Salmonella enterica subsp. enterica Welikade: guideline for phylogenetic analysis of serovars rarely involved in foodborne outbreaks

Abstract

Background: Salmonella spp. is a major foodborne pathogen with a wide variety of serovars associated with human cases and food sources. Nevertheless, in Europe a panel of ten serovars is responsible for up to 80% of confirmed human cases. Clustering studies by single nucleotide polymorphism (SNP) core-genome phylogenetic analysis of outbreaks due to these major serovars are simplified by the availability of many complete genomes in the free access databases. This is not the case for outbreaks due to less common serovars, such as Welikade, for which no reference genomes are available. In this study, we propose a method to solve this problem. We propose to perform a core genome MLST (cgMLST) analysis based on hierarchical clustering using the free-access EnteroBase to select the most suitable genome to use as a reference for SNP phylogenetic analysis. In this study, we applied this protocol to a retrospective analysis of a Salmonella enterica serovar Welikade (S. Welikade) foodborne outbreak that occurred in France in 2016. Finally, we compared the cgMLST and SNP analyses. SNP phylogenetic reconstruction was carried out considering the effect of recombination events identified by the ClonalFrameML tool. The accessory genome was also explored by phage content and virulome analyses.

Results: Our findings revealed high clustering concordance using cgMLST and SNP analyses. Nevertheless, SNP analysis allowed for better assessment of the genetic distance among strains. The results revealed epidemic clones of S. Welikade circulating within the poultry and dairy sectors in France, responsible for sporadic and non-sporadic human cases between 2012 and 2019.

Conclusions: This study increases knowledge on this poorly described serovar and enriches public genome databases with 42 genomes from human and non-human S. Welikade strains, including the isolate collected in 1956 in Sri Lanka, which gave the name to this serovar. This is the first genomic analysis of an outbreak due to S. Welikade described to date.

Keywords: Accessory genome analysis; ClonalFrameML; Core genome SNP analysis; EnteroBase analysis; Outbreak characterization; Phage analysis; Reference genome; Salmonella Welikade; Virulome analysis.

Fig. 1

EnteroBase GrapeTree clustering of cgMLST allelic distances between S. Welikade strains. Clustering was produced using EnteroBase GrapeTree with the MSTreeV2 algorithm. Allelic distances are indicated on branches. Different colored nodes indicate the Hierarchical Clustering (HC)-5 profiles

Fig. 2

EnteroBase dendrogram of non-repetitive SNPs of the 53 S. Welikade genomes and the S. Gaminara reference genome SA20063285. The dendrogram was produced using EnteroBase SNP project tools. Different colored nodes indicate the strains’ geographic localization. The number of strains per country is indicated between brackets [n]

Fig. 3

Boxplot representing breadth coverage data according to the reference genome used

Fig. 4

Phylogenetic tree based on the core-genome SNPs of S. Welikade strains, constructed with maximum likelihood according to the K3Pu + F + I model. Consensus tree was obtained after 3,000 bootstraps. SNP tree branch lengths were corrected taking in account the recombination events predicted by the ClonalFrame ML tool. The tree is rooted on the historical strain 839 K. The SNP average carried out on branches is noted in blue. Bootstrap values greater than 80% are noted with blue dots on nodes. The strains implicated in the goat’s cheese outbreak that occurred in France in 2016 are highlighted in the purple box. The blue box highlights the epidemic Gallus gallus cluster. The heatmap shows the presence (in black) or absence (in beige) of the phages. The accession numbers of each phage are: CTC2A [NC_ 030949], 186 [NC_001317], AA91 [NC_022750], Haemop_HP2 [ NC_003315], KO2 [NC_005857], 118970_sal3 [NC_031940], Fels-1 [NC_010391], Fels-2 [NC_010463], SEN34_[NC_028699], ST64B [NC_004313], Sf6 [NC_005344], X29 [NC_024369], F108 [NC_008193], RE-2010 _[NC_019488], Gifsy-1 _[NC_010392], Gifsy-2 _[NC_010393], GF-2 _[NC_026611], I2_2 _[NC_001332], P88 _[NC_026014], PsP3 _[NC_005340], and Mu _[NC_000929]

Fig. 5

Boxplot representing the average SNP distance calculated as a function of the reference genome used and accounting or not for recombination events. a inter-comparison of SNP distance average between lineages or clades. b intra-comparison of SNP distance average inside clusters. Two conditions were compared: SNP and SNP + CFML. SNP: SNP analysis using the iVARCall2 tool; SNP + CFML: SNP analysis using iVARCall2 and a ClonalFrameML analysis. Three reference genomes were compared: Gaminara SA200663285, Gaminara CFSAN070644, and Typhimurium LT2