were de novo assembled and analysed by core genome multilocus sequence typing (cgMLST) using Ridom SeqSphere Software (version 4.1.6). With the same software, the clonal relationship was illustrated with a minimum spanning tree. All read data have been deposited with the European Nucleotide Archive (ENA) under the project number PRJEB27159.

One S. aureus representing each spa type (n = 37) was selected for whole-genome sequencing (WGS) on an Illumina MiSeq or NextSeq platform (Illumina Inc., San Diego, USA) with a 250-/150 bp paired-end protocol aiming for 100 × coverage [24 ]. Subsequently, reads were de novo-assembled using the SKESA assembler integrated into the SeqSphere⁺ software (version 7.0, Ridom GmbH, Münster, Germany). The antimicrobial resistance and virulence genes and multilocus sequence types (ST) were predicted in silico using SeqSphere⁺ as recently described [25 ]. The staphylococcal cassette chromosome mec (SCCmec) types of the MRSA isolates were determined by the SCCmecFinder 1.2 [26 ] from the Centre for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/SCCmecFinder/; accessed on 22 April 2021). The raw reads of the representative isolates were deposited in the European Nucleotide Archive (https://www.ebi.ac.uk/ena) under the project accession number PRJEB44433. The Neighbor-Joining (NJ) tree was constructed using 1861 genes of the S. aureus core genome multilocus sequence typing (cgMLST) scheme (Task templates: S. aureus cgMLST v1.3, pairwise ignore missing values).

To reveal the genomic diversity and phylogenetic relation among E. coli strains carrying the mcr-1 gene, cluster analysis of a large set of E. coli genomes was performed on the basis of the core genome genes. The mcr-1-positive E. coli strains of poultry and human origin, isolated between 2016 and 2019, were selected from the database of the Bacterial and Viral Bioinformatics Resource Center (BV-BRC) (https://www.bv-brc.org/; accessed on 12 June 2023). Only E. coli strains with high-quality whole-genome sequencing data and with known sequence types (STs) were subjected to cgMLST analysis using the Ridom SeqSphere+ software v9.08 [42 (link)].
First, sequence types (STs) were determined by MLST based on the polymorphism of the seven housekeeping genes according to the Warwick MLST scheme for E. coli [43 (link)]. Then, the core genes of E. coli were analyzed by blasting all genome sequences against the E. coli reference strain K-12 MG1655 (GenBank accession no. NC_000913). The serotype of the strain Ec45-2020 was identified using SerotypeFinder 2.0 [44 (link)].