Bacteriocins attenuate Listeria monocytogenes-induced intestinal barrier dysfunction and inflammatory response.

Bacteriocins have the potential to effectively improve food-borne infections or gastrointestinal diseases and hold promise as viable alternatives to antibiotics. This study aimed to explore the antibacterial activity of three bacteriocins (nisin, enterocin Gr17, and plantaricin RX-8) and their ability to attenuate intestinal barrier dysfunction and inflammatory responses induced by Listeria monocytogenes, respectively. Bacteriocins have shown excellent antibacterial activity against L. monocytogenes without causing any cytotoxicity. Bacteriocins inhibited the adhesion and invasion of L. monocytogenes on Caco-2 cells, lactate dehydrogenase (LDH), trans-epithelial electrical resistance (TEER), and cell migration showed that bacteriocin improved the permeability of Caco-2 cells. These results were attributed to the promotion of tight junction proteins (TJP) assembly, specifically zonula occludens-1 (ZO-1), occludin, and claudin-1. Furthermore, bacteriocins could alleviate inflammation by inhibiting the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways and reducing the secretion of interleukin-6 (IL-6), interleukin-1 ß (IL-1ß) and tumor necrosis factor α (TNF-α). Among three bacteriocins, plantaricin RX-8 showed the best antibacterial activity against L. monocytogenes and the most pronounced protective effect on the intestinal barrier due to its unique structure. Based on our findings, we hypothesized that bacteriocins may inhibit the adhesion and invasion of L. monocytogenes by competing adhesion sites. Moreover, they may further enhance intestinal barrier function by inhibiting the expression of L. monocytogenes virulence factors, increasing the expression of TJP and decreasing the secretion of inflammatory factors. Therefore, bacteriocins will hopefully be an effective alternative to antibiotics, and this study provides valuable insights into food safety concerns. KEY POINTS: • Bacteriocins show excellent antibacterial activity against L. monocytogenes • Bacteriocins improve intestinal barrier damage and inflammatory response • Plantaricin RX-8 has the best protective effect on Caco-2 cells damage.

The rapidly progressing and fatal outcome of rhombencephalitis by listeriosis in a 61-year-old male.

Listeria monocytogenes is a Gram-positive facultative anaerobic bacterium that is ubiquitous in the environment and can cause severe infections in immunocompromised individuals, pregnant women, and newborns. Listeriosis can manifest as meningitis, encephalitis, or sepsis, and its diagnosis requires a high index of suspicion. The case is reported of a rare presentation of rhombencephalitis by listeriosis in a 61-year-old male who initially suffered from subacute gastric disturbances and fever. Neurological consultation showed abnormal functions of cranial nerves and meningeal signs were observed. MRI revealed a poorly demarcated focus of approximately 45 × 16 × 15mm, indicating possible inflammatory processes, necessitating a lumbar puncture. Assessment of the CSF indicated infection with the bacterium- Listeria Monocytogenes, with the final diagnosis of Listeriosis encephalitis. Despite antibiotic therapy of Ceftazidine and Ampicillin, the patient's condition deteriorated, followed by death.

The mitochondrial carboxylase PCCA interacts with Listeria monocytogenes phospholipase PlcB to modulate bacterial survival.

Listeria monocytogenes, a prominent foodborne pathogen responsible for zoonotic infections, owes a significant portion of its virulence to the presence of the phospholipase PlcB. In this study, we performed an in-depth examination of the intricate relationship between L. monocytogenes PlcB and host cell mitochondria, unveiling a novel participant in bacterial survival: the mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA). Our investigation uncovered previously unexplored levels of interaction and colocalization between PCCA and PlcB within host cells, with particular emphasis on the amino acids 504-508 of PCCA, which play a pivotal role in this partnership. To assess the effect of PCCA expression on L. monocytogenes proliferation, PCCA expression levels were manipulated by siRNA-si-PCCA or pCMV-N-HA-PCCA plasmid transfection. Our findings demonstrated a clear inverse correlation between PCCA expression levels and the proliferation of L. monocytogenes. Furthermore, the effect of L. monocytogenes infection on PCCA expression was investigated by assessing PCCA mRNA and protein expression in HeLa cells infected with L. monocytogenes. These results indicate that L. monocytogenes infection did not significantly alter PCCA expression. These findings led us to propose that PCCA represents a novel participant in L. monocytogenes survival, and its abundance has a detrimental impact on bacterial proliferation. This suggests that L. monocytogenes may employ PlcB-PCCA interactions to maintain stable PCCA expression, representing a unique pro-survival strategy distinct from that of other intracellular bacterial pathogens. IMPORTANCE: Mitochondria represent attractive targets for pathogenic bacteria seeking to modulate host cellular processes to promote their survival and replication. Our current study has uncovered mitochondrial carboxylase propionyl-coenzyme A carboxylase (PCCA) as a novel host cell protein that interacts with L. monocytogenes PlcB. The results demonstrate that PCCA plays a negative regulatory role in L. monocytogenes infection, as heightened PCCA levels are associated with reduced bacterial survival and persistence. However, L. monocytogenes may exploit the PlcB-PCCA interaction to maintain stable PCCA expression and establish a favorable intracellular milieu for bacterial infection. Our findings shed new light on the intricate interplay between bacterial pathogens and host cell mitochondria, while also highlighting the potential of mitochondrial metabolic enzymes as antimicrobial agents.

Designing a Multiepitope Vaccine against the Foodborne Pathogenic Bacteria Listeria monocytogenes Using Subtractive Immunoinformatics Approaches.

BACKGROUND: Listeria monocytogenes, a Gram-positive bacterium, is a prominent foodborne pathogen that causes listeriosis and poses substantial health hazards worldwide. The continuing risk of listeriosis outbreaks underlies the importance of designing an effective prevention strategy and developing a robust immune response by reverse vaccinology approaches. This study aimed to provide a critical approach for developing a potent multiepitope vaccine against this foodborne disease. METHODS: A chimeric peptide construct containing 5 B-cell epitopes, 16 major histocompatibility complex I (MHC-I) epitopes, and 18 MHC-II epitopes were used to create a subunit vaccination against L. monocytogenes. The vaccine safety was evaluated by several online methods, and molecular docking was performed using ClusPro to determine the binding affinity. Immune simulation was performed using the C-ImmSimm server to demonstrate the immune response. RESULTS: The results validated the antigenicity, non-allergenicity, and nontoxicity of the chimeric peptide construct, confirming its suitability as a subunit vaccine. Molecular docking showed a good score of 1276.5 and molecular dynamics simulations confirmed the construct's efficacy, demonstrating its promise as a good candidate for listeriosis prophylaxis. The population coverage was as high as 91.04% with a good immune response, indicating good antigen presentation with dendritic cells and production of memory cells. CONCLUSIONS: The findings of this study highlight the potential of the designed chimeric peptide construct as an effective subunit vaccine against Listeria, paving the way for future advances in preventive methods and vaccine design.

The Evaluation of a Lateral Flow Strip Based on the Covalently Fixed "End-On" Orientation of an Antibody for Listeria monocytogenes Detection.

In this study, the covalently fixed "end-on" orientation of a monoclonal Listeria monocytogenes antibody (mAb-Lis) to amino terminated oligo (ethylene glycol)-capped gold nanoparticles (NH2-TEG-AuNPs) was used to fabricate an in-house lateral flow strip (LFS), namely, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS. The aim was to evaluate the performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS in detecting L. monocytogenes. The proposed LFS enabled the sensitive detection of L. monocytogenes in 15 min with a visual limit of detection of 102 CFU/mL. Quantitative analysis indicated an LOD at 10 CFU/mL. The fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS showed no cross-reactivity with other pathogenic bacteria and practical performance across different food matrices, including human blood, milk, and mushroom samples. Furthermore, the clinical performance of the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS for detecting L. monocytogenes was evaluated by using 12 clinical samples validated by the hemoculture method. It demonstrated excellent concordance with the reference methods, with no false-positive or false-negative results observed. Therefore, the fixed "end-on" Lis-mAb-NH-TEG-AuNPs LFS serves as a promising candidate for a point-of-care test (POCT), enabling the rapid, precise, and highly sensitive detection of L. monocytogenes in clinical samples and contaminated food.

Listeria brain abscess: a therapeutically challenging rare presentation of listeriosis.

We report a very rare case of Listeria multiple brain abscesses manifested as delirium, which represented diagnostic and therapeutic challenges overcome only by the close cooperation between Infectious Diseases and Neuroradiology, without which a satisfactory outcome would not be achieved.An elderly man presented with confusion and drowsiness with a background of type-II diabetes mellitus. Although computed tomography of the brain only showed frontal lobe oedema, contrast magnetic resonance (MR) imaging showed numerous irregular rim-enhancing lesions containing central diffusion restriction, suggesting multiple pyogenic cerebral abscesses of unclear aetiology. Thereafter, Listeria monocytogenes was isolated from blood cultures, suggesting this as the causative organism. Deemed unsuitable for neurosurgical drainage, the patient received medical management with a protracted course of antibiotics. This case was extremely challenging, due to 1) the impossibility of source control, 2) the small number of effective antibiotics available to treat this condition, and 3) the inevitable antibiotic side-effects, derived from long-term exposure. A successful outcome was only possible thanks to strict close multidisciplinary follow up, requiring frequent MR imaging and a judicious antibiotic choice, including monitoring of their side-effects. Due to the rarity of this condition, there is lack of guidance on its management, hence the importance of multidisciplinary involvement with very close imaging and antibiotic monitoring.

Outbreak of Listeria monocytogenes in hospital linked to a fava bean product, Finland, 2015 to 2019.

Listeria monocytogenes (Lm) is a bacterium widely distributed in the environment. Listeriosis is a severe disease associated with high hospitalisation and mortality rates. In April 2019, listeriosis was diagnosed in two hospital patients in Finland. We conducted a descriptive study to identify the source of the infection and defined a case as a person with a laboratory-confirmed Lm serogroup IIa sequence type (ST) 37. Six cases with Lm ST 37 were notified to the Finnish Infectious Diseases Registry between 2015 and 2019. Patient interviews and hospital menus were used to target traceback investigation of the implicated foods. In 2021 and 2022, similar Lm ST 37 was detected from samples of a ready-to-eat plant-based food product including fava beans. Inspections by the manufacturer and the local food control authority indicated that the food products were contaminated with Lm after pasteurisation. Our investigation highlights the importance that companies producing plant-based food are subject to similar controls as those producing food of animal origin. Hospital menus can be a useful source of information that is not dependent on patient recall.

Role of ethanolamine utilization and bacterial microcompartment formation in Listeria monocytogenes intracellular infection.

Ethanolamine (EA) affects the colonization and pathogenicity of certain human bacterial pathogens in the gastrointestinal tract. However, EA can also affect the intracellular survival and replication of host cell invasive bacteria such as Listeria monocytogenes (LMO) and Salmonella enterica serovar Typhimurium (S. Typhimurium). The EA utilization (eut) genes can be categorized as regulatory, enzymatic, or structural, and previous work in LMO showed that loss of genes encoding functions for the enzymatic breakdown of EA inhibited LMO intracellular replication. In this work, we sought to further characterize the role of EA utilization during LMO infection of host cells. Unlike what was previously observed for S. Typhimurium, in LMO, an EA regulator mutant (ΔeutV) was equally deficient in intracellular replication compared to an EA metabolism mutant (ΔeutB), and this was consistent across Caco-2, RAW 264.7, and THP-1 cell lines. The structural genes encode proteins that self-assemble into bacterial microcompartments (BMCs) that encase the enzymes necessary for EA metabolism. For the first time, native EUT BMCs were fluorescently tagged, and EUT BMC formation was observed in vitro and in vivo. Interestingly, BMC formation was observed in bacteria infecting Caco-2 cells, but not the macrophage cell lines. Finally, the cellular immune response of Caco-2 cells to infection with eut mutants was examined, and it was discovered that ΔeutB and ΔeutV mutants similarly elevated the expression of inflammatory cytokines. In conclusion, EA sensing and utilization during LMO intracellular infection are important for optimal LMO replication and immune evasion but are not always concomitant with BMC formation.IMPORTANCEListeria monocytogenes (LMO) is a bacterial pathogen that can cause severe disease in immunocompromised individuals when consumed in contaminated food. It can replicate inside of mammalian cells, escaping detection by the immune system. Therefore, understanding the features of this human pathogen that contribute to its infectiousness and intracellular lifestyle is important. In this work we demonstrate that genes encoding both regulators and enzymes of EA metabolism are important for optimal growth inside mammalian cells. Moreover, the formation of specialized compartments to enable EA metabolism were visualized by tagging with a fluorescent protein and found to form when LMO infects some mammalian cell types, but not others. Interestingly, the formation of the compartments was associated with features consistent with an early stage of the intracellular infection. By characterizing bacterial metabolic pathways that contribute to survival in host environments, we hope to positively impact knowledge and facilitate new treatment strategies.

13C metabolite tracing reveals glutamine and acetate as critical in vivo fuels for CD8 T cells.

Infusion of 13C-labeled metabolites provides a gold standard for understanding the metabolic processes used by T cells during immune responses in vivo. Through infusion of 13C-labeled metabolites (glucose, glutamine, and acetate) in Listeria monocytogenes-infected mice, we demonstrate that CD8 T effector (Teff) cells use metabolites for specific pathways during specific phases of activation. Highly proliferative early Teff cells in vivo shunt glucose primarily toward nucleotide synthesis and leverage glutamine anaplerosis in the tricarboxylic acid (TCA) cycle to support adenosine triphosphate and de novo pyrimidine synthesis. In addition, early Teff cells rely on glutamic-oxaloacetic transaminase 1 (Got1)-which regulates de novo aspartate synthesis-for effector cell expansion in vivo. CD8 Teff cells change fuel preference over the course of infection, switching from glutamine- to acetate-dependent TCA cycle metabolism late in infection. This study provides insights into the dynamics of Teff metabolism, illuminating distinct pathways of fuel consumption associated with CD8 Teff cell function in vivo.

Impaired meningeal lymphatic drainage in Listeria monocytogenes infection.

Previous studies have demonstrated an association between lymphatic vessels and diseases caused by bacterial infections. Listeria monocytogenes (LM) bacterial infection can affect multiple organs, including the intestine, brain, liver and spleen, which can be fatal. However, the impacts of LM infection on morphological and functional changes of lymphatic vessels remain unexplored. In this study, we found that LM infection not only induces meningeal and mesenteric lymphangiogenesis in mice, but also impairs meningeal lymphatic vessels (MLVs)-mediated macromolecules drainage. Interestingly, we found that the genes associated with lymphatic vessel development and function, such as Gata2 and Foxc2, were downregulated, suggesting that LM infection may affect cellular polarization and valve development. On the other hand, photodynamic ablation of MLVs exacerbated inflammation and bacterial load in the brain of mice with LM infection. Overall, our findings indicate that LM infection induces lymphangiogenesis and may affect cell polarization, cavity formation, and valve development during lymphangiogenesis, ultimately impairing MLVs drainage.

Temporal and spatial dynamics of Listeria monocytogenes central nervous system infection in mice.

Listeria monocytogenes is a bacterial pathogen that can cause life-threatening central nervous system (CNS) infections. While mechanisms by which L. monocytogenes and other pathogens traffic to the brain have been studied, a quantitative understanding of the underlying dynamics of colonization and replication within the brain is still lacking. In this study, we used barcoded L. monocytogenes to quantify the bottlenecks and dissemination patterns that lead to cerebral infection. Following intravenous (IV) inoculation, multiple independent invasion events seeded all parts of the CNS from the blood, however, only one clone usually became dominant in the brain. Sequential IV inoculations and intracranial inoculations suggested that clones that had a temporal advantage (i.e., seeded the CNS first), rather than a spatial advantage (i.e., invaded a particular brain region), were the main drivers of clonal dominance. In a foodborne model of cerebral infection with immunocompromised mice, rare invasion events instead led to a highly infected yet monoclonal CNS. This restrictive bottleneck likely arose from pathogen transit into the blood, rather than directly from the blood to the brain. Collectively, our findings provide a detailed quantitative understanding of the L. monocytogenes population dynamics that lead to CNS infection and a framework for studying the dynamics of other cerebral infections.

Phenotypic and genotypic antimicrobial resistance of Listeria spp. in Spain.

Listeriosis is a zoonotic disease caused by Listeria monocytogenes and Listeria ivanovii. The genus Listeria currently includes 27 recognized species and is found throughout the environment. The number of systematic studies on antimicrobial resistance in L. monocytogenes isolates from domestic farms using antimicrobial substances is limited. Importantly, dairy ruminant farms are reservoir of hypervirulent lineage I L. monocytogenes isolates, previously associated with human clinical cases. Considering that the classes of antibiotics used in food-producing domestic animals are frequently the same or closely related to those used in human medicine, studies about the impact of antibiotic use on the acquisition of antibiotic resistance in Listeria spp. in domestic animal farms are, therefore, of high importance. Here, susceptibility to 25 antibiotics was determined. Eighty-one animal-related, 35 food and 21 human pathogenic Listeria spp. isolates and 114 animal-related non-pathogenic Listeria spp. isolates were tested. Whole genome sequencing data was used for molecular characterization. Regarding L. monocytogenes, 2 strains from the clinical-associated linage I showed resistance to erythromycin, both related to dairy ruminants. Acquired resistance to one antibiotic was exhibited in 1.5% of L. monocytogenes isolates compared with 14% of non-pathogenic Listeria spp. isolates. Resistance to tetracycline (7.9%), doxycycline (7.9%), penicillin (4.4%), and ampicillin (4.4%) were the most frequently observed in non-pathogenic Listeria spp. While resistance to two or more antibiotics (5.6%) was most common in Listeria spp., isolates, resistance to one antibiotic was also observed (1.6%). The present results show that non-pathogenic Listeria spp. harbour antimicrobial resistance genes.

Listeria monocytogenes-associated spontaneous bacterial peritonitis in France: a nationwide observational study of 208 cases.

BACKGROUND: Listeriosis is a foodborne infection caused by Listeria monocytogenes. Three main forms of listeriosis are well characterised, but little is known about L monocytogenes-associated spontaneous bacterial peritonitis. We used data from the French national surveillance of listeriosis to perform a nationwide retrospective study. METHODS: All patients with L monocytogenes isolated by culture from a peritoneal fluid sample in France between April 1, 1993, and Dec 31, 2022, were included. Individuals for whom bacterial peritonitis was not confirmed and those who also had another type of invasive listeriosis were excluded. A standardised checklist was used to collect demographic, clinical, and biological data as well as antibiotic treatment and follow-up data. The primary outcome was to determine the characteristics of L monocytogenes-associated spontaneous bacterial peritonitis. We did descriptive analyses and assessed risk factors for 1-month mortality using an exploratory multivariable Cox model analysis. FINDINGS: Among the 8768 L monocytogenes cases reported, 208 (2%) were patients with L monocytogenes-associated spontaneous bacterial peritonitis. Mean age was 65 years (SD 13), 50 (24%) of 208 patients were female, and 158 (76%) were male (no data on race or ethnicity were available). 200 (98%) of 205 patients with L monocytogenes-associated spontaneous bacterial peritonitis with available data had immunosuppressive comorbidities, including cirrhosis (148 [74%] of 201 with available data), ongoing alcoholism (58 [62%] of 94), and ongoing neoplasia (60 [31%] of 195). Causes of ascites included cirrhosis (146 [70%] of 208), ongoing neoplasia (26 [13%]), end-stage heart failure (13 [6%]), and peritoneal dialysis (11 [5%]). Among those with available data, presentation was pauci-symptomatic and non-specific; only 67 (50%) of 135 patients presented with fever, 49 (37%) of 132 with abdominal pain, and 27 (21%) of 129 with diarrhoea. 61 (29%) of 208 patients were dead at 1 month, 92 (44%) were dead at 3 months, and 109 (52%) were dead at 6 months after diagnosis. Ongoing neoplasia (hazard ratio 2·42 [95% CI 1·05-5·56]; p=0·039), septic shock (8·03 [2·66-24·02]; p=0·0021), and high blood leukocyte count (1·05 [1·00-1·09]; p=0·045) were independently associated with 1-month mortality. INTERPRETATION: Despite the non-specific and mild presentation of L monocytogenes-associated spontaneous bacterial peritonitis, the outcome is poor and similar to that of neurolisteriosis, and so identification of L monocytogenes in ascitic fluid samples requires urgent parenteral amoxicillin-based treatment to avoid a fatal outcome. FUNDING: Institut Pasteur, Inserm, and French Public Health Agency. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Closing the gap: Oxford Nanopore Technologies R10 sequencing allows comparable results to Illumina sequencing for SNP-based outbreak investigation of bacterial pathogens.

Whole-genome sequencing has become the method of choice for bacterial outbreak investigation, with most clinical and public health laboratories currently routinely using short-read Illumina sequencing. Recently, long-read Oxford Nanopore Technologies (ONT) sequencing has gained prominence and may offer advantages over short-read sequencing, particularly with the recent introduction of the R10 chemistry, which promises much lower error rates than the R9 chemistry. However, limited information is available on its performance for bacterial single-nucleotide polymorphism (SNP)-based outbreak investigation. We present an open-source workflow, Prokaryotic Awesome variant Calling Utility (PACU) (https://github.com/BioinformaticsPlatformWIV-ISP/PACU), for constructing SNP phylogenies using Illumina and/or ONT R9/R10 sequencing data. The workflow was evaluated using outbreak data sets of Shiga toxin-producing Escherichia coli and Listeria monocytogenes by comparing ONT R9 and R10 with Illumina data. The performance of each sequencing technology was evaluated not only separately but also by integrating samples sequenced by different technologies/chemistries into the same phylogenomic analysis. Additionally, the minimum sequencing time required to obtain accurate phylogenetic results using nanopore sequencing was evaluated. PACU allowed accurate identification of outbreak clusters for both species using all technologies/chemistries, but ONT R9 results deviated slightly more from the Illumina results. ONT R10 results showed trends very similar to Illumina, and we found that integrating data sets sequenced by either Illumina or ONT R10 for different isolates into the same analysis produced stable and highly accurate phylogenomic results. The resulting phylogenies for these two outbreaks stabilized after ~20 hours of sequencing for ONT R9 and ~8 hours for ONT R10. This study provides a proof of concept for using ONT R10, either in isolation or in combination with Illumina, for rapid and accurate bacterial SNP-based outbreak investigation.

Cross-regulation of Listeria monocytogenes and the host ubiquitin system in listeriosis.

The facultative intracellular bacterium Listeria (L.) monocytogenes may cause severe diseases in humans and animals. The control of listeriosis/L. monocytogenes requires the concerted action of cells of the innate and adaptive immune systems. In this regard, cell-intrinsic immunity of infected cells, activated by the immune responses, is crucial for the control and elimination intracellular L. monocytogenes. Both the immune response against L. monocytogenes and cell intrinsic pathogen control are critically regulated by post-translational modifications exerted by the host ubiquitin system and ubiquitin-like modifiers (Ubls). In this review, we discuss our current understanding of the role of the ubiquitin system and Ubls in listeriosis, as well as future directions of research.

Listeria monocytogenes: the silent assassin.

Listeriosis is a foodborne infection in humans caused by Listeria monocytogenes. Consumption of contaminated food can lead to severe infection in vulnerable patients, that can be fatal. Clinical manifestations include sepsis and meningitis, and in pregnancy-associated infection, miscarriage and stillbirth. Diagnosis is confirmed by culture and identification of the pathogen from blood, cerebrospinal fluid, vaginal swab, placenta or amniotic fluid. Treatment regimens recommend amoxicillin, ampicillin or an aminoglycoside. Virulence factors mediate bacterial adhesion and invasion of gut epithelial cells. Other factors mediate biofilm formation and tolerance to low temperatures and high salt concentrations facilitating persistence and survival in the environment.

Lymph node stromal cells vary in susceptibility to infection but can support the intracellular growth of Listeria monocytogenes.

Lymph node stromal cells (LNSCs) are an often overlooked component of the immune system but play a crucial role in maintaining tissue homeostasis and orchestrating immune responses. Our understanding of the functions these cells serve in the context of bacterial infections remains limited. We previously showed that Listeria monocytogenes, a facultative intracellular foodborne bacterial pathogen, must replicate within an as-yet-unidentified cell type in the mesenteric lymph node (MLN) to spread systemically. Here, we show that L. monocytogenes could invade, escape from the vacuole, replicate exponentially, and induce a type I interferon response in the cytosol of 2 LNSC populations infected in vitro, fibroblastic reticular cells (FRCs) and blood endothelial cells (BECs). Infected FRCs and BECs also produced a significant chemokine and proinflammatory cytokine response after in vitro infection. Flow cytometric analysis confirmed that GFP+  L. monocytogenes were associated with a small percentage of MLN stromal cells in vivo following foodborne infection of mice. Using fluorescent microscopy, we showed that these cell-associated bacteria were intracellular L. monocytogenes and that the number of infected FRCs and BECs changed over the course of a 3-day infection in mice. Ex vivo culturing of these infected LNSC populations revealed viable, replicating bacteria that grew on agar plates. These results highlight the unexplored potential of FRCs and BECs to serve as suitable growth niches for L. monocytogenes during foodborne infection and to contribute to the proinflammatory environment within the MLN that promotes clearance of listeriosis.

Molecular Characteristics and Virulence Profile of Clinical Listeria monocytogenes Isolates in Northern Taiwan, 2009-2019.

Listeria monocytogenes is a critical foodborne pathogen that causes severe invasive and noninvasive diseases and is associated with high mortality. Information on the prevalence of L. monocytogenes infections in Taiwan is very limited. This study aimed to analyze the molecular epidemiological surveillance and virulence gene distribution of 176 human clinical L. monocytogenes isolates collected between 2009 and 2019 in northern Taiwan. Our results showed that the isolates belonged to 4 serogroups (IIa, IIb, IVb, and IIc), with most isolates in serogroups IIa (81/176, 46%) and IIb (71/176, 40.3%). Multilocus sequence typing analysis revealed 18 sequence types (STs) and 13 clonal complexes (CCs). Eighty-four percent of all isolates belonged to six STs: CC87-ST87 (40/176, 22.7%), CC19-ST378 (36/176, 19.9%), CC155-ST155 (28/176, 15.5%), CC1-ST710 (16/176, 8.8%), CC5-ST5 (16/176, 8.8%), and CC101-ST101 (11/176, 6.1%). Furthermore, our analysis showed the distributions of four Listeria pathogenicity islands (LIPI) among all isolates. LIPI-1 and LIPI-2 existed in all isolates, whereas LIPI-3 and LIPI-4 only existed in specific STs and CCs. LIPI-3 existed in the STs, CC1-ST710, CC3-ST3, CC288-ST295, and CC191-ST1458, whereas LIPI-4 could be found in the STs, CC87-ST87 and CC87-ST1459. Strains containing LIPI-3 and LIPI-4 are potentially hypervirulent; thus, 68/176 isolates (39.1%) collected in this study were potentially hypervirulent. Since L. monocytogenes infections are considered highly correlated with diet, molecular epidemiological surveillance of Listeria in food is important; continued surveillance will provide critical information to prevent foodborne diseases.

Eosinophils promote CD8+ T cell memory generation to potentiate anti-bacterial immunity.

Memory CD8+ T cell generation is crucial for pathogen elimination and effective vaccination against infection. The cellular and molecular circuitry that underlies the generation of memory CD8+ T cells remains elusive. Eosinophils can modulate inflammatory allergic responses and interact with lymphocytes to regulate their functions in immune defense. Here we report that eosinophils are required for the generation of memory CD8+ T cells by inhibiting CD8+ T cell apoptosis. Eosinophil-deficient mice display significantly impaired memory CD8+ T cell response and weakened resistance against Listeria monocytogenes (L.m.) infection. Mechanistically, eosinophils secrete interleukin-4 (IL-4) to inhibit JNK/Caspase-3 dependent apoptosis of CD8+ T cells upon L.m. infection in vitro. Furthermore, active eosinophils are recruited into the spleen and secrete more IL-4 to suppress CD8+ T cell apoptosis during early stage of L.m. infection in vivo. Adoptive transfer of wild-type (WT) eosinophils but not IL-4-deficient eosinophils into eosinophil-deficient mice could rescue the impaired CD8+ T cell memory responses. Together, our findings suggest that eosinophil-derived IL-4 promotes the generation of CD8+ T cell memory and enhances immune defense against L.m. infection. Our study reveals a new adjuvant role of eosinophils in memory T cell generation and provides clues for enhancing the vaccine potency via targeting eosinophils and related cytokines.

Gut Microbiota Protects Listeria monocytogenes-Infected Mice by Reducing the Inflammatory Cytokines Storm and Cell Apoptosis.

Gut microbiota (GM) has been proven to resist pathogenic infection through nutritional competition, colonization resistance and promotion of the host immune response. However, in clinical practice, GM is mainly used in intestinal diseases, such as Clostridium difficile infection, and there are few reports on its application in the treatment of pathogenic bacterial infections. In this study, GM from healthy mice was transplanted into mice infected with Listeria monocytogenes using fecal microbiota transplantation (FMT) and the effects were observed. We found that GM from healthy mice could reduce the mortality of infected mice and decrease the counts of L. monocytogenes in their liver and spleen. In addition, FMT inhibited the expression of inflammatory factors in the liver and spleen of infected mice. In vitro cell experiments revealed that GM can reduce the count of L. monocytogenes invading Caco-2 cells and inhibit the L. monocytogenes-caused apoptosis. These results indicate that GM can be used to protect mice infected with L. monocytogenes by eliminating the amount of L. monocytogenes in the host and inhibiting the overexpression of inflammatory factors. Hence, this method can potentially replace antibiotics in the treatment of L. monocytogenes infection.