Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis

Abstract

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods.

FIG 1

Phylogeny and outbreak clusters inferred by WGST. Different lineages (I, II, III, IV, and V) are labeled. A total of 16 outbreak clusters (A through P) are identified and labeled. Bootstrapping values of branches leading to individual outbreak clusters are labeled. The designations of three isolates from sporadic cases (2012K-0619, 2012K-0738, and 2012K-0597) are underlined.

FIG 2

Clustering of outbreak isolates by MLVA, CRISPR-MVLST, and PFGE. Outbreaks are labeled A through P according to Table 1 data. Outbreaks that included isolates not clustered together are labeled with a single asterisk (*) and indicated by dashed lines. The designations of three isolates from sporadic cases (2012K-0619, 2012K-0738, and 2012K-0597) are underlined. The lineages to which each isolate belonged are also labeled. These dendrograms are intended to show the hierarchical clustering of isolates, and their branch lengths are not comparable between the different methods.