Dataset Information

Singleton Sequence Type 382, an Emerging Clonal Group of Listeria monocytogenes Associated with Three Multistate Outbreaks Linked to Contaminated Stone Fruit, Caramel Apples, and Leafy Green Salad.

ABSTRACT: Three multistate outbreaks between 2014 and 2016, involving case patients in and outside the United States, were linked to stone fruit, caramel apples, and packaged leafy green salad contaminated with Listeria monocytogenes singleton sequence type 382 (ST382), a serotype IVb-v1 clone with limited genomic divergence. Isolates from these outbreaks and other ST382 isolates not associated with these outbreaks were analyzed by whole-genome sequencing (WGS) analysis. The primary differences among ST382 strains were single nucleotide polymorphisms (SNPs). WGS analysis differentiated ST382 from a clonal complex 1 outbreak strain co-contaminating the caramel apples. WGS clustered food, environmental, and clinical isolates within each outbreak, and also differentiated among the three outbreak strains and epidemiologically unrelated ST382 isolates, which were indistinguishable by pulsed-field gel electrophoresis. ST382 appeared to be an emerging clone that began to diverge from its ancestor approximately 32 years before 2016. We estimated that there was 1.29 nucleotide substitution per genome (2.94 Mbp) per year for this clone.

SUBMITTER: Chen Y

PROVIDER: S-EPMC5328462 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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Singleton Sequence Type 382, an Emerging Clonal Group of Listeria monocytogenes Associated with Three Multistate Outbreaks Linked to Contaminated Stone Fruit, Caramel Apples, and Leafy Green Salad.

Chen Yi Y Luo Yan Y Pettengill James J Timme Ruth R Melka David D Doyle Matthew M Jackson Alikeh A Parish Mickey M Hammack Thomas S TS Allard Marc W MW Brown Eric W EW Strain Errol A EA

Journal of clinical microbiology 20170104 3

Three multistate outbreaks between 2014 and 2016, involving case patients in and outside the United States, were linked to stone fruit, caramel apples, and packaged leafy green salad contaminated with <i>Listeria monocytogenes</i> singleton sequence type 382 (ST382), a serotype IVb-v1 clone with limited genomic divergence. Isolates from these outbreaks and other ST382 isolates not associated with these outbreaks were analyzed by whole-genome sequencing (WGS) analysis. The primary differences amo ...[more]

PMID: 28053218

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Project description: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.ImportanceWGS 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.

Project description:Fresh apples are typically stored for up to 1 year commercially; different apple varieties require different storage temperatures to maintain their quality characteristics. There is sparse information available about Listeria monocytogenes survival on fresh apples under various storage temperatures. The objective of this study was to comprehensively evaluate the effect of storage temperature on apple fruit decay and L. monocytogenes survival. Unwaxed apple fruits of selected varieties (Fuji and Granny Smith) were dip inoculated in a three-strain L. monocytogenes cocktail to establish ∼3.5 and 6.0 Log10 CFU/apple. Twenty-four hours post-inoculation, apples were subjected to 1, 4, 10, or 22°C storage for up to 3 months. Apples under the different storage treatments were sampled at 1-, 4-, 7- and 14-day for short-term storage under all four tested temperatures, and 2-, 4-, 8-, and 12-week for long-term storage at 1, 4, and 10°C. A set of uninoculated and unwaxed apples were simultaneously subjected to the previously mentioned storage temperatures and sampled biweekly for their total bacterial count (TPC) and yeasts/molds (Y/M) count. During the 2-week short-term storage, L. monocytogenes population on organic Granny Smith apples stored at 1, 4, or 10°C was reduced by 0.2-0.3 Log. When apples were stored at 22°C, there was a 0.5-1.2 Log10 CFU/apple reduction 14-day post storage dependent on the initial inoculation level. During the 12-week cold storage under 1, 4, and 10°C, L. monocytogenes count on organic Granny Smith apples decreased by 0.5-1.5 Log10 CFU/apple for both inoculation levels. L. monocytogenes had similar survival pattern on conventional Granny Smith and Fuji apples with 0.8-2.0 Log10 CFU/apple reduction over a 3-month cold storage period. Interestingly, both TPC and Y/M count were stable regardless of apple variety or cultivation practice during the 12-week storage at all tested temperatures. In summary, while L. monocytogenes did not proliferate on apple surfaces during 12 weeks of refrigerated storage, only a limited reduction of L. monocytogenes was observed in this study. Therefore, the apple industry cannot rely on cold storage alone to control this pathogen. Additional interventions are needed to eradicate Listeria on fresh apples during long-term cold storage.

Project description:BACKGROUND:The more quickly bacterial pathogens responsible for foodborne illness outbreaks can be linked to a vehicle of transmission or a source, the more illnesses can be prevented. Whole genome sequencing (WGS) based approaches to source tracking have greatly increased the speed and resolution with which public health response can pinpoint the vehicle and source of outbreaks. Traditionally, WGS approaches have focused on the culture of an individual isolate before proceeding to DNA extraction and sequencing. For Listeria monocytogenes (Lm), generation of an individual isolate for sequencing typically takes about 6?days. Here we demonstrate that a hybrid, "quasimetagenomic" approach ie; direct sequencing of microbiological enrichments (first step in pathogen detection and recovery) can provide high resolution source tracking sequence data, 5 days earlier than response that focuses on culture and sequencing of an individual isolate. This expedited approach could save lives, prevent illnesses and potentially minimize unnecessary destruction of food. METHODS:Naturally contaminated ice cream (from a 2015 outbreak) was enriched to recover Listeria monocytogenes following protocols outlined in the Bacteriological Analytic Manual (BAM). DNA from enriching microbiota was extracted and sequenced at incremental time-points during the first 48?h of pre-enrichment using the Illumina MiSeq platform (2 by 250), to evaluate genomic coverage of target pathogen, Listeria monocytogenes. RESULTS:Quasimetagenomic sequence data acquired from hour 20 were sufficient to discern whether or not Lm strain/s were part of the ongoing outbreak or not. Genomic data from hours 24, 28, 32, 36, 40, 44, and 48 of pre-enrichments all provided identical phylogenetic source tracking utility to the WGS of individual isolates (which require an additional 5?days to culture). CONCLUSIONS:The speed of this approach (more than twice as fast as current methods) has the potential to reduce the number of illnesses associated with any given outbreak by as many as 75% percent of total cases and potentially with continued optimization of the entire chain of response, contribute to minimized food waste.

Dataset Information

Singleton Sequence Type 382, an Emerging Clonal Group of Listeria monocytogenes Associated with Three Multistate Outbreaks Linked to Contaminated Stone Fruit, Caramel Apples, and Leafy Green Salad.

Publications

Singleton Sequence Type 382, an Emerging Clonal Group of Listeria monocytogenes Associated with Three Multistate Outbreaks Linked to Contaminated Stone Fruit, Caramel Apples, and Leafy Green Salad.

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