Genome Sequencing of Listeria monocytogenes “Quargel” Listeriosis Outbreak Strains Reveals Two Different Strains with Distinct In Vitro 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.

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Heterogeneity of a Campylobacter jejuni Protein That Is Secreted through the Flagellar Filament

Infection and Immunity ◽

10.1128/iai.00159-07 ◽

2007 ◽

Vol 75 (8) ◽

pp. 3859-3867 ◽

Cited By ~ 74

Author(s):

Frédéric Poly ◽

Cheryl Ewing ◽

Scarlett Goon ◽

Thomas E. Hickey ◽

David Rockabrand ◽

...

Keyword(s):

Campylobacter Jejuni ◽

Virulence Factor ◽

Promoter Analysis ◽

Acidic Protein ◽

Rapid Induction ◽

Virulence Potential ◽

Flagellar Filament ◽

Induction Of Apoptosis ◽

Different Strains

ABSTRACTCj0859c, or FspA, is a small, acidic protein ofCampylobacter jejunithat is expressed by a σ28promoter. Analysis of thefspAgene in 41 isolates ofC. jejunirevealed two overall variants of the predicted protein, FspA1 and FspA2. Secretion of FspA occurs in broth-grown bacteria and requires a minimum flagellar structure. The addition of recombinant FspA2, but not FspA1, to INT407 cells in vitro resulted in a rapid induction of apoptosis. These data define a novelC. jejunivirulence factor, and the observed heterogeneity amongfspAalleles suggests alternate virulence potential among different strains.

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ON-rep-seq as a rapid and cost-effective alternative to whole-genome sequencing for species-level identification and strain-level discrimination of Listeria monocytogenes contamination in a salmon processing plant

10.22541/au.163543013.37719528/v1 ◽

2021 ◽

Author(s):

Gunn Merethe Thomassen ◽

Lukasz Krych ◽

Susanne Knøchel ◽

Lisbeth Mehli

Keyword(s):

Listeria Monocytogenes ◽

Whole Genome Sequencing ◽

Genome Sequencing ◽

Screening Method ◽

Cost Effective ◽

Strain Level ◽

Source Tracking ◽

Processing Plant ◽

Whole Genome ◽

Different Strains

Identification, source tracking, and surveillance of food pathogens is a crucial factor for the food-producing industry. Over the last decade, the techniques used for this have moved from conventional enrichment methods, through species-specific detection by PCR to sequencing-based methods, whole-genome sequencing (WGS) being the ultimate method. However, using WGS requires the right infrastructure, high computational power, and bioinformatics expertise. Therefore, there is a need for faster, more cost-effective, and more user-friendly methods. A newly developed method, ON-rep-seq, combines the classical rep-PCR method with nanopore sequencing, resulting in a highly discriminating set of sequences that can be used for species identification and also strain discrimination. This study is essentially a real industry case from a salmon processing plant. Twenty Listeria monocytogenes isolates were analyzed both by ON-rep-seq and WGS to identify and differentiate putative L. monocytogenes from a routine sampling of processing equipment and products, and finally, compare the strain-level discriminatory power of ON-rep-seq to different analyzing levels delivered from the WGS data. The analyses revealed that among the isolates tested there were three different strains. The isolates of the most frequently detected strain (n=15) were all detected in the problematic area in the processing plant. The strain level discrimination done by ON-rep-seq was in full accordance with the interpretation of WGS data. Our findings also demonstrate that ON-rep-seq may serve as a primary screening method alternative to WGS for identification and strain-level differentiation for surveillance of potential pathogens in a food-producing environment.

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Chitin Attenuates Expression of Listeria monocytogenes Virulence Genes in vitro

Frontiers in Microbiology ◽

10.3389/fmicb.2020.588906 ◽

2020 ◽

Vol 11 ◽

Author(s):

Miguel Villoria Recio ◽

Bo-Hyung Lee ◽

Eva Maria Sternkopf Lillebæk ◽

Birgitte H. Kallipolitis ◽

Cormac G. M. Gahan ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Disease Risk ◽

Foodborne Pathogen ◽

Post Translational Modification ◽

Competitive Environments ◽

Virulence Potential ◽

Alternative Approach ◽

External Signals ◽

First Time

External signals are crucial for bacteria to sense their immediate environment and fine-tune gene expression accordingly. The foodborne pathogen Listeria monocytogenes senses a range of environmental cues in order to activate or deactivate the virulence-inducing transcriptional factor PrfA during transition between infectious and saprophytic lifecycles. Chitin is an abundant biopolymer formed from linked β-(1–4)-N-acetyl-D-glucosamine residues associated with fungi, the exoskeleton of insects and often incorporated into foods as a thickener or stabilizer. L. monocytogenes evolved to hydrolyse chitin, presumably, to facilitate nutrient acquisition from competitive environments such as soil where the polymer is abundant. Since mammals do not produce chitin, we reasoned that the polymer could serve as an environmental signal contributing to repression of L. monocytogenes PrfA-dependent expression. This study shows a significant downregulation of the core PrfA-regulon during virulence-inducing conditions in vitro in the presence of chitin. Our data suggest this phenomenon occurs through a mechanism that differs from PTS-transport of oligosaccharides generated from either degradation or chitinase-mediated hydrolysis of the polymer. Importantly, an indication that chitin can repress virulence expression of a constitutively active PrfA∗ mutant is shown, possibly mediated via a post-translational modification inhibiting PrfA∗ activity. To our knowledge, this is the first time that chitin is reported as a molecule with anti-virulence properties against a pathogenic bacterium. Thus, our findings identify chitin as a signal which may downregulate the virulence potential of the pathogen and may provide an alternative approach toward reducing disease risk.

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Virulence of Listeria monocytogenes isolated from the cheese dairy environment, other foods and clinical cases

Journal of Medical Microbiology ◽

10.1099/jmm.0.47672-0 ◽

2008 ◽

Vol 57 (4) ◽

pp. 411-415 ◽

Cited By ~ 22

Author(s):

Elsa Neves ◽

Ana Carla Silva ◽

Sylvie M. Roche ◽

Philippe Velge ◽

Luisa Brito

Keyword(s):

Listeria Monocytogenes ◽

Virulent Strain ◽

Cell Monolayers ◽

Virulence Potential ◽

Production Environments ◽

Good Manufacturing Practices ◽

Highly Virulent ◽

Ht 29 ◽

Cheese Production

The virulence potential of 51 Listeria monocytogenes isolates, including strains from cheese, cheese production environments and from human cases of listeriosis, was evaluated in this study. The isolates were used to infect HT-29 cell monolayers in an in vitro test of virulence, based on a plaque-forming assay (PFA). Fifteen selected isolates were used for subcutaneous footpad inoculation in mice and subsequent recovery of the bacterium from the spleen 3 days after inoculation. In the PFA, two isolates from milk (serovar 1/2a) were not significantly different (P<0.05) from the low-virulence strain (442) used as reference. Thirty-three isolates were not significantly different (P<0.05) from the virulent strain (EGDe) used as reference. Nine isolates were significantly more virulent (highly virulent) than the EGDe strain and seven isolates were significantly less virulent. The nine highly virulent isolates were either from humans (four), from cheese dairy environments (two isolates of a strain were found persistently in two dairies), from cheese (one), from milk (one) and the reference strain for serovar 1/2b (CECT 936). The two milk isolates with low virulence in the PFA were found to be virulent in mice. In conclusion, all the isolates from food and food-related environments were potentially virulent or highly virulent. These results stress the risk of listeriosis associated with the consumption of cheese contaminated with L. monocytogenes, and once more emphasize the importance of good manufacturing practices (GMPs) together with sanitation standard operating procedures (SSOPs) throughout the food chain.

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Molecular serotyping and virulence potential of Listeria monocytogenes isolated from bovine, swine and human in the province of Quebec

10.31274/safepork-180809-254 ◽

2015 ◽

Author(s):

P. Fravalo ◽

T. Cherifi ◽

K. Neira ◽

A. Letellier ◽

J.-H. Fairbrother ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Molecular Serotyping ◽

Virulence Potential

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Combined application of phenolic acids and essential oil components against Salmonella Enteritidis and Listeria monocytogenes in vitro and in ready-to-eat cooked ham

LWT ◽

10.1016/j.lwt.2021.111881 ◽

2021 ◽

pp. 111881

Author(s):

Jessica Audrey Feijó Corrêa ◽

João Vitor Garcia dos Santos ◽

Alberto Gonçalves Evangelista ◽

Anne Caroline Schoch Marques Pinto ◽

Renata Ernlund Freitas de Macedo ◽

...

Keyword(s):

Listeria Monocytogenes ◽

Essential Oil ◽

Phenolic Acids ◽

Salmonella Enteritidis ◽

Combined Application ◽

Cooked Ham ◽

Oil Components

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Biallelic variants in COPB1 cause a novel, severe intellectual disability syndrome with cataracts and variable microcephaly

Genome Medicine ◽

10.1186/s13073-021-00850-w ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

William L. Macken ◽

Annie Godwin ◽

Gabrielle Wheway ◽

Karen Stals ◽

Liliya Nazlamova ◽

...

Keyword(s):

Genome Sequencing ◽

Donor Site ◽

Xenopus Tropicalis ◽

Splice Donor Site ◽

Homologous Region ◽

Disease Genes ◽

Coat Proteins ◽

Splice Donor ◽

Severe Intellectual Disability

Abstract Background Coat protein complex 1 (COPI) is integral in the sorting and retrograde trafficking of proteins and lipids from the Golgi apparatus to the endoplasmic reticulum (ER). In recent years, coat proteins have been implicated in human diseases known collectively as “coatopathies”. Methods Whole exome or genome sequencing of two families with a neuro-developmental syndrome, variable microcephaly and cataracts revealed biallelic variants in COPB1, which encodes the beta-subunit of COPI (β-COP). To investigate Family 1’s splice donor site variant, we undertook patient blood RNA studies and CRISPR/Cas9 modelling of this variant in a homologous region of the Xenopus tropicalis genome. To investigate Family 2’s missense variant, we studied cellular phenotypes of human retinal epithelium and embryonic kidney cell lines transfected with a COPB1 expression vector into which we had introduced Family 2’s mutation. Results We present a new recessive coatopathy typified by severe developmental delay and cataracts and variable microcephaly. A homozygous splice donor site variant in Family 1 results in two aberrant transcripts, one of which causes skipping of exon 8 in COPB1 pre-mRNA, and a 36 amino acid in-frame deletion, resulting in the loss of a motif at a small interaction interface between β-COP and β’-COP. Xenopus tropicalis animals with a homologous mutation, introduced by CRISPR/Cas9 genome editing, recapitulate features of the human syndrome including microcephaly and cataracts. In vitro modelling of the COPB1 c.1651T>G p.Phe551Val variant in Family 2 identifies defective Golgi to ER recycling of this mutant β-COP, with the mutant protein being retarded in the Golgi. Conclusions This adds to the growing body of evidence that COPI subunits are essential in brain development and human health and underlines the utility of exome and genome sequencing coupled with Xenopus tropicalis CRISPR/Cas modelling for the identification and characterisation of novel rare disease genes.

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Safety Assessment of Bacillus subtilis MB40 for Use in Foods and Dietary Supplements

Nutrients ◽

10.3390/nu13030733 ◽

2021 ◽

Vol 13 (3) ◽

pp. 733

Author(s):

Jessica L. Spears ◽

Richard Kramer ◽

Andrey I. Nikiforov ◽

Marisa O. Rihner ◽

Elizabeth A. Lambert

Keyword(s):

Antibiotic Resistance ◽

Bacillus Subtilis ◽

Dietary Supplements ◽

Genome Sequencing ◽

In Silico ◽

Safety Assessment ◽

In Vivo Studies ◽

Strain Identification ◽

Consumer Safety

With the growing popularity of probiotics in dietary supplements, foods, and beverages, it is important to substantiate not only the health benefits and efficacy of unique strains but also safety. In the interest of consumer safety and product transparency, strain identification should include whole-genome sequencing and safety assessment should include genotypic and phenotypic studies. Bacillus subtilis MB40, a unique strain marketed for use in dietary supplements, and food and beverage, was assessed for safety and tolerability across in silico, in vitro, and in vivo studies. MB40 was assessed for the absence of undesirable genetic elements encoding toxins and mobile antibiotic resistance. Tolerability was assessed in both rats and healthy human volunteers. In silico and in vitro testing confirmed the absence of enterotoxin and mobile antibiotic resistance genes of safety concern to humans. In rats, the no-observed-adverse-effect level (NOAEL) for MB40 after repeated oral administration for 14 days was determined to be 2000 mg/kg bw/day (equivalent to 3.7 × 1011 CFU/kg bw/day). In a 28 day human tolerability trial, 10 × 109 CFU/day of MB40 was well tolerated. Based on genome sequencing, strain characterization, screening for undesirable attributes and evidence of safety by appropriately designed safety evaluation studies in rats and humans, Bacillus subtilis MB40 does not pose any human health concerns under the conditions tested.

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Effect of Gaseous Ozone on Listeria monocytogenes Planktonic Cells and Biofilm: An In Vitro Study

Foods ◽

10.3390/foods10071484 ◽

2021 ◽

Vol 10 (7) ◽

pp. 1484

Author(s):

Felice Panebianco ◽

Selene Rubiola ◽

Francesco Chiesa ◽

Tiziana Civera ◽

Pierluigi Aldo Di Ciccio

Keyword(s):

Listeria Monocytogenes ◽

In Vitro Study ◽

Planktonic Cells ◽

Free Living ◽

Biofilm Inhibition ◽

Additional Tool ◽

Complete Inactivation ◽

Gaseous Ozone ◽

Food Borne

Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

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