Spontaneous virulence loss in natural populations of Listeria monocytogenes

International audienceThe pathogenesis of Listeria monocytogenes depends on the ability of this bacterium to escape from the phagosome of the host cells via the action of the pore-forming toxin listeriolysin O (LLO). Expression of the LLO-encoding gene (hly) requires the transcriptional activator PrfA, and both hly and prfA genes are essential for L. monocytogenes virulence. Here, we used the hemolytic activity of LLO as a phenotypic marker to screen for spontaneous virulence-attenuating mutations in L. monocytogenes. Sixty nonhemolytic isolates were identified among a collection of 57,820 confirmed L. monocytogenes strains isolated from a variety of sources (0.1%). In most cases (56/60; 93.3%), the nonhemolytic phenotype resulted from nonsense, missense, or frameshift mutations in prfA. Five strains carried hly mutations leading to a single amino acid substitution (G299V) or a premature stop codon causing strong virulence attenuation in mice. In one strain, both hly and gshF (encoding a glutathione synthase required for full PrfA activity) were missing due to genomic rearrangements likely caused by a transposable element. The PrfA/LLO loss-of-function (PrfA Ϫ /LLO Ϫ) mutants belonged to phylogenetically diverse clades of L. monocyto-genes, and most were identified among nonclinical strains (57/60). Consistent with the rare occurrence of loss-of-virulence mutations, we show that prfA and hly are under purifying selection. Although occurring at a low frequency, PrfA Ϫ /LLO Ϫ muta-tional events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen

MALDI-TOF mass spectrometry-based identification of Listeria species in surveillance: a prospective study

International audienceThis study aimed to evaluate MALDI-TOF MS for species discrimination of Listeria in the context of routine surveillance. MALDI-TOF MS yielded 100% accuracy for the identification of L. monocytogenes, L. innocua, L. ivanovii, L. fleischmannii, L. grayi, L. seeligeri, L. weihenstephanensis and L. welshimeri, as confirmed by whole genome sequence analyses

Phenotypic and genotypic antimicrobial resistance of Listeria monocytogenes: an observational study in France

Background: Large-scale studies are needed to clarify antimicrobial resistance in the foodborne pathogen Listeria monocytogenes (Lm) and the effectiveness of listeriosis treatment options. Here we examined the antimicrobial resistance patterns in Lm over time and assessed genotype-phenotype concordances. Methods: We analyzed 5,339 isolates (2,908 clinical and 2,431 food isolates) collected in France and overseas territories, between 2012 and 2019. Whole genome sequencing was performed for all isolates and antimicrobial resistance profiles inferred from draft assemblies. Antimicrobial susceptibility towards 22 antimicrobials was determined for all clinical isolates, and in food isolates with acquired resistance genes. Findings: All tested isolates were resistant to at least 3 different classes of antimicrobials, consistent with Lm intrinsic traits. Acquired antimicrobial resistance in Lm was rare (2•23% isolates) and more prevalent in food (mainly lineage II) compared to clinical isolates (mainly lineage I) (3•74% vs 0•98%, p99%), except for ciprofloxacin. Acquired antimicrobial phenotypes were towards tetracyclines (mostly due to tetM), trimethoprim (dfrD), lincosamides (lnuG), macrolides (ermB, mphB) and phenicols (fexA). Interpretation: The reference treatment for listeriosis (aminopenicillins/aminoglycosides) remains effective, with no acquired resistance observed. Continuous surveillance of antimicrobial resistance in clinical and food isolates is crucial to detect the emergence of novel resistance

Hypervirulent Listeria monocytogenes clones’ adaption to mammalian gut accounts for their association with dairy products

International audienceListeria monocytogenes (Lm) is a major human and animal foodborne pathogen. Here we show that hypervirulent Lm clones, particularly CC1, are strongly associated with dairy products, whereas hypovirulent clones, CC9 and CC121, are associated with meat products. Clone adaptation to distinct ecological niches and/or different food products contamination routes may account for this uneven distribution. Indeed, hypervirulent clones colonize better the intestinal lumen and invade more intestinal tissues than hypovirulent ones, reflecting their adaption to host environment. Conversely, hypovirulent clones are adapted to food processing environments, with a higher prevalence of stress resistance and benzalkonium chloride tolerance genes and a higher survival and biofilm formation capacity in presence of sub-lethal benzalkonium chloride concentrations. Lm virulence heterogeneity therefore reflects the diversity of the ecological niches in which it evolves. These results also have important public health implications and may help in reducing food contamination and improving food consumption recommendations to at-risk populations

Cutaneous listeriosis, a case series of 16 consecutive patients over 25 years

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Prevalence of Listeria spp. and characterization of Listeria monocytogenes isolated from food products in Tetouan, Morocco

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Optimized Multilocus Variable-Number Tandem-Repeat Analysis Assay and Its Complementarity with Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing for Listeria monocytogenes Clone Identification and Surveillance

International audiencePopulations of the food-borne pathogen Listeria monocytogenes are genetically structured into a small number of major clonal groups, some of which have been implicated in multiple outbreaks. The goal of this study was to develop and evaluate an optimized multilocus variable number of tandem repeat (VNTR) analysis (MLVA) subtyping scheme for strain discrimination and clonal group identification. We evaluated 18 VNTR loci and combined the 11 best ones into two multiplexed PCR assays (MLVA-11). A collection of 255 isolates representing the diversity of clonal groups within phylogenetic lineages I and II, including representatives of epidemic clones, were analyzed by MLVA-11, multilocus sequence typing (MLST), and pulsed-field gel electropho-resis (PFGE). MLVA-11 had less discriminatory power than PFGE, except for some clones, and was unable to distinguish some epidemiologically unrelated isolates. Yet it distinguished all major MLST clones and therefore constitutes a rapid method to identify epidemiologically relevant clonal groups. Given its high reproducibility and high throughput, MLVA represents a very attractive first-line screening method to alleviate the PFGE workload in outbreak investigations and listeriosis surveillance. L isteriosis is a food-borne infection caused by the bacterium Listeria monocytogenes. Invasive forms of human listeriosis include septicemia, meningitis, and maternal-fetal infections (1). Listeriosis is associated with high hospitalization and fatality rates (almost 100% and 25 to 30%, respectively). Populations at risk include pregnant women, immunocompromised individuals, and the elderly. L. monocytogenes is widely present in the environment, including soil, water, vegetation, and silage, as well as in animals and animal-derived food, and can contaminate food in processing plants and retail establishments. L. monocytogenes is recognized as a public health issue and a serious challenge for the food industry, and this has led to the establishment of national surveillance systems in several countries. L. monocytogenes also stands out as a model system in the fields of microbiology, cell biology, and im-munology and for the study of host-pathogen interactions (2-5). L. monocytogenes strain characterization on the basis of sero-typing and molecular typing methods is used for surveillance, epidemiological tracking, and outbreak investigation purposes (6, 7). Genetic variants of L. monocytogenes have diversified into four major phylogenetic lineages, with lineages 1 and 2 each containing multiple clonal groups of public health importance (8-14). As these groups appear to differ in virulence and epidemic potential (6, 15), it will be interesting to better define their epidemiological, clinical, and microbiological specificities. For this purpose, tools for the easy identification of clonal groups are needed to recognize such groups and determine their presence in a large variety of sources. Several typing methods are currently available for L. monocytogenes strains. Conventional serotyping (16) and its molecular proxy PCR serogrouping (17) discriminate major categories of strains that correlate strongly (albeit not totally) with lin-eages and clones (11, 12, 14), but these methods do not have the necessary discriminatory power in the context of outbreak investigations. Pulsed-field gel electrophoresis (PFGE) is established as the gold standard for L. monocytogenes strain subtyping and is widely used for listeriosis surveillance and outbreak investigations (18). Yet, PFGE presents several practical disadvantages, as it is time-consuming and requires stringent standardization for inter-laboratory data comparison. Multilocus sequence typing (MLST) is a well-established reference method for global epidemiology and population biology (19, 20), as it renders interlaboratory genotype comparisons easy and unambiguous and as sequence data can be used to infer useful population genetic information such as amounts of genetic diversity, recombination rates, and strain phy-logeny. MLST also provides backward compatibility with genome sequencing (21). However, MLST is neither rapid nor cheap and has limited discriminatory power within L. monocytogenes (12, 22). Given the current limitations of available methods for L. monocytogenes strain typing, a potentially useful complementary approach is multilocus variable number of tandem repeat

Listeria-associated lymphadenitis: a series of 11 consecutive cases and review of the literature

International audienceWe studied 11 cases of culture-proven Listeria-associated lymphadenitis reported to the French National Reference Center for Listeria from 1994 to 2019, and 8 additional published cases. Listeria-associated lymphadenitis is rare but associated with a mortality as high as for invasive listeriosis, and frequently diagnosed with concomitant neoplasia

Listeria valentina sp. nov., isolated from a water trough and the faeces of healthy sheep

International audienceIn the context of a study on the occurrence of Listeria species in an animal farm environment in Valencia, Spain, six Listeria-like isolates could not be assigned to any known species. Phylogenetic analysis based on the 16S rRNA gene and on 231 Listeria core genes grouped these isolates in a monophyletic clade within the genus Listeria, with highest similarity to Listeria thailandensis. Whole-genome sequence analyses based on in silico DNA-DNA hybridization, the average nucleotide blast and the pairwise amino acid identities against all currently known Listeria species confirmed that these isolates constituted a new taxon within the genus Listeria. Phenotypically, these isolates differed from other Listeria species mainly by the production of acid from inositol, the absence of acidification in presence of methyl α-d-glucoside, and the absence of α-mannosidase and nitrate reductase activities. The name Listeria valentina sp. nov. is proposed for this novel species, and the type strain is CLIP 2019/00642T (=CIP 111799T=DSM 110544T)

Listeria ilorinensis sp. nov. isolated from cow milk cheese in Nigeria

International audienceDuring microbial assessment of cow milk cheese products in the city of Ilorin, Nigeria, a Listeria-like isolate was detected which could not be assigned to any known species. Whole- genome sequence analyses against all currently known 26 Listeria species confirmed that this isolate constitutes a new taxon within the Listeria genus, with highest similarity to Listeria costaricensis (average nucleotide identity BLAST of 82.66%, in silico DNA-DNA hybridization of 28.3%). Phenotypically, it differs from L. costaricensis by the inability to ferment D-sucrose, L-fucose and starch. The absence of haemolysis and Listeria pathogenic islands suggest that this novel species is not pathogenic for humans and animals. The name Listeria ilorinensis sp. nov. is proposed, with the type strain CLIP 2019/01311T (=CIP111875T = DSM 111566T