scholarly journals Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

2021 ◽  
Vol 10 (1) ◽  
pp. 62
Author(s):  
Giuditta Fiorella Schiavano ◽  
Collins Njie Ateba ◽  
Annalisa Petruzzelli ◽  
Veronica Mele ◽  
Giulia Amagliani ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Clonal Complex ◽  
Human Colon ◽  
Whole Genome ◽  
Human Colon Carcinoma Cell ◽  
Level Of Invasion ◽  
Virulence Potential

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

scholarly journals Retrospective Use of Whole-Genome Sequencing Expands the Multicountry Outbreak Cluster of Listeria monocytogenes ST1247

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Mihkel Mäesaar ◽  
Rafael Mamede ◽  
Terje Elias ◽  
Mati Roasto
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Core Genome ◽  
Clonal Complex ◽  
Antibiotic Resistance Genes ◽  
Whole Genome ◽  
Outbreak Strain ◽  
Determining Factors ◽  
Fish Product

Listeria monocytogenes sequence type 1247 clonal complex 8 caused a prolonged multicountry outbreak in five EU countries: Denmark, Estonia, Finland, France, and Sweden. A total of 22 disease cases were identified with onset of symptoms between July 2014 and February 2019. Five patients died due to, or with, the disease. The retrospective analysis of L. monocytogenes isolate VLTRLM2013 revealed the presence of an outbreak-related strain (cgMLST type L2-SL8-ST1247-CT4158) in ready-to-eat fish product more than a year prior to the first outbreak-related cases. Reference outbreak strain and VLTRLM2013 strain were compared using core genome and whole-genome multilocus sequence typing analyses. Genomic level differences of the persistent L. monocytogenes strains associated with a prolonged multicountry foodborne listeriosis outbreak are described. It was concluded that the persistent nature of the multicountry outbreak-related L. monocytogenes strain VLTRLM2013 together with stress island, virulence, and antibiotic resistance genes could potentially be the determining factors for the extensive and prolonged outbreak affecting five European Union countries. Our results support the systematic application of whole-genome sequencing in food and public health surveillance and further encourages its wide adoption.

scholarly journals SureSelect targeted enrichment, a new cost effective method for the whole genome sequencing of Candidatus Liberibacter asiaticus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Weili Cai ◽  
Schyler Nunziata ◽  
John Rascoe ◽  
Michael J. Stulberg
Keyword(s):  
Whole Genome Sequencing ◽  
Molecular Characterization ◽  
Genome Sequencing ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Genome Coverage ◽  
Metagenomic Sample ◽  
Liberibacter Asiaticus

AbstractHuanglongbing (HLB) is a worldwide deadly citrus disease caused by the phloem-limited bacteria ‘Candidatus Liberibacter asiaticus’ (CLas) vectored by Asian citrus psyllids. In order to effectively manage this disease, it is crucial to understand the relationship among the bacterial isolates from different geographical locations. Whole genome sequencing approaches will provide more precise molecular characterization of the diversity among populations. Due to the lack of in vitro culture, obtaining the whole genome sequence of CLas is still a challenge, especially for medium to low titer samples. Hundreds of millions of sequencing reads are needed to get good coverage of CLas from an HLB positive citrus sample. In order to overcome this limitation, we present here a new method, Agilent SureSelect XT HS target enrichment, which can specifically enrich CLas from a metagenomic sample while greatly reducing cost and increasing whole genome coverage of the pathogen. In this study, the CLas genome was successfully sequenced with 99.3% genome coverage and over 72X sequencing coverage from low titer tissue samples (equivalent to 28.52 Cq using Li 16 S qPCR). More importantly, this method also effectively captures regions of diversity in the CLas genome, which provides precise molecular characterization of different strains.

Advancing RNA Virus Discovery and Biology with Whole Genome Sequencing

2021 ◽  
Author(s):  
◽  
Mariah Taylor ◽  
Keyword(s):  
Genetic Variation ◽  
Amino Acid ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Rna Virus ◽  
Animal Health ◽  
Functional Domains ◽  
Whole Genome ◽  
Coding Region

Two RNA virus families that pose a threat to human and animal health are Hantaviridae and Coronaviridae. These RNA viruses which originate in wildlife continue and will continue to cause disease, and hence, it is critical that scientific research define the mechanisms as to how these viruses spillover and adapt to new hosts to become endemic. One gap in our ability to define these mechanisms is the lack of whole genome sequences for many of these viruses. To address this specific gap, I developed a versatile amplicon-based whole-genome sequencing (WGS) approach to identify viral genomes of hantaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) within reservoir and spillover hosts. In my research studies, I used the amplicon-based WGS approach to define the genetic plasticity of viral RNA within pathogenic and nonpathogenic hantavirus species. The standing genetic variation of Andes orthohantavirus and Prospect Hill orthohantavirus was mapped out and amino acid changes occurring outside of functional domains were identified within the nucleocapsid and glycoprotein. I observed several amino acid changes in functional domains of the RNA-dependent RNA polymerase, as well as single nucleotide polymorphisms (SNPs) within the 3’ non-coding region (NCR) of the S-segment. To identify whether virus adaptation would occur within the S- and L-segments we attempted to adapt hantaviruses in vitro in a spillover host model through passaging experiments. In early passages we identified few mutations in the M-segment with the majority being identified in the S-segment 3’ NCR and the L-segment. This work suggests that hantavirus adaptation occurs in the S- and L-segments although the effect of these mutants on pathology is yet to be determined. While sequencing laboratory isolates is easily accomplished, sequencing low concentrations of virus within the reservoir is a formidable task. I further translated our amplicon-based WGS approach into a pan-oligonucleotide amplicon-based WGS approach to sequence hantavirus vRNA and mRNA from reservoir and spillover hosts in Ukraine. This approach successfully identified a novel Puumala orthohantavirus (PUUV) strain in Ukraine and using Bayesian phylogenetics we found this strain to be associated with the PUUV Latvian lineage. Early during the SARS-CoV-2 pandemic, I applied the knowledge gained in the hantavirus WGS efforts to sequencing of SARS-CoV-2 from nasopharyngeal swabs collected in April 2020. The genetic diversity of 45 SARS-CoV-2 isolates was evaluated with the methods I developed. We identified D614G, a notable mutation known for increasing transmission, in over 90% of our isolates. Two major lineages distinguish SARS-CoV-2 variants worldwide, lineages A and B. While most of our isolates were found within B lineage, we also identified one isolate within lineage A. We performed in vitro work which confirmed A lineage isolates as having poor replication in the trachea as compared to the nasal cavity. Five of these isolates presented a unique array of mutations which were assessed in the keratin 18 human angiotensin-converting enzyme 2 (K18-hACE2) mouse model for its response immunologically and pathogenically. We identified a distinction of pathogenesis between the A and B lineages with emphysema being common amongst A lineage isolates. Additionally, we discovered a small cohort of likely SNPs that defined the late induction of eosinophils during infection. In summary, this work will further define the dynamics of genetic variation and plasticity within virus populations that cause disease outbreaks and will allow a deeper understanding of the virus-host relationship.

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 Revealing the Virulence Potential of Clinical and Environmental Aspergillus fumigatus Isolates Using Whole-Genome Sequencing

2019 ◽  
Vol 10 ◽  
Author(s):  
Fabiola Puértolas-Balint ◽  
John W. A. Rossen ◽  
Claudy Oliveira dos Santos ◽  
Monika M. A. Chlebowicz ◽  
Erwin C. Raangs ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Whole Genome ◽  
Virulence Potential

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

2017 ◽  
Vol 5 (49) ◽  
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.

scholarly journals Short communication: Persistent contamination by Listeria monocytogenes of bovine raw milk investigated by whole-genome sequencing

2019 ◽  
Vol 102 (7) ◽  
pp. 6032-6036
Author(s):  
M. Ricchi ◽  
E. Scaltriti ◽  
G. Cammi ◽  
C. Garbarino ◽  
N. Arrigoni ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Short Communication ◽  
Raw Milk ◽  
Whole Genome

Utility of Whole Genome Sequencing To Describe the Persistence and Evolution of Listeria monocytogenes Strains within Crabmeat Processing Environments Linked to Two Outbreaks of Listeriosis

2018 ◽  
Vol 82 (1) ◽  
pp. 30-38 ◽  
Author(s):  
RICHARD ELSON ◽  
ADEDOYIN AWOFISAYO-OKUYELU ◽  
TREVOR GREENER ◽  
CRAIG SWIFT ◽  
ANAÏS PAINSET ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Surveillance Data ◽  
Control Measures ◽  
Whole Genome ◽  
Typing Method ◽  
Outbreak Investigations ◽  
Molecular Typing Method ◽  
Clinical Cases

ABSTRACT This article describes the identification and investigation of two extended outbreaks of listeriosis in which crabmeat was identified as the vehicle of infection. Comparing contemporary and retrospective typing data of Listeria monocytogenes isolates from clinical cases and from food and food processing environments allowed the detection of cases going back several years. This information, combined with the analysis of routinely collected enhanced surveillance data, helped to direct the investigation and identify the vehicle of infection. Retrospective whole genome sequencing (WGS) analysis of isolates provided robust microbiological evidence of links between cases, foods, and the environments in which they were produced and demonstrated that for some cases and foods, identified by fluorescent amplified fragment length polymorphism, the molecular typing method in routine use at the time, were not part of the outbreak. WGS analysis also showed that the strains causing illness had persisted in two food production environments for many years and in one producer had evolved into two strains over a period of around 8 years. This article demonstrates the value of reviewing L. monocytogenes typing data from clinical cases together with that from foods as a means of identifying potential vehicles and sources of infection in outbreaks of listeriosis. It illustrates the importance of reviewing retrospective L. monocytogenes typing alongside enhanced surveillance data to characterize extended outbreaks and inform control measures. Also, this article highlights the advantages of WGS analysis for strain discrimination and clarification of evolutionary relationships that refine outbreak investigations and improve our understanding of L. monocytogenes in the food chain.

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