Listeria monocytogenes is a model organism for cellular microbiology and host-pathogen interaction studies and an important food-borne pathogen widespread in the environment, thus representing an attractive model to study the evolution of virulence. The phylogenetic structure of L. monocytogenes was determined by sequencing internal portions of seven housekeeping genes (3,288 nucleotides) in 360 representative isolates. Fifty-eight of the 126 disclosed sequence types were grouped into seven well-demarcated clonal complexes (clones) that comprised almost 75% of clinical isolates. Each clone had a unique or dominant serotype (4b for clones 1, 2 and 4, 1/2b for clones 3 and 5, 1/2a for clone 7, and 1/2c for clone 9), with no association of clones with clinical forms of human listeriosis. Homologous recombination was extremely limited (r/m<1 for nucleotides), implying long-term genetic stability of multilocus genotypes over time. Bayesian analysis based on 438 SNPs recovered the three previously defined lineages, plus one unclassified isolate of mixed ancestry. The phylogenetic distribution of serotypes indicated that serotype 4b evolved once from 1/2b, the likely ancestral serotype of lineage I. Serotype 1/2c derived once from 1/2a, with reference strain EGDe (1/2a) likely representing an intermediate evolutionary state. In contrast to housekeeping genes, the virulence factor internalin (InlA) evolved by localized recombination resulting in a mosaic pattern, with convergent evolution indicative of natural selection towards a truncation of InlA protein. This work provides a reference evolutionary framework for future studies on L. monocytogenes epidemiology, ecology, and virulence.

Listeria monocytogenes is worldwide a pathogen, but the geographic distribution of clones remains largely unknown. Genotyping of 300 isolates from the 5 continents and diverse sources showed the existence of few prevalent and globally distributed clones, some of which include previously described epidemic clones. Cosmopolitan distribution indicates the need for genotyping standardization.

Microbial pathogenesis studies are typically performed with reference strains, thereby overlooking within-species heterogeneity in microbial virulence. Here we integrated human epidemiological and clinical data with bacterial population genomics to harness the biodiversity of the model foodborne pathogen Listeria monocytogenes and decipher the basis of its neural and placental tropisms. Taking advantage of the clonal structure of this bacterial species, we identify clones epidemiologically associated either with food or with human central nervous system (CNS) or maternal-neonatal (MN) listeriosis. The latter clones are also most prevalent in patients without immunosuppressive comorbidities. Strikingly, CNS- and MN-associated clones are hypervirulent in a humanized mouse model of listeriosis. By integrating epidemiological data and comparative genomics, we have uncovered multiple new putative virulence factors and demonstrate experimentally the contribution of the first gene cluster mediating L. monocytogenes neural and placental tropisms. This study illustrates the exceptional power in harnessing microbial biodiversity to identify clinically relevant microbial virulence attributes.

The 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-/LLO-) mutants belonged to phylogenetically diverse clades of L. monocytogenes, 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-/LLO- mutational events in L. monocytogenes lead to niche restriction and open an evolutionary path for obligate saprophytism in this facultative intracellular pathogen.

Listeria monocytogenes (Lm) is a ubiquitous bacterium able to survive and thrive within the environment and readily colonizes a wide range of substrates, often as a biofilm. It is also a facultative intracellular pathogen, which actively invades diverse hosts and induces listeriosis. So far, these two complementary facets of Lm biology have been studied independently. Here we demonstrate that the major Lm virulence determinant ActA, a PrfA-regulated gene product enabling actin polymerization and thereby promoting its intracellular motility and cell-to-cell spread, is critical for bacterial aggregation and biofilm formation. We show that ActA mediates Lm aggregation via direct ActA-ActA interactions and that the ActA C-terminal region, which is not involved in actin polymerization, is essential for aggregation in vitro. In mice permissive to orally-acquired listeriosis, ActA-mediated Lm aggregation is not observed in infected tissues but occurs in the gut lumen. Strikingly, ActA-dependent aggregating bacteria exhibit an increased ability to persist within the cecum and colon lumen of mice, and are shed in the feces three order of magnitude more efficiently and for twice as long than bacteria unable to aggregate. In conclusion, this study identifies a novel function for ActA and illustrates that in addition to contributing to its dissemination within the host, ActA plays a key role in Lm persistence within the host and in transmission from the host back to the environment.

Listeria (L.) monocytogenes is an opportunistic pathogen causing life-threatening infections in diverse mammalian species including humans and ruminants. As little is known on the link between strains and clinicopathological phenotypes, we studied potential strain-associated virulence and organ tropism in L. monocytogenes isolates from well-defined ruminant cases of clinical infections and the farm environment. The phylogeny of isolates and their virulence-associated genes were analyzed by multilocus sequence typing (MLST) and sequence analysis of virulence-associated genes. Additionally, a panel of representative isolates was subjected to in vitro infection assays. Our data suggest the environmental exposure of ruminants to a broad range of strains and yet the strong association of sequence type (ST) 1 from clonal complex (CC) 1 with rhombencephalitis, suggesting increased neurotropism of ST1 in ruminants, which is possibly related to its hypervirulence. This study emphasizes the importance of considering clonal background of L. monocytogenes isolates in surveillance, epidemiological investigation and disease control.

Listeria monocytogenes is a life-threatening foodborne pathogen. Here, we report the genomic characterization of a nationwide dataset of 411 clinical and 82 food isolates collected in Taiwan between 2014 and 2019. The observed incidence of listeriosis increased from 0.83 to 7 cases per million population upon implementation of mandatory notification in 2018. Pregnancy-associated cases accounted for 2.8% of human listeriosis and all-cause 7-day mortality was of 11.9% in nonmaternal-neonatal listeriosis. L. monocytogenes was isolated from 90% of raw pork and 34% of chicken products collected in supermarkets. Sublineages SL87, SL5, and SL378 accounted for the majority (65%) of clinical cases. SL87 and SL378 were also predominant (57%) in food products. Five cgMLST clusters accounted for 57% clinical cases, suggesting unnoticed outbreaks spanning up to 6 years. Mandatory notification allowed identifying the magnitude of listeriosis in Taiwan. Continuous real-time genomic surveillance will allow reducing contaminating sources and disease burden. IMPORTANCE Understanding the phylogenetic relationship between clinical and food isolates is important to identify the transmission routes of foodborne diseases. Here, we performed a nationwide study between 2014 and 2019 of both clinical and food Listeria monocytogenes isolates and sequenced their genomes. We show a 9-fold increase in listeriosis reporting upon implementation of mandatory notification. We found that sublineages SL87 and SL378 predominated among both clinical (50%) and food (57%) isolates, and identified five cgMLST clusters accounting for 57% of clinical cases, suggestive of potential protracted sources of contamination over up to 6 years in Taiwan. These findings highlight that mandatory declaration is critical in identifying the burden of listeriosis, and the importance of genome sequencing for a detailed characterization of the pathogenic L. monocytogenes genotypes circulating in Asia.

Listeria monocytogenes is a Gram-positive bacterium that can be found in a broad range of environments, including soil, food, animals, and humans. L. monocytogenes can cause a foodborne disease manifesting as sepsis and meningo-encephalitis. To evaluate signals of selection within the core genome of neuroinvasive L. monocytogenes strains, we sequenced 122 L. monocytogenes strains from cerebrospinal fluid (CSF) of Dutch meningitis patients and performed a genome-wide analysis using Tajima's D and ω (dN/dS). We also evaluated the residual variation intolerance score (RVIS), a computationally less demanding methodology, to identify loci under selection. Results show that the large genetic distance between the listerial lineages influences the Tajima's D and ω (dN/dS) outcome. Within genetic lineages we detected signals of selection in 6 of 2327 loci (<1%), which were replicated in an external cohort of 105 listerial CSF isolates from France. Functions of identified loci under selection were within metabolism pathways (lmo2476, encoding aldose 1-epimerase), putative antimicrobial resistance mechanisms (lmo1855, encoding PBPD3), and virulence factors (lmo0549, internalin-like protein; lmo1482, encoding comEC). RVIS over the two genetic lineages showed signals of selection in internalin-like proteins loci potentially involved in pathogen-host interaction (lmo0549, lmo0610, and lmo1290). Our results show that RVIS can be used to detect bacterial loci under selection.

During 2015-2016, we evaluated the performance of whole-genome sequencing (WGS) as a routine typing tool. Its added value for microbiological and epidemiologic surveillance of listeriosis was compared with that for pulsed-field gel electrophoresis (PFGE), the current standard method. A total of 2,743 Listeria monocytogenes isolates collected as part of routine surveillance were characterized in parallel by PFGE and core genome multilocus sequence typing (cgMLST) extracted from WGS. We investigated PFGE and cgMLST clusters containing human isolates. Discrimination of isolates was significantly higher by cgMLST than by PFGE (p<0.001). cgMLST discriminated unrelated isolates that shared identical PFGE profiles and phylogenetically closely related isolates with distinct PFGE profiles. This procedure also refined epidemiologic investigations to include only phylogenetically closely related isolates, improved source identification, and facilitated epidemiologic investigations, enabling identification of more outbreaks at earlier stages. WGS-based typing should replace PFGE as the primary typing method for L. monocytogenes.

Listeria genus comprises two pathogenic species, L. monocytogenes (Lm) and L. ivanovii, and non-pathogenic species. All can thrive as saprophytes, whereas only pathogenic species cause systemic infections. Identifying Listeria species' respective biotopes is critical to understand the ecological contribution of Listeria virulence. In order to investigate the prevalence and abundance of Listeria species in various sources, we retrieved and analyzed 16S rRNA datasets from MG-RAST metagenomic database. 26% of datasets contain Listeria sensu stricto sequences, and Lm is the most prevalent species, most abundant in soil and host-associated environments, including 5% of human stools. Lm is also detected in 10% of human stool samples from an independent cohort of 900 healthy asymptomatic donors. A specific microbiota signature is associated with Lm faecal carriage, both in humans and experimentally inoculated mice, in which it precedes Lm faecal carriage. These results indicate that Lm faecal carriage is common and depends on the gut microbiota, and suggest that Lm faecal carriage is a crucial yet overlooked consequence of its virulence.

Invasion of nonphagocytic cells, a critical property of Listeria monocytogenes (Lm) that enables it to cross host barriers, is mediated by the interaction of two bacterial surface proteins, InlA and InlB, with their respective receptors E-cadherin and c-Met. Although InlA-E-cadherin interaction is necessary and sufficient for Lm crossing of the intestinal barrier, both InlA and InlB are required for Lm crossing of the placental barrier. The mechanisms underlying these differences are unknown. Phosphoinositide 3-kinase (PI3-K) is involved in both InlA- and InlB-dependent pathways. Indeed, InlA-dependent entry requires PI3-K activity but does not activate it, whereas InlB-c-Met interaction activates PI3-K. We show that Lm intestinal target cells exhibit a constitutive PI3-K activity, rendering InlB dispensable for InlA-dependent Lm intestinal barrier crossing. In contrast, the placental barrier does not exhibit constitutive PI3-K activity, making InlB necessary for InlA-dependent Lm placental invasion. Here, we provide the molecular explanation for the respective contributions of InlA and InlB to Lm host barrier invasion, and reveal the critical role of InlB in rendering cells permissive to InlA-mediated invasion. This study shows that PI3-K activity is critical to host barrier permissiveness to microbes, and that pathogens exploit both similarities and differences of host barriers to disseminate.

Listeriosis is a severe foodborne infection caused by Listeria monocytogenes, an important foodborne pathogen of animals and humans. Listeriosis is a rare disease in cats.

Understanding the role of the microbiota components in either preventing or favoring enteric infections is critical. Here, we report the discovery of a Listeria bacteriocin, Lmo2776, which limits Listeria intestinal colonization. Oral infection of conventional mice with a Δlmo2776 mutant leads to a thinner intestinal mucus layer and higher Listeria loads both in the intestinal content and deeper tissues compared to WT Listeria. This latter difference is microbiota dependent, as it is not observed in germ-free mice. Strikingly, it is phenocopied by pre-colonization of germ-free mice before Listeria infection with Prevotella copri, an abundant gut-commensal bacteria, but not with the other commensals tested. We further show that Lmo2776 targets P. copri and reduces its abundance. Together, these data unveil a role for P.copri in exacerbating intestinal infection, highlighting that pathogens such as Listeria may selectively deplete microbiota bacterial species to avoid excessive inflammation.

At present, little is known regarding Listeria monocytogenes-associated biliary tract infection, a rare form of listeriosis.In this article, we will study 12 culture-proven cases reported to the French National Reference Center for Listeria from 1996 to 2013 and review the 8 previously published cases.Twenty cases were studied: 17 cholecystitis, 2 cholangitis, and 1 biliary cyst infection. Half were men with a median age of 69 years (32-85). Comorbidities were present in 80%, including cirrhosis, rheumatoid arthritis, and diabetes. Five patients received immunosuppressive therapy, including corticosteroids and anti-tumor necrosis factor biotherapies. Half were afebrile. Blood cultures were positive in 60% (3/5). Gallbladder histological lesions were analyzed in 3 patients and evidenced acute, chronic, or necrotic exacerbation of chronic infection. Genoserogroup of the 12 available strains were IVb (n=6), IIb (n=5), and IIa (n=1). Their survival in the bile was not enhanced when compared with isolates from other listeriosis cases. Adverse outcome was reported in 33% (5/15): 3 deaths, 1 recurrence; 75% of the patients with adverse outcome received inadequate antimicrobial therapy (P=0.033).Biliary tract listeriosis is a severe infection associated with high mortality in patients not treated with appropriate therapy. This study provides medical relevance to in vitro and animal studies that had shown Listeria monocytogenes ability to survive in bile and induce overt biliary infections.

Listeria monocytogenes (Lm) is an invasive foodborne pathogen that leads to severe central nervous system and maternal-fetal infections. Lm ability to actively cross the intestinal barrier is one of its key pathogenic properties. Lm crosses the intestinal epithelium upon the interaction of its surface protein internalin (InlA) with its host receptor E-cadherin (Ecad). InlA-Ecad interaction is species-specific, does not occur in wild-type mice, but does in transgenic mice expressing human Ecad and knock-in mice expressing humanized mouse Ecad. To study listeriosis in wild-type mice, InlA has been "murinized" to interact with mouse Ecad. Here, we demonstrate that, unexpectedly, murinized InlA (InlA(m)) mediates not only Ecad-dependent internalization, but also N-cadherin-dependent internalization. Consequently, InlA(m)-expressing Lm targets not only goblet cells expressing luminally-accessible Ecad, as does Lm in humanized mice, but also targets villous M cells, which express luminally-accessible N-cadherin. This aberrant Lm portal of entry results in enhanced innate immune responses and intestinal barrier damage, both of which are not observed in wild-type Lm-infected humanized mice. Murinization of InlA therefore not only extends the host range of Lm, but also broadens its receptor repertoire, providing Lm with artifactual pathogenic properties. These results challenge the relevance of using InlA(m)-expressing Lm to study human listeriosis and in vivo host responses to this human pathogen.

Listeria 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.

Listeria monocytogenes (Lm) is a foodborne pathogen that crosses the intestinal barrier upon interaction between its surface protein InlA and its species-specific host receptor E-cadherin (Ecad). Ecad, the key constituent of adherens junctions, is typically situated below tight junctions and therefore considered inaccessible from the intestinal lumen. In this study, we investigated how Lm specifically targets its receptor on intestinal villi and crosses the intestinal epithelium to disseminate systemically. We demonstrate that Ecad is luminally accessible around mucus-expelling goblet cells (GCs), around extruding enterocytes at the tip and lateral sides of villi, and in villus epithelial folds. We show that upon preferential adherence to accessible Ecad on GCs, Lm is internalized, rapidly transcytosed across the intestinal epithelium, and released in the lamina propria by exocytosis from where it disseminates systemically. Together, these results show that Lm exploits intrinsic tissue heterogeneity to access its receptor and reveal transcytosis as a novel and unanticipated pathway that is hijacked by Lm to breach the intestinal epithelium and cause systemic infection.

Listeriolysin S (LLS) is a thiazole/oxazole-modified microcin (TOMM) produced by hypervirulent clones of Listeria monocytogenes LLS targets specific gram-positive bacteria and modulates the host intestinal microbiota composition. To characterize the mechanism of LLS transfer to target bacteria and its bactericidal function, we first investigated its subcellular distribution in LLS-producer bacteria. Using subcellular fractionation assays, transmission electron microscopy, and single-molecule superresolution microscopy, we identified that LLS remains associated with the bacterial cell membrane and cytoplasm and is not secreted to the bacterial extracellular space. Only living LLS-producer bacteria (and not purified LLS-positive bacterial membranes) display bactericidal activity. Applying transwell coculture systems and microfluidic-coupled microscopy, we determined that LLS requires direct contact between LLS-producer and -target bacteria in order to display bactericidal activity, and thus behaves as a contact-dependent bacteriocin. Contact-dependent exposure to LLS leads to permeabilization/depolarization of the target bacterial cell membrane and adenosine triphosphate (ATP) release. Additionally, we show that lipoteichoic acids (LTAs) can interact with LLS and that LTA decorations influence bacterial susceptibility to LLS. Overall, our results suggest that LLS is a TOMM that displays a contact-dependent inhibition mechanism.

Listeria monocytogenes is one of the major foodborne pathogens. Isolates of PCR-serogroups IIb (n = 17) and IVb (n = 31) recovered from food (n = 33) and food processing environment (n = 15) in Poland were characterized using whole genome sequencing. Most isolates belonged to Multi-Locus Sequence Type (MLST) ST2 (31.3%) and ST5 (22.9%). Core genome MLST (cgMLST) analysis classified isolates into seven sublineages (SL) and 25 different cgMLST types (CT). Consistent with the MLST results, most sublineages were SL2 and SL5. Eleven isolates harbored aacA4 encoding resistance to aminoglycosides, three isolates harbored emrC (n = 3) and one brcABC (n = 1) encoding tolerance to benzalkonium chloride. Isolates belonging to SL5 CT2323 carried a so far unreported inlB allele with a deletion of 141 nucleotides encoding the β-repeat sheet and partially the GW1 domain of InlB. Comparison with publicly available genome sequences from L. monocytogenes isolated from human listeriosis cases in Poland from 2004 to 2013 revealed five common CTs, suggesting a possible epidemiological link with these strains. The present study contributes to characterize the diversity of L. monocytogenes in ready-to-eat (RTE) meat and meat processing environments in Poland and unravels previously unnoticed links with clinical cases in Europe.

For nearly 3 decades, listeriologists and immunologists have used mainly three strains of the same serovar (1/2a) to analyze the virulence of the bacterial pathogen Listeria monocytogenes. The genomes of two of these strains, EGD-e and 10403S, were released in 2001 and 2008, respectively. Here we report the genome sequence of the third reference strain, EGD, and extensive genomic and phenotypic comparisons of the three strains. Strikingly, EGD-e is genetically highly distinct from EGD (29,016 single nucleotide polymorphisms [SNPs]) and 10403S (30,296 SNPs), and is more related to serovar 1/2c than 1/2a strains. We also found that while EGD and 10403S strains are genetically very close (317 SNPs), EGD has a point mutation in the transcriptional regulator PrfA (PrfA*), leading to constitutive expression of several major virulence genes. We generated an EGD-e PrfA* mutant and showed that EGD behaves like this strain in vitro, with slower growth in broth and higher invasiveness in human cells than those of EGD-e and 10403S. In contrast, bacterial counts in blood, liver, and spleen during infection in mice revealed that EGD and 10403S are less virulent than EGD-e, which is itself less virulent than EGD-e PrfA*. Thus, constitutive expression of PrfA-regulated virulence genes does not appear to provide a significant advantage to the EGD strain during infection in vivo, highlighting the fact that in vitro invasion assays are not sufficient for evaluating the pathogenic potential of L. monocytogenes strains. Together, our results pave the way for deciphering unexplained differences or discrepancies in experiments using different L. monocytogenes strains. IMPORTANCE Over the past 3 decades, Listeria has become a model organism for host-pathogen interactions, leading to critical discoveries in a broad range of fields, including bacterial gene regulation, cell biology, and bacterial pathophysiology. Scientists studying Listeria use primarily three pathogenic strains: EGD, EGD-e, and 10403S. Despite many studies on EGD, it is the only one of the three strains whose genome has not been sequenced. Here we report the sequence of its genome and a series of important genomic and phenotypic differences between the three strains, in particular, a critical mutation in EGD's PrfA, the main regulator of Listeria virulence. Our results show that the three strains display differences which may play an important role in the virulence differences observed between the strains. Our findings will be of critical relevance to listeriologists and immunologists who have used or may use Listeria as a tool to study the pathophysiology of listeriosis and immune responses.