Whole genome sequencing analyses of Listeria monocytogenes that persisted in a milkshake machine for a year and caused illnesses in Washington State

Yu Wang,Eric W Brown,Ailyn Pérez-Osorio,Yi Chen,Kaye Eckmann,Zhen Li,Marc W Allard,William A Glover

doi:10.1186/s12866-017-1043-1

Abstract

BackgroundIn 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. It was a hospital-acquired outbreak linked to milkshakes, made from contaminated ice cream mixes and milkshake maker, served to patients. Here we performed multiple analyses on isolates associated with this outbreak: pulsed-field gel electrophoresis (PFGE), whole genome single nucleotide polymorphism (SNP) analysis, species-specific core genome multilocus sequence typing (cgMLST), lineage-specific cgMLST and whole genome-specific MLST (wgsMLST)/outbreak-specific cgMLST. We also analyzed the prophages and virulence genes.ResultsThe outbreak isolates belonged to sequence type 1038, clonal complex 101, genetic lineage II. There were no pre-mature stop codons in inlA. Isolates contained Listeria Pathogenicity Island 1 and multiple internalins. PFGE and multiple whole genome sequencing (WGS) analyses all clustered together food, environmental and clinical isolates when compared to outgroup from the same clonal complex, which supported the finding that L. monocytogenes likely persisted in the soft serve ice cream/milkshake maker from November 2014 to November 2015 and caused 3 illnesses, and that the outbreak strain was transmitted between two ice cream production facilities. The whole genome SNP analysis, one of the two species-specific cgMLST, the lineage II-specific cgMLST and the wgsMLST/outbreak-specific cgMLST showed that L. monocytogenes cells persistent in the milkshake maker for a year formed a unique clade inside the outbreak cluster. This clustering was consistent with the cleaning practice after the outbreak was initially recognized in late 2014 and early 2015. Putative prophages were conserved among prophage-containing isolates. The loss of a putative prophage in two isolates resulted in the loss of the AscI restriction site in the prophage, which contributed to their AscI-PFGE banding pattern differences from other isolates.ConclusionsThe high resolution of WGS analyses allowed the differentiation of epidemiologically unrelated isolates, as well as the elucidation of the microevolution and persistence of isolates within the scope of one outbreak. We applied a wgsMLST scheme which is essentially the outbreak-specific cgMLST. This scheme can be combined with lineage-specific cgMLST and species-specific cgMLST to maximize the resolution of WGS.

Highlights

In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States
In late 2014, a different listeriosis outbreak in Washington State, unrelated to the 2010–2015 multistate outbreak, occurred in a hospital (Hospital X) in the Pacific Northwest of the United States, involving patients hospitalized for other medical conditions prior to exposure to milkshakes made from contaminated ice cream mixes manufactured in a company (Company A) [7]
Hospital X was using a different brand of ice cream mix from the 2014 outbreak, which was tested negative for L. monocytogenes; but isolates recovered from the milkshake samples and swab samples from the milkshake machine matched the outbreak-associated isolates collected in 2014 [8], confirming that this third patient was associated with this outbreak

Summary

Introduction

In 2015, in addition to a United States multistate outbreak linked to contaminated ice cream, another outbreak linked to ice cream was reported in the Pacific Northwest of the United States. L. monocytogenes exists in different environments due to its hardiness in harsh conditions, such as a wide pH range, high salt concentrations and ability to grow and persist at refrigeration temperatures [2]. A 2010–2015 multistate listeriosis outbreak was linked to contaminated ice cream manufactured in the southern United States [6]. In late 2014, a different listeriosis outbreak in Washington State, unrelated to the 2010–2015 multistate outbreak, occurred in a hospital (Hospital X) in the Pacific Northwest of the United States, involving patients hospitalized for other medical conditions prior to exposure to milkshakes made from contaminated ice cream mixes manufactured in a company (Company A) [7]. Hospital X was using a different brand of ice cream mix from the 2014 outbreak, which was tested negative for L. monocytogenes; but isolates recovered from the milkshake samples and swab samples from the milkshake machine matched the outbreak-associated isolates collected in 2014 [8], confirming that this third patient was associated with this outbreak

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