Characterization of Mobile Genetic Elements Using Long-Read Sequencing for Tracking Listeria monocytogenes from Food Processing Environments (original) (raw)
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Frontiers in Microbiology
The prfA-virulence gene cluster (pVGC) is the main pathogenicity island in Listeria monocytogenes, comprising the prfA, plcA, hly, mpl, actA, and plcB genes. In this study, the pVGC of 36 L. monocytogenes isolates with respect to different serotypes (1/2a or 4b), geographical origin (Australia, Greece or Ireland) and isolation source (food-associated or clinical) was characterized. The most conserved genes were prfA and hly, with the lowest nucleotide diversity (π) among all genes (P < 0.05), and the lowest number of alleles, substitutions and non-synonymous substitutions for prfA. Conversely, the most diverse gene was actA, which presented the highest number of alleles (n = 20) and showed the highest nucleotide diversity. Grouping by serotype had a significantly lower π value (P < 0.0001) compared to isolation source or geographical origin, suggesting a distinct and well-defined unit compared to other groupings. Among all tested genes, only hly and mpl were those with lower nucleotide diversity in 1/2a serotype than 4b serotype, reflecting a high within-1/2a serotype divergence compared to 4b serotype. Geographical divergence was noted with respect to the hly gene, where serotype 4b Irish strains were distinct from Greek and Australian strains. Australian strains showed less diversity in plcB and mpl relative to Irish or Greek strains. Notable differences regarding sequence mutations were identified between food-associated and clinical isolates in prfA, actA, and plcB sequences. Overall, these results indicate that virulence genes follow different evolutionary pathways, which are affected by a strain's origin and serotype and may influence virulence and/or epidemiological dominance of certain subgroups.
2020
A 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. .
We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 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 in L. mono-cytogenes and 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.
Journal of Microbial & Biochemical Technology
Listeria monocytogenes is an important foodborne pathogen and, relatedly, a persistent contaminant in many food processing facilities. Strain typing is critical to detecting and investigating outbreaks, and can be used to track down sources of contamination. The typing systems in current use have limitations ranging from low resolution and data portability to high cost and technical complexity. The aim of this study was to develop an outsourcing option for L. monocytogenes strain typing that addresses these limitations. The NCBI Genomes database representing 109 strains was screened for highly informative, tandem repeat-containing loci. The most promising, termed LmMT1 (0.8-1 kbp) and LmMT2 (0.7-0.8 kbp), exhibited complex patterns of polymorphism (insertions/deletions and single nucleotide polymorphisms), diversity indeces of 0.99 (LmMT1) and 0.98 (LmMT2), and were present in all L. monocytogenes and from one (LmMT1) to four (LmMT2) additional Listeria species represented in NCBI databases. Using a distinct set of strains, Miya et al. (J. Microbiol. Methods, 2012, 90:285-291) previously reported diversity indices of 0.95 and 0.91 for more limited regions (0.3-0.5 kbp) of these same two loci. Phylogenetic analysis of LmMT1 and LmMT2 sequences revealed distinct clusters corresponding to serotype (4b, 1/2a, and 4a complexes) and evolutionary lineage (I, II, and III/IV). Comparisons to PFGE, the current gold standard, suggest that LmMT1 typing is comparably discriminatory. Importantly, strains from four outbreaks formed corresponding clusters, although those from a 2002 outbreak were resolved into related environmental and food/human isolates that challenges their epidemiological connection. In the laboratory, LmMT1 and LmMT2 typing proved to be robust, generating high quality sequence from colonies submitted as non-hazardous heat-inactivated suspensions. Analysis of LmMT1 sequences from 62 diverse strains demonstrated overall agreement with single nucleotide polymorphism-based typing. Journal of Microbial & Biochemical Technology J o u rn al of M ic ro b ia l & Bioc h e m ic a l Te chno lo g y
Genomic methods for enhanced surveillance and persistence investigations of foodborne pathogens
2018
The globalized food supply chain became a vast and complex network leading to an increased risk of spread of known and emerging foodborne pathogens, Listeria monocytogenes and Salmonella enterica serovar Typhimurium variant 4,[5],12:i:-. Based on a previous study of persistence of L. monocytogenes, 27 ST14 and 6 ST121 newly sequenced genomes collected over one year on the same rabbit meat processing plant were investigated in comparison to a selection of public genomes. cgMLST analysis of sequenced genomes showed higher discriminatory power in comparison to conventional typing methods. wgSNPs phylogeny inferred on 273 newly sequenced and publicly available ST121 and ST14 genomes confirmed that a persistent clone was circulating in the Italian rabbit-meat plant along with not persistent strains. Mass screening of a novel dataset of genes involved in physiological adaptation to food-processing environment showed a significant enrichment in ST121 genomes concerned genetic features rela...
Microorganisms
Listeria monocytogenes is a foodborne pathogen with a highly clonal population structure comprising multiple phylogenetic sub-groups that can persist within food processing environments and contaminate food. The epidemiology of L. monocytogenes is well-described in some developed countries; however, little is known about the prevalence and population structure of this pathogen in food and food processing environments located in less developed regions. The aim of this study was to determine the genetic characteristics and clonal relatedness of L. monocytogenes that were isolated from two Jamaican meat processing facilities. Of the 37 isolates collected between 2011 and 2015, only a single lineage II isolate was recovered (serotype 1/2c), and the remaining were lineage I isolates representing serotypes 4b, 1/2b, 3b, and two untypeable isolates. Pulsed-field gel electrophoresis (PFGE) delineated isolates into seven pulsotypes, and whole-genome sequencing (WGS) categorized most isolates...
BMC Genomics, 2010
Background: A large, multi-province outbreak of listeriosis associated with ready-to-eat meat products contaminated with Listeria monocytogenes serotype 1/2a occurred in Canada in 2008. Subtyping of outbreakassociated isolates using pulsed-field gel electrophoresis (PFGE) revealed two similar but distinct AscI PFGE patterns. High-throughput pyrosequencing of two L. monocytogenes isolates was used to rapidly provide the genome sequence of the primary outbreak strain and to investigate the extent of genetic diversity associated with a change of a single restriction enzyme fragment during PFGE. Results: The chromosomes were collinear, but differences included 28 single nucleotide polymorphisms (SNPs) and three indels, including a 33 kbp prophage that accounted for the observed difference in AscI PFGE patterns. The distribution of these traits was assessed within further clinical, environmental and food isolates associated with the outbreak, and this comparison indicated that three distinct, but highly related strains may have been involved in this nationwide outbreak. Notably, these two isolates were found to harbor a 50 kbp putative mobile genomic island encoding translocation and efflux functions that has not been observed in other Listeria genomes. Conclusions: High-throughput genome sequencing provided a more detailed real-time assessment of genetic traits characteristic of the outbreak strains than could be achieved with routine subtyping methods. This study confirms that the latest generation of DNA sequencing technologies can be applied during high priority public health events, and laboratories need to prepare for this inevitability and assess how to properly analyze and interpret whole genome sequences in the context of molecular epidemiology.
Applied and Environmental Microbiology, 2007
Listeria monocytogenes is a facultative intracellular pathogen responsible for food-borne disease with high mortality rates in humans and is the leading microbiological cause of food recalls. Lineage I isolates of L. monocytogenes are a particular public health concern because they are responsible for most sporadic cases of listeriosis and the vast majority of epidemic outbreaks. Rapid, reproducible, and sensitive methods for differentiating pathogens below the species level are required for effective pathogen control programs, and the CDC PulseNet Task Force has called for the development and validation of DNA sequence-based methods for subtyping food-borne pathogens. Therefore, we developed a multilocus genotyping (MLGT) assay for L. monocytogenes lineage I isolates based on nucleotide variation identified by sequencing 23,251 bp of DNA from 22 genes distributed across seven genomic regions in 65 L. monocytogenes isolates. This single-well assay of 60 allele-specific probes captured 100% of the haplotype information contained in approximately 1.5 Mb of comparative DNA sequence and was used to reproducibly type a total of 241 lineage I isolates. The MLGT assay provided high discriminatory power (Simpson's index value, 0.91), uniquely identified isolates from the eight listeriosis outbreaks examined, and differentiated serotypes 1/2b and 4b as well as epidemic clone I (ECI), ECIa, and ECII. In addition, the assay included probes for a previously characterized truncation mutation in inlA, providing for the identification of a specific virulence-attenuated subtype. These results demonstrate that MLGT represents a significant new tool for use in pathogen surveillance, outbreak detection, risk assessment, population analyses, and epidemiological investigations.
Applied and Environmental Microbiology, 2015
While 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, supp...
Emergence and global spread of Listeria monocytogenes main clinical clonal complex
Science Advances, 2021
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