Whole-Genome Sequencing of Recent
                    Listeria monocytogenes
                    Isolates from Germany Reveals Population Structure and Disease Clusters

ABSTRACT Listeria monocytogenes causes foodborne outbreaks with high mortality. For improvement of outbreak cluster detection, the German consiliary laboratory for listeriosis implemented whole-genome sequencing (WGS) in 2015. A total of 424 human L. monocytogenes isolates collected in 2007 to 2017 were subjected to WGS and core-genome multilocus sequence typing (cgMLST). cgMLST grouped the isolates into 38 complexes, reflecting 4 known and 34 unknown disease clusters. Most of these complexes were confirmed by single nucleotide polymorphism (SNP) calling, but some were further differentiated. Interestingly, several cgMLST cluster types were further subtyped by pulsed-field gel electrophoresis, partly due to phage insertions in the accessory genome. Our results highlight the usefulness of cgMLST for routine cluster detection but also show that cgMLST complexes require validation by methods providing higher typing resolution. Twelve cgMLST clusters included recent cases, suggesting activity of the source. Therefore, the cgMLST nomenclature data presented here may support future public health actions.

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Whole-Genome Sequencing Allows for Improved Identification of Persistent Listeria monocytogenes in Food-Associated Environments

Applied and Environmental Microbiology ◽

10.1128/aem.01049-15 ◽

2015 ◽

Vol 81 (17) ◽

pp. 6024-6037 ◽

Cited By ~ 76

Author(s):

Matthew J. Stasiewicz ◽

Haley F. Oliver ◽

Martin Wiedmann ◽

Henk C. den Bakker

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Whole Genome Sequence ◽

Whole Genome ◽

Genetic Determinants ◽

Single Nucleotide ◽

Content Type ◽

Food Borne ◽

Clonal Spread

ABSTRACTWhile the food-borne pathogenListeria monocytogenescan persist in food associated environments, there are no whole-genome sequence (WGS) based methods to differentiate persistent from sporadic strains. Whole-genome sequencing of 188 isolates from a longitudinal study ofL. monocytogenesin retail delis was used to (i) apply single-nucleotide polymorphism (SNP)-based phylogenetics for subtyping ofL. monocytogenes, (ii) use SNP counts to differentiate persistent from repeatedly reintroduced strains, and (iii) identify genetic determinants ofL. monocytogenespersistence. WGS analysis revealed three prophage regions that explained differences between three pairs of phylogenetically similar populations with pulsed-field gel electrophoresis types that differed by ≤3 bands. WGS-SNP-based phylogenetics found that putatively persistentL. monocytogenesrepresent SNP patterns (i) unique to a single retail deli, supporting persistence within the deli (11 clades), (ii) unique to a single state, supporting clonal spread within a state (7 clades), or (iii) spanning multiple states (5 clades). Isolates that formed one of 11 deli-specific clades differed by a median of 10 SNPs or fewer. Isolates from 12 putative persistence events had significantly fewer SNPs (median, 2 to 22 SNPs) than between isolates of the same subtype from other delis (median up to 77 SNPs), supporting persistence of the strain. In 13 events, nearly indistinguishable isolates (0 to 1 SNP) were found across multiple delis. No individual genes were enriched among persistent isolates compared to sporadic isolates. Our data show that WGS analysis improves food-borne pathogen subtyping and identification of persistent bacterial pathogens in food associated environments.

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Genome Sequence of Listeria monocytogenes Strain F4244, a 4b Serotype

Genome Announcements ◽

10.1128/genomea.01324-17 ◽

2017 ◽

Vol 5 (49) ◽

Cited By ~ 3

Author(s):

Taylor W. Bailey ◽

Naila C. do Nascimento ◽

Arun K. Bhunia

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Genome Sequence ◽

Illumina Sequencing ◽

Foodborne Pathogen ◽

Whole Genome ◽

Content Type ◽

Serotype 1 ◽

Serotype 4B

ABSTRACT Listeria monocytogenes is an opportunistic invasive foodborne pathogen. Here, we performed whole-genome sequencing of L. monocytogenes strain F4244 (serotype 4b) using Illumina sequencing. The sequence showed 94.5% identity with strain F2365, serotype 4b, and 90.6% with EGD-e, serotype 1/2a.

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Evaluation of whole-genome sequencing-based subtyping methods for the surveillance of Shigella spp. and the confounding effect of mobile genetic elements in long-term outbreaks

Microbial Genomics ◽

10.1099/mgen.0.000672 ◽

2021 ◽

Vol 7 (11) ◽

Author(s):

Isabelle Bernaquez ◽

Christiane Gaudreau ◽

Pierre A. Pilon ◽

Sadjia Bekal

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Type Species ◽

Core Genome ◽

Outbreak Detection ◽

Whole Genome ◽

Content Type ◽

Link Type ◽

Interpretation Criteria

Many public health laboratories across the world have implemented whole-genome sequencing (WGS) for the surveillance and outbreak detection of foodborne pathogens. PulseNet-affiliated laboratories have determined that most single-strain foodborne outbreaks are contained within 0–10 multi-locus sequence typing (MLST)-based allele differences and/or core genome single-nucleotide variants (SNVs). In addition to being a food- and travel-associated outbreak pathogen, most Shigella spp. cases occur through continuous person-to-person transmission, predominantly involving men who have sex with men (MSM), leading to long-term and recurrent outbreaks. Continuous transmission patterns coupled to genetic evolution under antibiotic treatment pressure require an assessment of existing WGS-based subtyping methods and interpretation criteria for cluster inclusion/exclusion. An evaluation of 4 WGS-based subtyping methods [SNVPhyl, coreMLST, core genome MLST (cgMLST) and whole-genome MLST (wgMLST)] was performed on 9 foodborne-, travel- and MSM-related retrospective outbreaks from a collection of 91 Shigella flexneri and 232 Shigella sonnei isolates to determine the methods’ epidemiological concordance, discriminatory power, robustness and ability to generate stable interpretation criteria. The discriminatory powers were ranked as follows: coreMLST<SNVPhyl<cgMLST<wgMLST (range: 0.970–1.000). The genetic differences observed for non-MSM-related Shigella spp. outbreaks respect the standard 0–10 allele/SNV guideline; however, mobile genetic element (MGE)-encoded loci caused inflated genetic variation and discrepant phylogenies for prolonged MSM-related S. sonnei outbreaks via wgMLST. The S. sonnei correlation coefficients of wgMLST were also the lowest at 0.680, 0.703 and 0.712 for SNVPhyl, coreMLST and cgMLST, respectively. Plasmid maintenance, mobilization and conjugation-associated genes were found to be the main source of genetic distance inflation in addition to prophage-related genes. Duplicated alleles arising from the repeated nature of IS elements were also responsible for many false cg/wgMLST differences. The coreMLST approach was shown to be the most robust, followed by SNVPhyl and wgMLST for inter-laboratory comparability. Our results highlight the need for validating species-specific subtyping methods based on microbial genome plasticity and outbreak dynamics in addition to the importance of filtering confounding MGEs for cluster detection.

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Prospective Whole-Genome Sequencing Enhances National Surveillance of Listeria monocytogenes

Journal of Clinical Microbiology ◽

10.1128/jcm.02344-15 ◽

2015 ◽

Vol 54 (2) ◽

pp. 333-342 ◽

Cited By ~ 130

Author(s):

Jason C. Kwong ◽

Karolina Mercoulia ◽

Takehiro Tomita ◽

Marion Easton ◽

Hua Y. Li ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Variable Number Tandem Repeat ◽

Variable Number ◽

Outbreak Detection ◽

Whole Genome ◽

Genetic Population ◽

Content Type ◽

Epidemiologic Surveillance

Whole-genome sequencing (WGS) has emerged as a powerful tool for comparing bacterial isolates in outbreak detection and investigation. Here we demonstrate that WGS performed prospectively for national epidemiologic surveillance ofListeria monocytogeneshas the capacity to be superior to our current approaches using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), multilocus variable-number tandem-repeat analysis (MLVA), binary typing, and serotyping. Initially 423L. monocytogenesisolates underwent WGS, and comparisons uncovered a diverse genetic population structure derived from three distinct lineages. MLST, binary typing, and serotyping results inferredin silicofrom the WGS data were highly concordant (>99%) with laboratory typing performed in parallel. However, WGS was able to identify distinct nested clusters within groups of isolates that were otherwise indistinguishable using our current typing methods. Routine WGS was then used for prospective epidemiologic surveillance on a further 97L. monocytogenesisolates over a 12-month period, which provided a greater level of discrimination than that of conventional typing for inferring linkage to point source outbreaks. A risk-based alert system based on WGS similarity was used to inform epidemiologists required to act on the data. Our experience shows that WGS can be adopted for prospectiveL. monocytogenessurveillance and investigated for other pathogens relevant to public health.

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Clostridioides difficile Whole-Genome Sequencing Reveals Limited Within-Host Genetic Diversity in a Pediatric Cohort

Journal of Clinical Microbiology ◽

10.1128/jcm.00559-19 ◽

2019 ◽

Vol 57 (9) ◽

Author(s):

Aakash Balaji ◽

Egon A. Ozer ◽

Larry K. Kociolek

Keyword(s):

Genetic Diversity ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Core Genome ◽

Genetic Relatedness ◽

The Other ◽

Whole Genome ◽

Content Type ◽

Clostridioides Difficile ◽

Host Genetic

ABSTRACT Whole-genome sequencing (WGS) is a highly sensitive method for identifying genetic relatedness and transmission of Clostridioides difficile strains. Previous studies suggest that as few as 3 core genome single-nucleotide variants (SNVs) discriminate between genetically distinct isolates. Because a single C. difficile colony is selected from culture for WGS, significant within-host genetic diversity could preclude identification of transmission events. To evaluate the likelihood of missed transmission events using WGS of single colonies from culture, we examined within-host genetic diversity among C. difficile isolates collected from children. We performed WGS using an Illumina MiSeq instrument on 8 C. difficile colonies randomly selected from each culture performed on stool collected from 10 children (8 children diagnosed with C. difficile infection and 2 children with asymptomatic carriage); 77/80 (96%) isolate sequences were successfully assembled. Among 8/10 (80%) children, all isolates were the same sequence type (ST). The other 2 children each had mixed infection with two STs, although one ST predominated. Among 9/10 (90%) children, isotypic isolates differed by ≤2 SNVs; an isotypic isolate in the remaining child differed by 3 to SNVs relative to the other isolates from that child. Overall, among the 77 isolates collected from 10 stool cultures, 74/77 (96%) were clonal (i.e., same ST and ≤2 core genome SNVs) to other isolates in stool culture. In summary, we identified rare C. difficile within-host genetic diversity in children, suggesting that WGS of a single colony from stool is likely to appropriately characterize isolate clonality and putative transmission events in the majority of cases.

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In-Depth Longitudinal Study of Listeria monocytogenes ST9 Isolates from the Meat Processing Industry: Resolving Diversity and Transmission Patterns Using Whole-Genome Sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.00579-20 ◽

2020 ◽

Vol 86 (14) ◽

Author(s):

Annette Fagerlund ◽

Solveig Langsrud ◽

Trond Møretrø

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Raw Materials ◽

Source Tracking ◽

Common Source ◽

Whole Genome ◽

Meat Processing ◽

Production Environment ◽

Content Type

ABSTRACT Listeria monocytogenes is a pathogen mostly associated with the consumption of ready-to-eat foods and can cause severe disease and death. It can be introduced into food chains from raw materials, but often the contamination source is the food production environment, where certain clones can persist for years. In the meat chain, ST9 is one of the most commonly encountered L. monocytogenes sequence types, and for effective source tracking, the divergence and spread of ST9 must be understood. In this study, whole-genome sequencing (WGS) was used to characterize and track 252 L. monocytogenes ST9 isolates collected from four Norwegian meat processing plants between 2009 and 2017. The isolates formed distinct clusters relative to genomes found in public databases, and all but three isolates clustered into two major clonal populations. Different contamination patterns were revealed, e.g., evidence of contamination of two factories with a clone that diverged from its ancestor in the late 1990s through a common source of raw materials; breach of hygienic barriers within a factory, leading to repeated detection of two clones in the high-risk zone during a 4- to 6-year period; entry through the purchase and installation of second-hand equipment harboring a previously established clonal population; and spreading and diversification of two clones from two reservoirs within the same production room over a 9-year period. The present work provides data on the diversity of ST9, which is crucial for epidemiological investigations and highlights how WGS can be used for source tracking within food processing factories. IMPORTANCE Listeria monocytogenes is a deadly foodborne pathogen that is widespread in the environment, and certain types can be established in food factories. The sequence type ST9 dominates in meat processing environments, and this work was undertaken to obtain data needed for the tracking of this subtype. By using whole-genome sequencing (WGS), we revealed the presence of cross-contamination routes between meat factories as well as within a single factory, including the spread from different reservoirs within the same room. It was also possible to estimate the time frame of persistence in the factory, as well as when and how new clones had entered. The present work contributes valuable information about the diversity of ST9 and exemplifies the potential power of WGS in food safety management, allowing the determination of relationships between strains both in an international context and locally between and within factories.

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Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection

Journal of Clinical Microbiology ◽

10.1128/jcm.01280-15 ◽

2015 ◽

Vol 53 (10) ◽

pp. 3334-3340 ◽

Cited By ~ 77

Author(s):

Angela J. Taylor ◽

Victoria Lappi ◽

William J. Wolfgang ◽

Pascal Lapierre ◽

Michael J. Palumbo ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Salmonella Enterica ◽

Outbreak Detection ◽

Whole Genome ◽

Nucleotide Polymorphism ◽

Single Nucleotide ◽

Molecular Subtyping ◽

Foodborne Outbreaks

Salmonella entericaserovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis ofS. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmentalS. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of theS. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method forS. Enteritidis.

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Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing

Applied and Environmental Microbiology ◽

10.1128/aem.02440-15 ◽

2015 ◽

Vol 82 (3) ◽

pp. 928-938 ◽

Cited By ~ 30

Author(s):

Teresa M. Bergholz ◽

Henk C. den Bakker ◽

Lee S. Katz ◽

Benjamin J. Silk ◽

Kelly A. Jackson ◽

...

Keyword(s):

Genetic Variation ◽

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Evolutionary Relationships ◽

Whole Genome ◽

Evolutionary Analysis ◽

Production Chain ◽

Content Type ◽

Accessory Genome

ABSTRACTWe used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates ofListeria monocytogenesserotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or “epidemic clones” that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread inL. monocytogenesand is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.

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A Whole-Genome Sequencing Approach To Study Cefoxitin-Resistant Salmonella enterica Serovar Heidelberg Isolates from Various Sources

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01919-16 ◽

2017 ◽

Vol 61 (4) ◽

Cited By ~ 33

Author(s):

Romaine Edirmanasinghe ◽

Rita Finley ◽

E. Jane Parmley ◽

Brent P. Avery ◽

Carolee Carson ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Sequencing ◽

Salmonella Enterica ◽

Whole Genome ◽

Single Nucleotide ◽

Content Type ◽

Amoxicillin Clavulanic Acid ◽

Molecular Relationships ◽

Small Clusters ◽

Integrated Program

ABSTRACT This study characterized cefoxitin-resistant and -susceptible Salmonella enterica serovar Heidelberg strains from humans, abattoir poultry, and retail poultry to assess the molecular relationships of isolates from these sources in Québec in 2012. Isolates were collected as part of the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). All isolates were subjected to antimicrobial susceptibility testing, PCR for CMY-2, pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing (WGS). A total of 113 S. Heidelberg isolates from humans (n = 51), abattoir poultry (n = 18), and retail poultry (n = 44) were studied. All cefoxitin-resistant isolates (n = 65) were also resistant to amoxicillin-clavulanic acid, ampicillin, ceftiofur, and ceftriaxone, and all contained the CMY-2 gene. PFGE analysis showed that 111/113 (98.2%) isolates clustered together with ≥90% similarity. Core genome analysis using WGS identified 13 small clusters of isolates with 0 to 4 single nucleotide variations (SNVs), consisting of cefoxitin-resistant and -susceptible human, abattoir poultry, and retail poultry isolates. CMY-2 plasmids from cefoxitin-resistant isolates all belonged to incompatibility group I1. Analysis of IncI1 plasmid sequences revealed high identity (95 to 99%) to a previously described plasmid (pCVM29188_101) found in Salmonella Kentucky. When compared to pCVM29188_101, all sequenced cefoxitin-resistant isolates were found to carry 1 of 10 possible variant plasmids. Transmission of S. Heidelberg may be occurring between human, abattoir poultry, and retail poultry sources, and transmission of a common CMY-2 plasmid may be occurring among S. Heidelberg strains with variable genetic backgrounds.

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Novel Single Nucleotide Polymorphism-Based Assay for Genotyping Mycobacterium avium subsp. paratuberculosis

Journal of Clinical Microbiology ◽

10.1128/jcm.01958-15 ◽

2015 ◽

Vol 54 (3) ◽

pp. 556-564 ◽

Cited By ~ 8

Author(s):

Célia Leão ◽

Robert J. Goldstone ◽

Josephine Bryant ◽

Joyce McLuckie ◽

João Inácio ◽

...

Keyword(s):

Single Nucleotide Polymorphism ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Mycobacterium Avium ◽

Discriminatory Power ◽

Whole Genome ◽

Nucleotide Polymorphism ◽

Phylogenetic Groups ◽

Single Nucleotide ◽

Content Type

Typing ofMycobacterium aviumsubspeciesparatuberculosisstrains presents a challenge, since they are genetically monomorphic and traditional molecular techniques have limited discriminatory power. The recent advances and availability of whole-genome sequencing have extended possibilities for the characterization ofMycobacterium aviumsubspeciesparatuberculosis, and whole-genome sequencing can provide a phylogenetic context to facilitate global epidemiology studies. In this study, we developed a single nucleotide polymorphism (SNP) assay based on PCR and restriction enzyme digestion or sequencing of the amplified product. The SNP analysis was performed using genome sequence data from 133Mycobacterium aviumsubspeciesparatuberculosisisolates with different genotypes from 8 different host species and 17 distinct geographic regions around the world. A total of 28,402 SNPs were identified among all of the isolates. The minimum number of SNPs required to distinguish between all of the 133 genomes was 93 and between only the type C isolates was 41. To reduce the number of SNPs and PCRs required, we adopted an approach based on sequential detection of SNPs and a decision tree. By the analysis of 14 SNPsMycobacterium aviumsubspeciesparatuberculosisisolates can be characterized within 14 phylogenetic groups with a higher discriminatory power than mycobacterial interspersed repetitive unit–variable number tandem repeat assay and other typing methods. Continuous updating of genome sequences is needed in order to better characterize new phylogenetic groups and SNP profiles. The novel SNP assay is a discriminative, simple, reproducible method and requires only basic laboratory equipment for the large-scale global typing ofMycobacterium aviumsubspeciesparatuberculosisisolates.

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