Listeria monocytogenes in Stone Fruits Linked to a Multistate Outbreak: Enumeration of Cells and Whole-Genome Sequencing

Abstract

In 2014, the identification of stone fruits contaminated with Listeria monocytogenes led to the subsequent identification of a multistate outbreak. Simultaneous detection and enumeration of L. monocytogenes were performed on 105 fruits, each weighing 127 to 145 g, collected from 7 contaminated lots. The results showed that 53.3% of the fruits yielded L. monocytogenes (lower limit of detection, 5 CFU/fruit), and the levels ranged from 5 to 2,850 CFU/fruit, with a geometric mean of 11.3 CFU/fruit (0.1 CFU/g of fruit). Two serotypes, IVb-v1 and 1/2b, were identified by a combination of PCR- and antiserum-based serotyping among isolates from fruits and their packing environment; certain fruits contained a mixture of both serotypes. Single nucleotide polymorphism (SNP)-based whole-genome sequencing (WGS) analysis clustered isolates from two case-patients with the serotype IVb-v1 isolates and distinguished outbreak-associated isolates from pulsed-field gel electrophoresis (PFGE)-matched, but epidemiologically unrelated, clinical isolates. The outbreak-associated isolates differed by up to 42 SNPs. All but one serotype 1/2b isolate formed another WGS cluster and differed by up to 17 SNPs. Fully closed genomes of isolates from the stone fruits were used as references to maximize the resolution and to increase our confidence in prophage analysis. Putative prophages were conserved among isolates of each WGS cluster. All serotype IVb-v1 isolates belonged to singleton sequence type 382 (ST382); all but one serotype 1/2b isolate belonged to clonal complex 5.

Importance: WGS proved to be an excellent tool to assist in the epidemiologic investigation of listeriosis outbreaks. The comparison at the genome level contributed to our understanding of the genetic diversity and variations among isolates involved in an outbreak or isolates associated with food and environmental samples from one facility. Fully closed genomes increased our confidence in the identification and comparison of accessory genomes. The diversity among the outbreak-associated isolates and the inclusion of PFGE-matched, but epidemiologically unrelated, isolates demonstrate the high resolution of WGS. The prevalence and enumeration data could contribute to our further understanding of the risk associated with Listeria monocytogenes contamination, especially among high-risk populations.

FIG 1

Number of fruits containing L. monocytogenes at various levels. Each fruit weighed 127 to 145 g. The numbers are listed on top of each bar. Different cultivars of the same variety are combined for this analysis. The total number of fruits of each variety is listed in parentheses following the variety name.

FIG 2

Phylogenetic tree of serotype IVb-v1 isolates constructed from SNPs identified by the CFSAN SNP Pipeline, using CFSAN023463 as the reference. All isolates in the tree were indistinguishable by PFGE. Clinical isolate identification (ID) is followed by the abbreviation of the state where it was isolated. For other isolates, the isolate ID is followed by the food and environmental source of the isolate. Isolates were from patients (black), white peaches (green), yellow peaches (blue), white nectarines (purple), yellow nectarines (orange), nectarines of unidentified variety (red), and environment (gray). The outbreak-associated clinical isolates are highlighted in red boxes. The median, minimum, and maximum pairwise SNP differences among isolates of major clades are shown near the root of each clade, with the minimum and maximum in parentheses. Bootstrap values are shown near major nodes.

FIG 3

Phylogenetic tree of serotype 1/2b isolates, except CFSAN024093, constructed from SNPs identified by the CFSAN SNP Pipeline, using CFSAN023459 as the reference. The isolate ID is followed by the food and environmental source of the isolate. Isolates were from white peaches (green), yellow peaches (blue), white nectarines (purple), yellow nectarines (orange) and environment (gray). The median, minimum, and maximum pairwise SNP differences among isolates of major clades are shown near the root of each clade, with the minimum and maximum in parentheses. Bootstrap values are shown near major nodes.