Lunatic instrument

Before WGS, all the Cronobacter spp. and Salmonella spp. strains were cultured in Columbia blood agar plates (bioMérieux, Marcy-l’Étoile, France) at 37°C for 24 h. DNA was isolated from bacterial cultures with the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The amount of DNA was quantified on a Lunatic instrument (Unchained Labs, Pleasanton, CA, United States). Nextera XT chemistry (Illumina Inc., San Diego, CA, United States) was used to prepare sequencing libraries for a 2 × 300 bp paired-end sequencing run on an Illumina MiSeq sequencer. Samples were sequenced to achieve a minimum of 80-fold coverage using standard protocols by Illumina. The resulting FASTQ files were quality trimmed and de novo assembled with the SPAdes version 3.9.0. Contigs were filtered for a minimum of 5-fold coverage and 200 bp minimum length with Ridom SeqSphere+ software v. 7.8.0 (Ridom, Münster, Germany; Jünemann et al., 2013 (link)).

DNA was isolated from bacterial cultures using the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany) using the protocol for Gram-positive bacteria according to the instructions of the manufacturer. The amount of input DNA was quantified on a Lunatic instrument (Unchained Labs, Pleasanton, CA, USA). Sequencing libraries were prepared using Nextera XT chemistry (Illumina Inc., San Diego, CA, USA) for a 300-bp paired-end sequencing run on an Illumina MiSeq sequencer. Samples were sequenced to aim for minimum coverage of 80-fold using Illumina’s recommended standard protocols. The resulting FASTQ files were first quality trimmed and then de novo assembled using SPAdes version 3.9.0. Contigs were filtered for a minimum coverage of 5× and a minimum length of 200 bp using SeqSphere+ software v6.0.0 (Ridom, Münster, Germany).

Prior to WGS, a primary species identification from single colonies was performed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker, Billerica, MA, United States) and MBT Compass IVD software 4.1.60 (Bruker) as described by Halbedel et al. (2020) (link).
As for WGS, DNA was isolated from bacterial cultures with the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions for Gram-positive bacteria. The amount of input DNA was quantified on a Lunatic instrument (Unchained Labs, Pleasanton, CA, United States). Nextera XT chemistry (Illumina Inc., San Diego, CA, United States) was used to prepare sequencing libraries for a 300 bp paired-end sequencing run on an Illumina MiSeq sequencer. Samples were sequenced to achieve a minimum 80-fold coverage using recommended standard protocols by Illumina. The resulting FASTQ files were quality trimmed and de novo assembled with the SPAdes version 3.9.0. Contigs were filtered for a minimum of fivefold coverage and 200 bp minimum length with SeqSphere+ software v. 7.8.0 (Ridom, Münster, Germany) (Jünemann et al., 2013 (link)).