High resolution clustering of Salmonella enterica serovar Montevideo strains using a next-generation sequencing approach

Abstract

Background: Next-Generation Sequencing (NGS) is increasingly being used as a molecular epidemiologic tool for discerning ancestry and traceback of the most complicated, difficult to resolve bacterial pathogens. Making a linkage between possible food sources and clinical isolates requires distinguishing the suspected pathogen from an environmental background and placing the variation observed into the wider context of variation occurring within a serovar and among other closely related foodborne pathogens. Equally important is the need to validate these high resolution molecular tools for use in molecular epidemiologic traceback. Such efforts include the examination of strain cluster stability as well as the cumulative genetic effects of sub-culturing on these clusters. Numerous isolates of S. Montevideo were shot-gun sequenced including diverse lineage representatives as well as numerous replicate clones to determine how much variability is due to bias, sequencing error, and or the culturing of isolates. All new draft genomes were compared to 34 S. Montevideo isolates previously published during an NGS-based molecular epidemiological case study.

Results: Intraserovar lineages of S. Montevideo differ by thousands of SNPs, that are only slightly less than the number of SNPs observed between S. Montevideo and other distinct serovars. Much less variability was discovered within an individual S. Montevideo clade implicated in a recent foodborne outbreak as well as among individual NGS replicates. These findings were similar to previous reports documenting homopolymeric and deletion error rates with the Roche 454 GS Titanium technology. In no case, however, did variability associated with sequencing methods or sample preparations create inconsistencies with our current phylogenetic results or the subsequent molecular epidemiological evidence gleaned from these data.

Conclusions: Implementation of a validated pipeline for NGS data acquisition and analysis provides highly reproducible results that are stable and predictable for molecular epidemiological applications. When draft genomes are collected at 15×-20× coverage and passed through a quality filter as part of a data analysis pipeline, including sub-passaged replicates defined by a few SNPs, they can be accurately placed in a phylogenetic context. This reproducibility applies to all levels within and between serovars of Salmonella suggesting that investigators using these methods can have confidence in their conclusions.

Figure 1

Phylogenetic diversity of Salmonella Montevideo based on a GARLI analysis of 72,063 variable SNP sites of which 63,987 were identified as parsimony informative. The tree was rooted with four outgroups including S. Schwarzengrund, S. Pomona, and S. Javiana. Terminal names correspond to samples in Table 1. The numbers at the base of each node are bootstrap scores with most of the deepest nodes supported at 100%. The scale bar units are nucleotide substitutions per site and these are proportional across the branch lengths with longer branches having greater substitutions. S. Montevideo strains partitioned into four clades designated I-IV.

Figure 2

Genome size variation and estimated N50 sizes within Salmonella Montevideo draft genome sequences. The estimated N50 value is a rough estimate of the quality and coverage of the draft genomes which was sequenced to approximately 15-20× coverage. The N50 value represents the average contig size after assembly with the Newbler software. Isolate names correspond to samples in Table 1. Genome length ranged from less than 4.45 mbp to about 4.75 mbp, with most isolates approximately 4.65 mbp in size (unlabeled boxes). Only the larger or smaller genomes are listed.

Figure 3

Phylogenetic diversity and relationships among a single S. Montevideo clone. GARLI phylogenetic analysis of the outbreak isolates was performed on a set of 43 concatenated ORFs containing informative SNPs (Table 3). Terminal names, scale bar, branch lengths and bootstrap scores are as in Figure 1. Numbers above the branches represent unique SNPs that define these internal branches. The phylogenetic analysis reported here partitions the S. Montevideo clone into 6 lineages (A-F) and expands upon a previous tree [5] with the inclusion of 5 more strains and the noted expansion of outbreak strains into clade E. To the right of the tree, each isolate is labeled with the Not1 pattern that was determined using PFGE with each unique number identifying a new Not1 pattern.

Figure 4

NGS discovery of unique SNPs and insertional genetic attributes found in a highly homogeneous strain of S. Montevideo from California (157_Clinical_CA). (A) Isolate names correspond to samples in Table 1, and gene names correspond to the ORFs containing informative SNPs among a single S. Montevideo outbreak clone in Table 3. A representative nucleotide site observed across 5 isolates is listed for each ORF. ORFs are mapped against a reference of S. Typhimurium strain LT2 with lines going to approximate chromosomal positions relative to the reference (numbers in mbp). (B) A comparative MAUVE analysis of isolate 157_Clinical_CA revealed the presence of a 100 kb insertion with homology to Enterobacterial phage D6. Here we compared the isolate to another more complete homologous relative, phage P1 to document the insertion site. Graphic is standard MAUVE format showing putative genes as boxes with arrows documenting insertions and rearrangements. Forward and reverse strands are on opposite sides of the mid-line.

Figure 5

Phylogenetic GARLI tree from resequencing of matching human-food isolate pairs, individual colonies, and sub-passages of a single strain of S. Montevideo. Terminal names, scale bar, and branch lengths are as in Figure 1. The tree was rooted with two outgroup isolates, both of which were obtained from Pistachio. The laboratory-generated isolates were indistinguishable in a phylogenetic analysis with all replicates clustering together. Some of the biological, laboratory, or technical replicate genomes yielded nucleotide differences and these are seen as longer terminal branches for several isolates on the tree. These few changes did not alter relatedness or inclusivity/exclusivity among the matching food/human isolates.