As a common foodborne pathogen, infection with L. monocytogenes poses a significant threat to human life and health. The objective of this study was to employ comparative genomics to unveil the biodiversity and evolutionary characteristics of L. monocytogenes strains from different regions, screening for potential target genes and mining novel target genes, thus providing significant reference value for the specific molecular detection and therapeutic targets of L. monocytogenes strains. Pan-genomic analysis revealed that L. monocytogenes from different regions have open genomes, providing a solid genetic basis for adaptation to different environments. These strains contain numerous virulence genes that contribute to their high pathogenicity. They also exhibit relatively high resistance to phosphonic acid, glycopeptide, lincosamide, and peptide antibiotics. The results of mobile genetic elements indicate that, despite being located in different geographical locations, there is a certain degree of similarity in bacterial genome evolution and adaptation to specific environmental pressures. The potential target genes identified through pan-genomics are primarily associated with the fundamental life activities and infection invasion of L. monocytogenes, including known targets such as inlB, which can be utilized for molecular detection and therapeutic purposes. After screening a large number of potential target genes, we further screened them using hub gene selection methods to mining novel target genes. The present study employed eight different hub gene screening methods, ultimately identifying ten highly connected hub genes (bglF_1, davD, menE_1, tilS, dapX, iolC, gshAB, cysG, trpA, and hisC), which play crucial roles in the pathogenesis of L. monocytogenes. The results of pan-genomic analysis showed that L. monocytogenes from different regions exhibit high similarity in bacterial genome evolution. The PCR results demonstrated the excellent specificity of the bglF_1 and davD genes for L. monocytogenes. Therefore, the bglF_1 and davD genes hold promise as specific molecular detection and therapeutic targets for L. monocytogenes strains from different regions.

Controlling Listeria in produce packinghouses can be challenging due to the large number of potential contamination routes. For example, repeated isolation of the same Listeria subtype in a packinghouse could indicate persistence in the packinghouse or reintroduction of the same Listeria from an upstream source. To improve understanding of Listeria transmission patterns in packinghouses, we performed a longitudinal study in four apple packinghouses, including testing of 1,339 environmental sponges and whole genome sequencing (WGS)-based characterization of 280 isolates. Root cause analysis and subsequent intervention implementation was also performed and assessed for effectiveness. Listeria prevalence among environmental sponges collected from the four packinghouses was 20% (range of 5 to 31% for individual packinghouses). Sites that showed high Listeria prevalence included drains, forklift tires and forks, forklift stops, and waxing area equipment frames. A total of 240/280 WGS-characterized isolates were represented in 41 clusters, each containing two or more isolates that differed by ≤50 high-quality single nucleotide polymorphisms (hqSNPs); 21 clusters were isolated from one packinghouse over ≥2 samplings (suggesting persistence or possibly reintroduction), while 11 clusters included isolates from >2 packinghouses, suggesting common upstream sources. Some interventions successfully (i) reduced Listeria detection on forklift tires and forks (across packinghouses) and (ii) mitigated packinghouse-specific Listeria issues (e.g., in catch pans). However, interventions that lacked enhanced equipment disassembly when persistence was suspected typically appeared to be unsuccessful. Overall, while our data suggest a combination of intensive environmental sampling with subtyping and root cause analysis can help identify effective interventions, implementation of effective interventions continues to be a challenge in packinghouses.

The objective of this study was to evaluate the effect of fat on thermal resistance of L. monocytogenes, E. coli O157:H7, and Salmonella spp. A 4-strain cocktail of each microorganism was inoculated to beef tallow and heated isothermally at temperatures between 55 and 80℃. All survival curves did not follow the 1st-order inactivation kinetics but conformed to a two-stage linear pattern. The first stage was markedly less heat-resistant than the second, as manifested by significantly lower D values. The z values of E. coli O157 H7 and Salmonella spp. were 11.8 °C and 12.3 °C in the first stage (z1) but increased to 23.7 °C and 20.8 °C in the second stage (z2), respectively. For L. monocytogenes, while the z values were similar for both stages (z1 = 19.6 °C and z2 = 18.5 °C), the second stage D values are 3.6-5.9 times of those in the first stage. One-step analysis was used to fit the nonlinear curves to the Weibull model, yielding < 1 exponents for the model (0.495, 0.362, and 0.282, respectively, for L. monocytogenes, E. coli O157:H7, and Salmonella spp.), suggesting gradually increased thermal resistance during heating. The experimental results showed that these microorganisms could resist heating for longer time and at higher temperatures in tallow than they do in regular meats containing lower levels of fat. The kinetic models can be used to develop thermal processes to properly inactivate pathogens contaminated in the fat portions of meat products or other high fat products.

The engineering of a home-made portable double-layer filtration and concentration device with the common syringe for rapid analysis of water samples is reported. The core elements of the device were two installed filtration membranes with different pore sizes for respective functions. The upper filtration membrane was used for preliminary intercepting large interfering impurities (interception membrane), while the lower filtration membrane was used for collecting multiple target pathogens (enrichment membrane) for determination. This combination can make the contaminated environmental water, exemplified by surface water, filtrated quickly through the device and just retained the target bacteria of Escherichia coli O157:H7, Staphylococcus aureus, and Listeria monocytogenes on the lower enrichment membrane. Integrating with surface-enhanced Raman spectra (SERS) platform to decode the SERS-Tags (SERS-TagCVa, SERS-TagR6G, and SERS-TagMB) already labeled on each of the enriched bacteria based the antibody-mediated immuno-recognition effect, fast separation, concentration, and detection of multiple pathogenic bacteria from the bulk of contaminated environmental water were realized. Results show that within 30 min, all target bacteria in the lake water can be simultaneously and accurately measured in the range from 101 to 106 CFU mL-1 with detection limit of 10.0 CFU mL-1 without any pre-culture procedures. This work highlights the simplicity, rapidness, cheapness, selectivity, and the robustness of the constructed method for simultaneous detecting multiple pathogens in aqueous samples. This protocol opens a new avenue for facilitating the development of versatile analytical tools for drinking water and food safety monitoring in underdeveloped or developing countries.

Transposon sequencing (Tn-seq) is a powerful genome-wide technique to assess bacterial fitness under varying growth conditions. However, screening via Tn-seq in vivo is challenging. Dose limitations and host restrictions create bottlenecks that diminish the transposon mutant pool being screened. Here, we have developed a murine model with a disruption in Akr1c13 that renders the resulting RECON-/- mouse resistant to high-dose infection. We leveraged this model to perform a Tn-seq screen of the human pathogen Listeria monocytogenes in vivo. We identified 135 genes which were required for L. monocytogenes growth in mice including novel genes not previously identified for host survival. We identified organ-specific requirements for L. monocytogenes survival and investigated the role of the folate enzyme FolD in L. monocytogenes liver pathogenesis. A mutant lacking folD was impaired for growth in murine livers by 2.5-log10 compared to wild type and failed to spread cell-to-cell in fibroblasts. In contrast, a mutant in alsR, which encodes a transcription factor that represses an operon involved in D-allose catabolism, was attenuated in both livers and spleens of mice by 4-log10 and 3-log10, respectively, but showed modest phenotypes in in vitro models. We confirmed that dysregulation of the D-allose catabolism operon is responsible for the in vivo growth defect, as deletion of the operon in the ∆alsR background rescued virulence. By undertaking an unbiased, genome-wide screen in mice, we have identified novel fitness determinants for L. monocytogenes host infection, which highlights the utility of the RECON-/- mouse model for future screening efforts.
OBJECTIVE: Listeria monocytogenes is the gram-positive bacterium responsible for the food-borne disease listeriosis. Although infections with L. monocytogenes are limiting in healthy hosts, vulnerable populations, including pregnant and elderly people, can experience high rates of mortality. Thus, understanding the breadth of genetic requirements for L. monocytogenes in vivo survival will present new opportunities for treatment and prevention of listeriosis. We developed a murine model of infection using a RECON-/- mouse that is restrictive to systemic L. monocytogenes infection. We utilized this model to screen for L. monocytogenes genes required in vivo via transposon sequencing. We identified the liver-specific gene folD and a repressor, alsR, that only exhibits an in vivo growth defect. AlsR controls the expression of the D-allose operon which is a marker in diagnostic techniques to identify pathogenic Listeria. A better understanding of the role of the D-allose operon in human disease may further inform diagnostic and prevention measures.

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.

Patients with multiple myeloma (MM) have an increased risk of sepsis due to underlying disease- and treatment-related immunosuppression. However, data on sepsis incidence, causative pathogens, and impact on outcomes in newly diagnosed MM (NDMM) are limited. We conducted a retrospective observational study of 92 NDMM patients who developed sepsis between 2022 and 2023 at a tertiary care center in Italy. Patient characteristics, sepsis criteria [Quick Sequential Organ Failure Assessment, Systemic Inflammatory Response Syndrome (SIRS)], microbiology results, and associations with progression-free survival (PFS) were analyzed. In this cohort of 92 critically-ill patients, pathogenic organisms were identified via microbiological culture in 74 cases. However, among the remaining 18 culture-negative patients, 9 exhibited a SIRS score of 2 and another 9 had a SIRS score of 4, suggestive of a clinical presentation consistent with sepsis despite negative cultures. Common comorbidities included renal failure (60%), anemia (71%), and bone disease (83%). Gram-negative (28%) and Gram-positive (23%) bacteria were frequent causative organisms, along with fungi (20%). Cox Univariate analyses for PFS showed statically significant HR in patients with albumin ≥ 3.5 vs < 3.5 (HR = 5.04, p < 0.001), Karnofsky performance status ≥ 80 vs < 80 (HR = 2.01, p = 0.002), and early-stage vs late-stage disease by International Staging System (HR = 4.76 and HR = 12.52, both p < 0.001) and Revised International Staging System (R-ISS III vs R-ISS I, HR = 7.38, p < 0.001). Sepsis is common in NDMM and associated with poor outcomes. Risk stratification incorporating sepsis severity, comorbidities, and disease stage may help guide preventive strategies and optimize MM management.

Listeria monocytogenes is a ubiquitous bacterial pathogen that threatens the food chain and human health. In this study, whole-genome sequencing (WGS) was used for the genomic characterization of L. monocytogenes (n = 24) from beef and beef-based products. Multilocus Sequence Type (MLST) analysis revealed that ST204 of CC204 was the most common sequence type (ST). Other sequence types detected included ST1 and ST876 of CC1, ST5 of CC5, ST9 of CC9, ST88 of CC88, ST2 and ST1430 of CC2, and ST321 of CC321. Genes encoding for virulence factors included complete LIPI-1 (pfrA-hly-plcA-plcB-mpl-actA) from 54% (13/24) of the isolates of ST204, ST321, ST1430, and ST9 and internalin genes inlABC that were present in all the STs. All the L. monocytogenes STs carried four intrinsic/natural resistance genes, fosX, lin, norB, and mprF, conferring resistance to fosfomycin, lincosamide, quinolones, and cationic peptides, respectively. Plasmids pLGUG1 and J1776 were the most detected (54% each), followed by pLI100 (13%) and pLM5578 (7%). The prophage profile, vB_LmoS_188, was overrepresented amongst the isolates, followed by LP_101, LmoS_293_028989, LP_030_2_021539, A006, and LP_HM00113468. Listeria genomic island 2 (LGI-2) was found to be present in all the isolates, while Listeria genomic island 3 (LGI-3) was present in a subset of isolates (25%). The type VII secretion system was found in 42% of the isolates, and sortase A was present in all L. monocytogenes genomes. Mobile genetic elements and genomic islands did not harbor any virulence, resistance, or environmental adaptation genes that may benefit L. monocytogenes. All the STs did not carry genes that confer resistance to first-line antibiotics used for the treatment of listeriosis. The characterization of L. monocytogenes in our study highlighted the environmental resistance and virulence potential of L. monocytogenes and the risk posed to the public, as this bacterium is frequently found in food and food processing environments.

Extensive research has been conducted to identify key proteins governing stress responses, virulence, and antimicrobial resistance, as well as to elucidate their interactions within Listeria monocytogenes. While these proteins hold promise as potential targets for novel strategies to control L. monocytogenes, given their critical roles in regulating the pathogen\'s metabolism, additional analysis is needed to further assess their druggability-the chance of being effectively bound by small-molecule inhibitors. In this work, 535 binding pockets of 46 protein targets for known drugs (mainly antimicrobials) were first analyzed to extract 13 structural features (e.g., hydrophobicity) in a ligand-protein docking platform called Molsoft ICM Pro. The extracted features were used as inputs to develop a logistic regression model to assess the druggability of protein binding pockets, with a value of one if ligands can bind to the protein pocket. The developed druggability model was then used to evaluate 23 key proteins from L. monocytogenes that have been identified in the literature. The following proteins are predicted to be high-potential druggable targets: GroEL, FliH/FliI complex, FliG, FlhB, FlgL, FlgK, InlA, MogR, and PrfA. These findings serve as an initial point for future research to identify specific compounds that can inhibit druggable target proteins and to design experimental work to confirm their effectiveness as drug targets.

Listeria monocytogenes, a foodborne pathogen causing listeriosis, poses substantial societal, economic, and public health challenges due to its resistance, persistence, and biofilm formation in the food industry. Exploring subinhibitory concentrations of compounds to target virulence inhibition and increase susceptibility to adverse conditions presents a promising strategy to mitigate its impact of L. monocytogenes and unveils new potential applications. Thus, this study aims to explore the effect of linalool on virulence factors of L. monocytogenes and potential use in the reduction in its tolerance to stressful conditions. This action was analysed considering the use of two sub-inhibitory concentrations of linalool, 0.312 and 0.625 mg/mL. We found that even with the lowest tested concentrations, a 65% inhibition of violacein production by Chromobacterium violaceum, 55% inhibition in biofilm formation by L. monocytogenes and 62% reduction on haemolysis caused by this bacterium were observed. In addition to its impact on virulence factors, linalool diminished the tolerance to osmotic stress (up to 4.3 log reduction after 24 h with 12% NaCl), as well as to high (up to 3.8 log reduction after 15 min at 55 °C) and low temperatures (up to 4.6 log reduction after 84 days with 12% NaCl at 4 °C). Thus, this study paves the way to further investigation into the potential utilization of linalool to mitigate the threat posed by L. monocytogenes in the field of food safety and public health.