Clc main workbench 21

The Epicentre MasterPure DNA purification Kit (Illumina Inc., San Diego, CA, USA) was used for DNA preparation prior to WGS using the MiSeq platform (Illumina Inc.), followed by de novo assembly by CLC Genomics Workbench version 21 (QIAGEN, Hilden, Germany). Acquired resistance genes were identified in contigs using the ResFinder database 4.0 and verified using CLC Main Workbench 21 (QIAGEN).^{13 (link)} A database including gene sequences of porins, efflux pumps and associated transcription regulators, as well as genes associated with polymyxin B resistance, was created using K. pneumoniae MGH 78578 (NCBI Reference Sequence NC_009648.1) as a reference strain. Alignments were created using CLC Main Workbench 21 (QIAGEN).

All RNA samples with CT< 37 on rRT-PCR were further subjected to RT-nested PCR targeting a 520 nucleotide portion of the VP1 coding gene as previously described [42 (link)]. Five microliters of PCR products were analysed on Gelgreen® (Invitrogen, Carlsbad, CA, USA) stained agarose gel and visualized under an ultraviolet transilluminator.
Resulting amplicons were purified using the QIAquick PCR pufication kit (Qiagen, Courtaboeuf, France) and subjected to direct sequencing of both strands using the BigDye terminator v3.1 kit (Applied Biosystems, Foster City, CA, USA) and nested PCR primers on an ABI Prism 3140 automated sequencer. Sequence contigs, consensus sequences and multiple sequence alignments were generated using CLC Main Workbench 21.0.3 software (Qiagen, France). Newly determined sequences were submitted to the GenBank database under the nucleotide sequence accession numbers OR078518 and OR078529. Maximum likelihood phylogenetic analysis was performed with MEGA version 7.0 software using the best fit GTR+G+I+4 model. The reliability of individual tree topology was estimated using 1,000 bootstrap pseudo-replicates.

The deduced BafA orthologs in the B. bacilliformis genome (NCBI accession number NC_008783.1) were searched against the sequence of the passenger domain of B. henselae-derived BafA autotransporter using local BLAST program in CLC Main Workbench 21.0.3 software (Qiagen, Hilden, Germany) with an E value cutoff of <1 × 10⁻⁵⁰. Conserved domain analyses in these candidates were conducted using the Pfam 34.0 server (http://pfam.xfam.org/). Signal peptides were predicted by the SignalP-5.0 server (https://services.healthtech.dtu.dk/service.php?SignalP-5.0). The passenger domains of BafA_Bhe, RS02470, and RS02475 were aligned and compared by CLC Main Workbench. The details of synteny analysis are described in Text S1 in the supplemental material.