Computational protocol: Whole genome sequencing for typing and characterisation of Listeria monocytogenes isolated in a rabbit meat processing plant

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[…] L. monocytogenes isolates included in the present study are a subset of isolates collected over one year within a previous study on prevalence of L. monocytogenes in rabbit meat processing plants (De Cesare et al., ). In particular, isolates sharing the same genotype and collected more than six times over a period of more than six months from different sources (rabbit carcasses, rabbit meat cuts, rabbit meat products and food processing environment) in the same plant were considered as potentially persistent. With this definition, 33 isolates of L. monocytogenes isolates of lineage II, serotype 1/2a and belonging to two 7-locus Multi Locus Sequence Types (MLST) ST14 and ST121 were selected as potentially persistent. An additional sporadic isolate, belonging to lineage I, serotype 1/2b, ST224 was also included for backward comparability. Isolates sharing the same 7– locus MLST were indistinguishable also by ApaI-Pulsed Field Eletrophoresis (PFGE), automated ribotyping and Multi Locus Variable number tandem repeat Analysis (MLVA) (De Cesare et al., ).Whole-genomic DNA was extracted using the MagAttract HMW DNA Kit (Qiagen, Hilden, Germany). The purified DNA concentration and the quality parameter ratio 260/280 were measured by BioSpectrometer fluorescence (Eppendorf). The whole genome of selected 34 L. monocytogenes isolates was sequenced using Illumina MiSeq platform (TrueSeq library, paired-end reads). Reads were quality checked and de novo assembled using the INNUca pipeline (https://github.com /INNUENDOCON/INNUca).Briefly, INNUca calculates if the sample raw data fulfill the expected coverage (minimum default 15X). Then, after a read quality analysis using FASTQC and trimming using TRIMMOMATIC, de novo draft genome assembly is performed with SPAdes, which is subsequently improved using PILON to correct bases and fix misassemblies.Core genome Multi Locus Sequence Typing (cgMLST) was inferred based on in silico sequence alignment of 1748 loci of corresponding core genes. Briefly, after alignment, similarly to 7-locus MLST, an allele number is assigned to each locus. A cluster type, representative of all allele numbers, is then assigned (Moura et al., ).The Multi Virulence Locus Sequence Type (MVLST) was inferred based on in silico sequence alignment of seven virulence determinant gene loci: clpP, dal, inlB, inlC, lisR, prfA (https://sites.google.com /site/mvlstdatabase ) (Zhang et al., 2004).Analyses of virulence was performed using VirulenceFinder 1.5 (https://cge.cbs.dtu.dk/services/VirulenceFinder) (Joensen et al., ). With this tool a BLAST search of a database of 82 L. monocytogenes virulence determinant genes was applied to all ST14 and ST121 draft genomes included in the present study, as well as to 23 publicly available genomes of L. monocytogenes belonging to ST14 and ST121 and isolated from humans or food processing plants. Publicly available genome of EGD-e (Genbank accession number NC_003210.1) was included as reference. The default parameters used were 90% ID threshold and 60% of minimum length. Upon detection of no gene the analysis was repeated with 85% ID threshold and 20% minimum length. Alignment of inlA and prfA genes was performed by Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo).The discriminatory power of each typing method was assessed by the Simpson’s Index of Diversity (ID). ID values with P<0.05 were considered statistical significant different (Hunter and Gaston, 1988). […]

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