scholarly journals An Open-Source Program (Haplo-ST) for Whole-Genome Sequence Typing Shows Extensive Diversity among Listeria monocytogenes Isolates in Outdoor Environments and Poultry Processing Plants

2020 ◽  
Vol 87 (1) ◽  
Author(s):  
Swarnali Louha ◽  
Richard J. Meinersmann ◽  
Zaid Abdo ◽  
Mark E. Berrang ◽  
Travis C. Glenn
Keyword(s):  
Listeria Monocytogenes ◽  
Open Source ◽  
Strain Identification ◽  
Specific Gene ◽  
Whole Genome ◽  
Content Type ◽  
Open Source Tool ◽  
Processing Plants ◽  
Poultry Processing ◽  
Reference Strains

ABSTRACT A reliable and standardized classification of Listeria monocytogenes is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS)-based approaches for strain characterization are either difficult to standardize, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool, Haplo-ST, to improve standardization and provide maximum discriminatory potential to WGS data tied to a multilocus sequence typing (MLST) framework. Haplo-ST performs whole-genome MLST (wgMLST) for L. monocytogenes while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user-specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, and (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely used BIGSdb-Lm database. Tests of Haplo-ST’s performance with simulated reads from L. monocytogenes reference strains demonstrated high sensitivity (97.5%), and coverage depths of ≥20× were found to be sufficient for wgMLST profiling. We then used Haplo-ST to characterize and differentiate between two groups of L. monocytogenes isolates derived from the natural environment and poultry processing plants. Phylogenetic reconstruction identified lineages within each group, and no lineage specificity was observed with isolate phenotypes (transient versus persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of L. monocytogenes within poultry processing plants. IMPORTANCE We have developed an open-source tool (https://github.com/swarnalilouha/Haplo-ST) that provides allele-based subtyping of L. monocytogenes isolates at the whole-genome level. Along with allelic profiles, this tool also generates allele sequences and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low-resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of L. monocytogenes isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlight the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates in studies of bacterial population genetics.

scholarly journals Whole-Genome Sequence Typing shows extensive diversity of Listeria monocytogenes in the outdoor environment and poultry processing plants

2020 ◽  
Author(s):  
Swarnali Louha ◽  
Richard J. Meinersmann ◽  
Zaid Abdo ◽  
Mark E. Berrang ◽  
Travis C. Glenn
Keyword(s):  
Listeria Monocytogenes ◽  
Open Source ◽  
Outdoor Environment ◽  
Strain Identification ◽  
Whole Genome ◽  
Open Source Tool ◽  
Processing Plants ◽  
Outbreak Investigations ◽  
Poultry Processing ◽  
Reference Strains

ABSTRACTA reliable and standardized classification of Listeria monocytogenes (Lm) is important for accurate strain identification during outbreak investigations. Current whole-genome sequencing (WGS) based approaches for strain characterization either lack standardization, rendering them less suitable for data exchange, or are not freely available. Thus, we developed a portable and open-source tool Haplo-ST to improve standardization and provide maximum discriminatory potential to WGS data tied to an MLST (multi locus sequence typing) framework. Haplo-ST performs whole-genome MLST (wgMLST) for Lm while allowing for data exchangeability worldwide. This tool takes in (i) raw WGS reads as input, (ii) cleans the raw data according to user specified parameters, (iii) assembles genes across loci by mapping to genes from reference strains, (iv) assigns allelic profiles to assembled genes and provides a wgMLST subtyping for each isolate. Data exchangeability relies on the tool assigning allelic profiles based on a centralized nomenclature defined by the widely-used BIGSdb-Lm database. Tests on Haplo-ST’s performance with simulated reads from Lm reference strains yielded a high sensitivity of 97.5%, and coverage depths of ≥ 20× was found to be sufficient for wgMLST profiling. We used Haplo-ST to characterize and differentiate between two groups of Lm isolates, derived from the natural environment and poultry processing plants. Phylogenetic reconstruction showed sharp delineation of lineages within each group and no lineage-specificity was observed with isolate phenotypes (transient vs. persistent) or origins. Genetic differentiation analyses between isolate groups identified 21 significantly differentiated loci, potentially enriched for adaptation and persistence of Lm within poultry processing plants.IMPORTANCEWe have developed an open-source tool that provides allele-based subtyping of Lm isolates at the whole genome level. Along with allelic profiles, this tool also generates allele sequences, and identifies paralogs, which is useful for phylogenetic tree reconstruction and deciphering relationships between closely related isolates. More broadly, Haplo-ST is flexible and can be adapted to characterize the genome of any haploid organism simply by installing an organism-specific gene database. Haplo-ST also allows for scalable subtyping of isolates; fewer reference genes can be used for low resolution typing, whereas higher resolution can be achieved by increasing the number of genes used in the analysis. Our tool enabled clustering of Lm isolates into lineages and detection of potential loci for adaptation and persistence in food processing environments. Findings from these analyses highlights the effectiveness of Haplo-ST in subtyping and evaluating relationships among isolates for routine surveillance, outbreak investigations and source tracking.

scholarly journals Defining and Evaluating a Core Genome Multilocus Sequence Typing Scheme for Whole-Genome Sequence-Based Typing of Listeria monocytogenes

2015 ◽  
Vol 53 (9) ◽  
pp. 2869-2876 ◽  
Author(s):  
Werner Ruppitsch ◽  
Ariane Pietzka ◽  
Karola Prior ◽  
Stefan Bletz ◽  
Haizpea Lasa Fernandez ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Genome Sequence ◽  
Core Genome ◽  
Target Genes ◽  
Sequence Data ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Content Type ◽  
Typing Scheme ◽  
Reference Strains

Whole-genome sequencing (WGS) has emerged today as an ultimate typing tool to characterizeListeria monocytogenesoutbreaks. However, data analysis and interlaboratory comparability of WGS data are still challenging for most public health laboratories. Therefore, we have developed and evaluated a newL. monocytogenestyping scheme based on genome-wide gene-by-gene comparisons (core genome multilocus the sequence typing [cgMLST]) to allow for a unique typing nomenclature. Initially, we determined the breadth of theL. monocytogenespopulation based on MLST data with a Bayesian approach. Based on the genome sequence data of representative isolates for the whole population, cgMLST target genes were defined and reappraised with 67L. monocytogenesisolates from two outbreaks and serotype reference strains. The Bayesian population analysis generated fiveL. monocytogenesgroups. Using all available NCBI RefSeq genomes (n= 36) and six additionally sequenced strains, all genetic groups were covered. Pairwise comparisons of these 42 genome sequences resulted in 1,701 cgMLST targets present in all 42 genomes with 100% overlap and ≥90% sequence similarity. Overall, ≥99.1% of the cgMLST targets were present in 67 outbreak and serotype reference strains, underlining the representativeness of the cgMLST scheme. Moreover, cgMLST enabled clustering of outbreak isolates with ≤10 alleles difference and unambiguous separation from unrelated outgroup isolates. In conclusion, the novel cgMLST scheme not only improves outbreak investigations but also enables, due to the availability of the automatically curated cgMLST nomenclature, interlaboratory exchange of data that are crucial, especially for rapid responses during transsectorial outbreaks.

scholarly journals comKProphage Junction Fragments as Markers for Listeria monocytogenes Genotypes Unique to Individual Meat and Poultry Processing Plants and a Model for Rapid Niche-Specific Adaptation, Biofilm Formation, and Persistence

2011 ◽  
Vol 77 (10) ◽  
pp. 3279-3292 ◽  
Author(s):  
Bindhu Verghese ◽  
Mei Lok ◽  
Jia Wen ◽  
Valentina Alessandria ◽  
Yi Chen ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Food Processing ◽  
Biofilm Formation ◽  
Content Type ◽  
Niche Adaptation ◽  
Processing Plants ◽  
Poultry Processing ◽  
Conditioning Film ◽  
Conditioning Films ◽  
Different Strains

ABSTRACTDifferent strains ofListeria monocytogenesare well known to persist in individual food processing plants and to contaminate foods for many years; however, the specific genotypic and phenotypic mechanisms responsible for persistence of these unique strains remain largely unknown. Based on sequences incomKprophage junction fragments, different strains of epidemic clones (ECs), which included ECII, ECIII, and ECV, were identified and shown to be specific to individual meat and poultry processing plants. ThecomKprophage-containing strains showed significantly higher cell densities after incubation at 30°C for 48 h on meat and poultry food-conditioning films than did strains lacking thecomKprophage (P< 0.05). Overall, the type of strain, the type of conditioning film, and the interaction between the two were all highly significant (P< 0.001). Recombination analysis indicated that thecomKprophage junction fragments in these strains had evolved due to extensive recombination. Based on the results of the present study, we propose a novel model in which the concept of defectivecomKprophage was replaced with the rapid adaptation island (RAI). Genes within the RAI were recharacterized as “adaptons,” as these genes may allowL. monocytogenesto rapidly adapt to different food processing facilities and foods. If confirmed, the model presented would help explainListeria's rapid niche adaptation, biofilm formation, persistence, and subsequent transmission to foods. Also,comKprophage junction fragment sequences may permit accurate tracking of persistent strains back to and within individual food processing operations and thus allow the design of more effective intervention strategies to reduce contamination and enhance food safety.

scholarly journals Draft Whole-Genome Sequences of Seven Listeria monocytogenes Strains with Variations in Virulence and Stress Responses

2018 ◽  
Vol 7 (13) ◽  
Author(s):  
Yanhong Liu ◽  
Aixia Xu ◽  
Pina M. Fratamico ◽  
Christopher H. Sommers ◽  
Luca Rotundo ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Stress Responses ◽  
Draft Genome ◽  
Foodborne Pathogen ◽  
Whole Genome ◽  
Genome Sequences ◽  
Content Type

Listeria monocytogenes is an important foodborne pathogen that causes listeriosis. Here, we report the draft genome sequences of seven L. monocytogenes strains isolated from food, environmental, and clinical sources.

scholarly journals Whole-Genome Sequences of Six Listeria monocytogenes Strains Isolated from Food

2018 ◽  
Vol 7 (14) ◽  
Author(s):  
Jule Anna Horlbog ◽  
Hyein Jang ◽  
Gopal Gopinath ◽  
Roger Stephan ◽  
Claudia Guldimann
Keyword(s):  
Listeria Monocytogenes ◽  
Amino Acid ◽  
Whole Genome ◽  
Genome Sequences ◽  
Content Type ◽  
Milk Products ◽  
Stop Codons

Here, we report the whole-genome sequences of six Listeria monocytogenes strains isolated from meat and milk products in Switzerland. All of these strains carry premature stop codons or amino acid deletions in inlA.

scholarly journals chewBBACA: A complete suite for gene-by-gene schema creation and strain identification

2017 ◽  
Author(s):  
Mickael Silva ◽  
Miguel Machado ◽  
Diogo N. Silva ◽  
Mirko Rossi ◽  
Jacob Moran-Gilad ◽  
...  
Keyword(s):  
Open Source ◽  
Core Genome ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Strain Identification ◽  
List Type ◽  
Whole Genome ◽  
Link Type ◽  
The Creation ◽  
Allele Calling

ABSTRACTGene-by-gene approaches are becoming increasingly popular in bacterial genomic epidemiology and outbreak detection. However, there is a lack of open-source scalable software for schema definition and allele calling for these methodologies. The chewBBACA suite was designed to assist users in the creation and evaluation of novel whole-genome or core-genome gene-by-gene typing schemas and subsequent allele calling in bacterial strains of interest. The software can run in a laptop or in high performance clusters making it useful for both small laboratories and large reference centers. ChewBBACA is available athttps://github.com/B-UMMI/chewBBACAor as a docker image athttps://hub.docker.com/r/ummidock/chewbbaca/.DATA SUMMARYAssembled genomes used for the tutorial were downloaded from NCBI in August 2016 by selecting those submitted asStreptococcus agalactiaetaxon or sub-taxa. All the assemblies have been deposited as a zip file in FigShare (https://figshare.com/s/9cbe1d422805db54cd52), where a file with the original ftp link for each NCBI directory is also available.Code for the chewBBACA suite is available athttps://github.com/B-UMMI/chewBBACAwhile the tutorial example is found athttps://github.com/B-UMMI/chewBBACA_tutorial.I/We confirm all supporting data, code and protocols have been provided within the article or through supplementary data files. ⊠IMPACT STATEMENTThe chewBBACA software offers a computational solution for the creation, evaluation and use of whole genome (wg) and core genome (cg) multilocus sequence typing (MLST) schemas. It allows researchers to develop wg/cgMLST schemes for any bacterial species from a set of genomes of interest. The alleles identified by chewBBACA correspond to potential coding sequences, possibly offering insights into the correspondence between the genetic variability identified and phenotypic variability. The software performs allele calling in a matter of seconds to minutes per strain in a laptop but is easily scalable for the analysis of large datasets of hundreds of thousands of strains using multiprocessing options. The chewBBACA software thus provides an efficient and freely available open source solution for gene-by-gene methods. Moreover, the ability to perform these tasks locally is desirable when the submission of raw data to a central repository or web services is hindered by data protection policies or ethical or legal concerns.

scholarly journals Whole-Genome Sequences of Seven Listeria monocytogenes Strains from Different Stages of a Poultry Meat Production Chain

2019 ◽  
Vol 8 (11) ◽  
Author(s):  
Victoria López-Alonso ◽  
Sagrario Ortiz ◽  
Joaquín V. Martínez-Suárez
Keyword(s):  
Listeria Monocytogenes ◽  
Draft Genome ◽  
Poultry Meat ◽  
Meat Production ◽  
Whole Genome ◽  
Genome Sequences ◽  
Production Chain ◽  
Content Type ◽  
Three Stages

Here, we present the draft genome sequences of seven Listeria monocytogenes strains isolated during three independent studies carried out in three stages of a poultry meat production chain. The genome sequences of these strains obtained from different stages can help to understand the possible transmission of L. monocytogenes.

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

2015 ◽  
Vol 81 (17) ◽  
pp. 6024-6037 ◽  
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.

scholarly journals Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes

2020 ◽  
Vol 202 (9) ◽  
Author(s):  
Duarte N. Guerreiro ◽  
Jialun Wu ◽  
Charlotte Dessaux ◽  
Ana H. Oliveira ◽  
Teresa Tiensuu ◽  
...  
Keyword(s):  
Heat Stress ◽  
Listeria Monocytogenes ◽  
Laboratory Culture ◽  
Stress Conditions ◽  
Mild Stress ◽  
Whole Genome ◽  
Content Type ◽  
Acid Sensitivity ◽  
Stop Codons ◽  
Mild Heat

ABSTRACT In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype. IMPORTANCE In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.

scholarly journals Molecular Tracking, through Processing, of Campylobacter Strains Colonizing Broiler Flocks

2011 ◽  
Vol 77 (16) ◽  
pp. 5722-5729 ◽  
Author(s):  
Karen T. Elvers ◽  
Victoria K. Morris ◽  
Diane G. Newell ◽  
Vivien M. Allen
Keyword(s):  
United Kingdom ◽  
Oligonucleotide Probe ◽  
Content Type ◽  
The United Kingdom ◽  
Processing Plants ◽  
Processing Line ◽  
Poultry Processing ◽  
Commercial Poultry ◽  
Molecular Tracking ◽  
Carcass Contamination

ABSTRACTMany of the poultry flocks produced in the United Kingdom are colonized withCampylobacter, and the intensive nature of poultry processing usually results in contaminated carcasses. In this study, a previously reported molecular oligonucleotide probe method was used to track a specific flock-colonizing strain(s) on broiler carcasses during processing in two United Kingdom commercial poultry processing plants. FiveCampylobacter-positive flocks were sampled at four points along the processing line, postbleed, postpluck, prechill, and postchill, and twoCampylobacter-negative flocks processed immediately after positive flocks were sampled prechill.flaAwas sequenced fromCampylobacterstrains isolated from these flocks, and strain-specific probes were synthesized. Skin and cecal samples were plated onto selective agar to give individual colonies, which were transferred onto membranes. These were then hybridized with the strain- and genus-specific probes. For all the 5 positive flocks, there was a significant reduction in campylobacters postbleed compared to postpluck but no subsequent fall on sampling pre- and postchill, and the strain(s) predominating on the carcasses throughout processing came from the flock being processed. This indicates that strains from the abattoir environment were not a significant cause of carcass contamination in flocks with well-established campylobacter colonization. However, negative flocks that were preceded by positive flocks were contaminated by strains that did not generally originate from the predominating strains recovered from the ceca of the previous positive flocks. This suggests that the abattoir environment has a significant role in the contamination of carcasses from negative but not fully colonized flocks.

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