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Listeria monocytogenes is a significant concern in the food industry due to its association with outbreaks of listeriosis, particularly affecting vulnerable populations. High-throughput technologies such as RNA sequencing (RNA-seq) and proteomics offer valuable insights into the molecular responses of L. monocytogenes to stress environments. In this study, a combined transcriptomic and immunoproteomic approach was applied to explore the stress response mechanisms of the L. monocytogenes strain ST7, which was responsible for an outbreak in central Italy. The bacterium was exposed to both optimal conditions and a stress environment representative of pork product matrices (pH 5.5; 7 % NaCl) and thermal abuse prior to consumption (12 °C).Transcriptomic analysis revealed variations in gene expression related to pathogenesis, stress responses, and virulence factors under different environmental conditions. Transcriptomic analysis of Listeria involves studying the complete set of RNA transcripts produced by the bacterium under various conditions or during different stages of its lifecycle. It can provide insights into its pathogenicity and virulence mechanisms. Immunoproteomic analysis identified proteins involved in stress response pathways, including oxidoreductases and DNA repair enzymes, uniquely expressed under stress conditions. Furthermore, the study highlighted proteins linked to antibiotic resistance and cell wall biosynthesis. By delineating specific proteins crucial in the stress response pathways, these findings not only deepen our comprehension of L. monocytogenes biology but also pave the way for designing more targeted mitigation strategies to safeguard food safety and public health effectively.

Listeria monocytogenes is a pathogen frequently associated with ready-to-eat (RTE) meat and poultry products. Nitrite is a key antimicrobial additive that can offer some degree of protection against L. monocytogenes when included in meat product formulations. The objectives of this study were to determine the potential of nitrite-embedded film to affect the growth of L. monocytogenes following postthermal processing of conventionally-cured and nitrite-free bologna. Two bologna treatment formulations, a conventionally-cured control formulation (CON) and a nitrite-free formulation (UCC), were manufactured, packaged in conventional (CF) or nitrite-embedded (NEF) film, inoculated with 3.5 log CFU/cm2 of a cocktail of L. monocytogenes strains, and stored at 10 ± 1 °C. CON-NEF and UCC-NEF treatments had significantly slower (P

The foodborne pathogen Listeria monocytogenes can persist in produce processing environments, which increases the risk for food contamination. Increased resistance to antimicrobials commonly used in cleaning and sanitizing procedures may contribute to L. monocytogenes’ persistence in these environments. This study aimed to evaluate sanitizer resistance in L. monocytogenes isolates collected from three tree fruit packing facilities (F1, F2, and F3) during packing seasons 2020–2021 (Y1) and 2021–2022 (Y2), and to assess evidence of persistence based on the genomic similarity of isolates to historical isolates collected in previous years. L. monocytogenes isolates collected in 2020–2022 (n = 44) were tested for resistance to peroxyacetic acid (PAA) and a proprietary biofilm−removing agent using a broth microdilution assay. Further, L. monocytogenes isolates were whole genome sequenced and screened for the presence of antimicrobial resistance and virulence genes, as well as to assess the genomic similarity of isolates using the CFSAN SNP bioinformatic pipeline. Over half (57%) of the tested isolates had a PAA minimum inhibitory concentration (MIC) of 250 ppm, which was similar to the applied concentration of the PAA sanitizer in the three facilities (230 ppm). In contrast, 80% of tested isolates had a biofilm remover MIC of 0.13 ppm, which was substantially below the concentration applied in the facilities (137 ppm). Genomes of all tested isolates carried antimicrobial resistance (fosX, lin, mdrL, mprF, and norB) and virulence (inlA, inlB, plcA, plcB, prfA, hly, mpl, and iap) genes. L. monocytogenes isolates collected between 2020 and 2022 belonged to three distinct lineages, with 22 multilocus sequence types (MLSTs) belonging to 22 different clonal complexes. Genomic similarity analysis with historical isolates collected from the same facilities in 2016–2017 demonstrated a 5-year persistence of the genotypes ST 1003 and ST 554 in F2, which were no longer detected in 2022. Overall, our results highlight the need to re-evaluate sanitizer concentrations to effectively control persistent L. monocytogenes strains in tree fruit packing facilities.

The safety of uncooked fermented, dried sausages relies upon controlled fermentation and drying that inactivates pathogenic bacteria. Current guidelines for the production of fermented sausages by the United States Department of Agriculture (USDA) Food Safety Inspection Services (FSIS) and related research highlight specific safety parameters. The confidence that processing steps, which do not include cooking, inherently mitigate microbial risks, is challenged by the resilience of pathogens in the dry and acidic environments of these food products. The aim of this work was to examine the length of drying required to achieve a target pathogen reduction across a range of sausage diameters. This study investigated the relationship between product diameter and time required to achieve target reductions of Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes, as well as the attainment of specific water activity (aw). The research utilized salami and summer sausage with diameters of 18 mm, 30 mm, 60 mm, 90 mm, and 110 mm. Sausage batter was inoculated with 5 strains each of E. coli O157:H7, L. monocytogenes, and S. enterica. Inoculated sausages were processed with fermentation and drying protocols for each sausage type. Smaller diameter sausages reached both the desired pathogen reduction and target aw of 0.85 sooner than larger ones. However, the time to achieve the target aw did not align with the time to achieve the pathogen reduction targets, suggesting that aw alone is not a reliable indicator of safety. Another finding was larger sausages achieved the target pathogen reduction without reaching the target aw, suggesting complex relationship between aw, diameter, and pathogen inactivation. These data support the need for food safety guidelines that consider drying duration, aw, and pathogen behavior for varying sausage diameters. This research contributes to developing more precise safety protocols for producing dry and semi-dry fermented sausages.

Field-packing of cantaloupes involves numerous food contact surfaces that can contamination melons with foodborne pathogens; the soil on these surfaces increases throughout the harvest day. Data are lacking on the cross-contamination risk from contaminated food contact surfaces under the dry conditions typical of cantaloupe field-packing operations. This study sought to evaluate the survival of Salmonella and Listeria monocytogenes on cantaloupe field-pack food contact surfaces using both a wet and dry inoculum to provide insights into managing foodborne pathogen contamination risks. Five clean or fouled materials (cotton gloves, nitrile gloves, rubber gloves, cotton rags, and stainless steel) were inoculated with a cocktail of either Salmonella or L. monocytogenes. A wet inoculum was spot inoculated (100 µL) onto coupons. A dry inoculum was prepared by mixing wet inoculum with 100 g of sterile sand and shaking the coupons with the inoculated sand for 2 min. Coupons were held at 35°C (35% RH) and enumerated at 0, 2, 4, 6, and 8 h. Significant differences in pathogen concentrations over time were calculated, and the GInaFiT add-in tool for Excel was used to build Log-linear, Weibull, and Biphasic die-off models. Depending on the material type, coupon condition, and inoculum type, Salmonella and L. monocytogenes reductions over 8 h ranged from 0.3 to 3.3 and −0.4 to 4.2 log10 CFU/coupon, respectively. For all material types, Salmonella reductions were highest on wet-inoculated clean coupons; L. monocytogenes varied by material type. Weibull and biphasic models were a better fit of respective pathogen die-off curves than linear models. Overall, faster die-off rates were seen for wet inoculated and clean materials. Since pathogen populations remained viable over the study duration and both inoculum type and coupon condition impacted survival, frequent sanitation or replacement of food contact surfaces during the operational day is needed to reduce the risk of cross-contamination.

The objective of this study was to reveal the antibacterial mode of action of garviecin LG34 against S. aureus CICC 21600 and L. monocytogenes CICC 21633 and measure the inhibitions on these two foodborne pathogenic bacteria in milk. Antibacterial mechanism of garviecin LG34 was ascertained by its effect on the efflux of Potassium (K+) ions, extracellular electrical conductivity, UV-absorbing substances, potential across the membrane (ΔΨ), and cell permeability. The inhibition of garviecin LG34 against S. aureus CICC 21600 and L. monocytogenes CICC 21600 in milk was studied by viable counting method. Supplementation with 160 AU/ml of garviecin LG34 had a bactericidal effect on S. aureus CICC 21600 and L. monocytogenes CICC 21633. A total of 80, 160, and 320 AU/ml of garviecin LG34 resulted in the effusion of potassium ion and UV-absorbing substances, the leakage of cellular electrolytes, and the dissipation of electrical potential across the membrane of these two food-borne bacteria and showed a dose-dependent. Moreover, the increase in cell permeability of both strains was observed by flow cytometer after cells treated with 160 AU/ml of garviecin LG34. Garviecin LG34 significantly inhibited the growth of these two food-borne bacteria in milk, especially in skimmed milk. Garviecin LG34 could cause pore formation, intracellular materials release, and permeability increase of S. aureus CICC 21600 and L. monocytogenes CICC 21633, and could be applied to milk as bio-preservative.

Listeria monocytogenes (Lm) is a Gram-positive bacterium causing listeriosis, a rare but severe foodborne infection, particularly impactful during pregnancy. Maternal-fetal transmission can lead to adverse fetal outcomes, yet symptoms in mothers may be nonspecific, delaying intervention. Despite the severity, the mechanisms of vertical transmission remain unclear. This report describes a case of rapid Lm diagnosis in a preterm newborn using cord blood and placental swabs. A 31-week pregnant woman presented with abdominal pain, diarrhea, and reduced fetal movements after consuming raw sushi. Laboratory findings indicated infection, and she vaginally delivered a live infant with placental and fetal abscesses. Cultures confirmed Lm, with swift diagnosis aided by molecular syndromic testing. The neonate received appropriate antibiotics and was asymptomatic by the end of treatment. This case underscores the need for the rapid diagnosis of maternal-fetal listeriosis, as it poses significant risks during pregnancy, including preterm birth and neonatal complications. Current diagnostic methods often delay treatment. This report emphasizes the use of innovative molecular techniques for early diagnosis, which is crucial in managing neonatal infections, especially in preterm newborns.

Listeria monocytogenes (L. monocytogenes) is a foodborne pathogen with high morbidity and mortality rates, necessitating rapid detection methods. Current techniques, while reliable, are labor-intensive and not amenable to on-site testing. We report the design and synthesis of a novel imprinted upconversion fluorescence probe through Pickering emulsion polymerization for the specific detection of L. monocytogenes. The probe employs trimethylolpropane trimethacrylate and divinylbenzene as cross-linkers, acryloyl-modified chitosan as a functional monomer, and the bacterium itself as the template. The developed probe demonstrated high specificity and sensitivity in detecting L. monocytogenes, with a limit of detection of 72 CFU/mL. It effectively identified the pathogen in contaminated salmon and chicken samples, with minimal background interference. The integration of molecular imprinting and upconversion fluorescence materials presents a potent and reliable approach for the rapid and specific detection of L. monocytogenes, offering considerable potential for on-site food safety testing.

Listeria monocytogenes is a foodborne pathogen that lives in nature as a saprophyte. Two of the three most common serotypes that cause foodborne listeriosis are 1/2a and 4b. Within serotype 4b, there is a variant called 4bv-1. In the last decade, several produce-related outbreaks (linked to leafy salad, caramel apples, and stone fruit) were linked to 4bv-1 strains, specifically those of Sequence Type 382. This study assessed the fitness of ST 382 strains on lettuce leaf sections to determine if they are more fit on produce than strains of other serotypes. Strains of serotypes 1/2a, 4b, and ST 382 were inoculated as mixtures onto lettuce and incubated at 4 °C for 7 days or 25 °C for 24 h. Thirty L. monocytogenes colonies resulting from the growth on each lettuce piece were characterized for serotype by multiplex PCR, and the percentages of each serotype recovered were compared. In the individual mixtures with three strains, none of the ST 382 strains showed better fitness for growth on lettuce at either 4 °C or 25 °C. Overall, ST 382 strains showed better recovery from lettuce sections grown at 4 °C than at 25 °C. Statistical analysis of the recovery of twelve strains tested in competition experiments indicated that ST 382 strains were less fit for lettuce growth when competing against the other serotypes. The data indicate that ST 382 strains do not have a competitive fitness advantage on cut lettuce sections.

Listeria monocytogenes and Cronobacter sakazakii are two important foodborne bacterial pathogens. Bacterial endophytes, which reside in plant cells, can produce antimicrobial compounds to protect the host organism or inhibit pathogens. This study investigated the bacterial community of tropical fruits for their potential to inactivate L. monocytogenes or C. sakazakii in cantaloupe and liquid infant formula, respectively. Tropical fruits including papayas, dragon fruits, and sugar apples, were sourced from several countries. Candidate bacterial endophytes were recovered from these tropical fruits using blood agar and Reasoner’s 2A (R2A) agar and tested for potential inhibition against L. monocytogenes and C. sakazakii. A total of 196 bacterial endophytes were recovered from papayas, dragon fruits, and sugar apples. Among these bacterial endophytes, 33 (16.8%) and 13 (6.6%) of them demonstrated an inhibition zone against L. monocytogenes and C. sakazakii, respectively. The inhibitory strains were identified using 16S rRNA sequencing as Bacillus spp., Enterobacter spp., Klebsiella spp., Microbacterium spp., Pantoea spp., and Pseudomonas spp. A cocktail of Pantoea spp. and Enterobacter spp. was used in challenge studies with cantaloupe and significantly reduced the number of L. monocytogenes by approximately 2.5 log10 CFU/g. In addition, P. stewartii demonstrated antagonistic activity against C. sakazakii in liquid infant formula, i.e., it significantly decreased the number of C. sakazakii by at least 1 log10 CFU/mL. Thus, the use of bacterial endophytes recovered from fruits and vegetables could be a promising area of research. Their use as potential biocontrol agents to control bacterial pathogens in ready-to-eat foods warrants further investigation.

The Association for Dressings and Sauces’ (ADS) members have conducted challenge studies on salad dressing products to assess pathogen survival. Data from 79 different challenge studies provided by ADS were used in this analysis. The acid-moisture ratio, pH, incubation temperature, and ingredient details were provided for each study. Linear regression models were used to predict the time to 3-log, 4-log, and 5-log reduction as a function of study parameters. A statistically based approach also was used to estimate the concentration of pathogens in ingredients based on testing history. This was combined with decline modeling to estimate pathogen concentration over time. The time-to-five log reduction for each of the target pathogens were highly skewed. A logarithmic transformation of time to 5 log reduction resulted in approximately normal distributions. Incubation temperature and formulation pH were highly significant (p

Listeria monocytogenes infection often leads to listeriosis in immunocompromised individuals, and screening for potential nutrients that improve immunity may be beneficial. Here, we found that pre-incubation with heptadecanoic acid (C17:0), an odd-chain saturated fatty acid, significantly inhibited L.monocytogenes replication in differentiated THP-1 and RAW264.7, as evidenced by comparison of listeriolysin expression and colony count. Mechanistically, the administration of C17:0 triggered the activation of the IFN and NF-κB signaling pathways. Furthermore, C17:0 upregulated ACOD1 expression and the generation of itaconate, an anti-bacterial metabolite in macrophages. Based on these results, our comparative analysis showed that sheep tail fat (STF), ruminant lipid rich in C17:0, had similar anti-L.monocytogenes effect. Collectively, our data highlighted the function of C17:0 in boosting the immune response, shedding light on the application of C17:0 and STF as potential candidates for strengthening anti-L.monocytogenes response.

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 were also performed and assessed for effectiveness. Listeria prevalence among environmental sponges collected from the four packinghouses was 20% (range of 5–31% 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.

Listeria monocytogenes are considered to be the major foodborne pathogen worldwide. To understand the prevalence and potential risk of L. monocytogenes in retail foods, a total of 1243 retail foods in 12 food categories were sampled and screened for L. monocytogenes from 2020 to 2022 in Huzhou, China. A total of 46 out of 1234 samples were confirmed to be L. monocytogenes positive with a total rate of 3.7%. The contamination rate of seasoned raw meat (15.2%) was the highest, followed by raw poultry meat and raw livestock meat (9.9%) and salmon sashimi (9.5%). The L. monocytogenes isolates belonged to four serotypes, 1/2a,1/2b, 1/2c, and 4b, with the most prevalent serotype being 1/2a (47.9%). All isolates were grouped into 15 sequence types (STs) belonging to 14 clonal complexes (CCs) via multilocus sequence typing (MLST). The most prevalent ST was ST9/CC9 (23.9%), followed by ST3/CC3 (19.6%) and ST121/CC121 (17.4%). Notably, 11 STs were detected from ready-to-eat (RTE) foods, some of them have been verified to be strongly associated with clinical origin listeriosis cases, such as ST3, ST2, ST5, ST8, and ST87. Listeria pathogenicity islands 1 (LIPI-1) and LIPI-2 were detected in approximately all L. monocytogenes isolates, whereas the distribution of both LIPI-3 genes and LIPI-4 genes exhibited association with specific ST, with LIPI-3 in ST3 and ST288, and LIPI-4 in ST87. The strains carrying LIPI-3 and LIPI-4 virulence genes in this study were all isolated from RTE foods. Antimicrobial susceptibility tests showed that >90% of isolates were susceptible to PEN, AMP, ERY, CIP, SXT, VAN, CHL, and GEN, indicating the antibiotic treatment might be still efficient for most of the L. monocytogenes strains. However, for the three clinical first-line antibiotics (PEN, AMP, and GEN), we also observed three and four strains showing MIC values greater than the susceptibility standards for PEN and AMP, respectively, and one strain showing resistance to GEN.

Cooked, uncured meat products packaged under reduced oxygen packaging conditions require the control of anaerobic and facultative anaerobic pathogens if they are held at temperatures greater than 3°C at retail or consumer level. The objective of this study was to determine the inhibition of Listeria monocytogenes and Clostridium botulinum in cooked, uncured shredded turkey and pork formulated with synthetic or clean-label antimicrobials. Treatments of shredded meat products were prepared with or without antimicrobials using turkey thigh or breast that were cooked to 85°C, shredded, and chilled before inoculation with the target pathogen. L. monocytogenes inoculated samples were stored at 7.2°C, whereas C. botulinum samples were stored at 12.8°C; triplicate samples were assayed every 2 weeks. In the first set of experiments, L. monocytogenes populations increased 2 to 3 logs within 2 weeks of storage at 7.2°C in both meat control treatments without antimicrobials and in pork with 4% lactate-diacetate blend (LD). A 1-log increase was observed in turkey with 4% LD and Pork with 2% cultured dextrose-vinegar-rosemary (CDVR) under the same storage conditions; a 1-log increase was observed in turkey with CDVR at 4 weeks. The second set of experiments tested the effect of pH reduction (to less than 5.5 by the addition of 0.5% citric acid) in combination with 2% CDVR when added to the brine precook or postcook during shredding. Populations of L. monocytogenes increased 4-log within 2 and 4 weeks at 7.2°C for the control turkey and pork formulations, respectively. No growth was observed in 12 weeks for any antimicrobial CDVR-CA treatments regardless of how antimicrobial was added. Similarly, botulinum toxin was detected in both control treatments at week 2 at 12.8°C, but no toxicity was observed in either antimicrobial treatment through 12 weeks. These data suggest that a combination of 2% cultured dextrose-vinegar-rosemary extract plus 0.5% citric acid to reduce pH inhibits the growth of L. monocytogenes and toxin production of C. botulinum in uncured shredded turkey and pork products stored under mild temperature abuse conditions for up to 12 weeks in reduced oxygen packaging.

Two recent outbreaks of listeriosis have been linked to the consumption of enoki mushrooms. After the first outbreak, import sampling by the U.S. FDA identified that 43% of the samples evaluated were positive for Listeria monocytogenes (Lm). These observations raised questions about the potential sources of Lm contamination of enoki mushrooms. One potential source of contamination is during enoki mushroom cultivation, as growing conditions are comparatively cool and moist to induce mushroom germination, to which Lm is well adapted. Two varieties of enoki mushrooms were evaluated to determine the potential for Lm to contaminate enoki cultures when introduced at various points during cultivation (inoculation, scraping, pinning, and collaring). The results of two trials showed that Lm established contamination and grew to similar levels in the substrate regardless of when Lm was introduced and, with one exception, did not alter the rate of mushroom generation to below the control. Enumeration of Lm in enoki mushroom cultures at harvest found an average contamination of 103 cfu/g, though the results were variable. Refrigerated storage for six weeks was found to result in an increase in Lm. Additionally, no statistically significant difference in the levels of Lm was observed based on proximity to the substrate, though levels of Lm in the different enoki samples correlated with levels of Lm in the substrate at harvest, but not at scraping. The ability of Lm to grow independently in the media used to culture enoki was assessed, and Lm was found to be unable to grow but could sporadically survive in Masters Mix. No growth of Lm was observed in potato dextrose broth, though growth could occur on the agar. Overall, the data indicate a high potential for the establishment of Lm contamination at any point during enoki cultivation to result in Lm-contaminated mushrooms. These data indicate a need for active control mechanisms to prevent the introduction of Lm to enoki cultures.

The effect of fermentation and drying temperatures, caliber, and sodium lactate on Listeria monocytogenes inactivation was studied in salami, produced in a pilot scale, inoculated with 107 CFU/g of Listeria innocua ATCC® 33090 as a surrogate microorganism for L. monocytogenes. Fermentation temperature varied between 24 and 30°C, drying temperature between 14 and 20°C, caliber between 5.1 and 13.2 cm, and sodium lactate initial concentrations in salamis were 0 and 2%. L. innocua counts, pH and water activity were determined in salamis over time. Sodium lactate (2%) decreased pH drop and Listeria inactivation during fermentation. Baranyi & Roberts equation was used to fit the experimental data and to estimate, for each test condition, inactivation rate (k), initial (Y0), and final counts of L. innocua (YEND). Total inactivation was calculated as Y0 minus YEND (Y0-YEND). Then, using a Box Benkhen experimental design, a quadratic model for k and a two-factor interaction model (2FI) for Y0 − YEND were obtained as functions of fermentation temperature, drying temperature, and caliber size. The models predicted that maximum k and Y0 −YEND, −2.62 ± 0.14 log10 CFU/g/day and 4.5 ± 0.1 log10 CFU/g, respectively, would be obtained fermenting at 30°C and drying at 20°C regardless of caliber. Drying at 14°C allowed Listeria growth until a water activity (aw) of 0.92 was reached. Therefore, if initial Listeria contamination is high (3 log10 CFU/g), drying at low temperatures will compromise product safety.

Listeria monocytogenes may survive and persist in food processing environments due to formation of complex multi-species biofilms of environmental microbiota that co-exists in these environments. This study aimed to determine the effect of selected environmental microbiota on biofilm formation and tolerance of L. monocytogenes to benzalkonium chloride in formed biofilms. The studied microbiota included bacterial families previously shown to co-occur with L. monocytogenes in tree fruit packing facilities, including Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae. Biofilm formation ability and the effect of formed biofilms on the tolerance of L. monocytogenes to benzalkonium chloride was measured in single- and multi-family assemblages. Biofilms were grown statically on polystyrene pegs submerged in a R2A broth. Biofilm formation was quantified using a crystal violet assay, spread-plating, confocal laser scanning microscopy, and its composition was assessed using amplicon sequencing. The concentration of L. monocytogenes in biofilms was determined using the most probable number method. Biofilms were exposed to the sanitizer benzalkonium chloride, and the death kinetics of L. monocytogenes were quantified using a most probable number method. A total of 8, 8, 6, and 3 strains of Pseudomonadaceae, Xanthomonadaceae, Microbacteriaceae, and Flavobacteriaceae, respectively, were isolated from the environmental microbiota of tree fruit packing facilities and were used in this study. Biofilms formed by Pseudomonadaceae, Xanthomonadaceae, and all multi-family assemblages had significantly higher concentration of bacteria, as well as L. monocytogenes, compared to biofilms formed by L. monocytogenes alone. Furthermore, multi-family assemblage biofilms increased the tolerance of L. monocytogenes to benzalkonium chloride compared to L. monocytogenes mono-species biofilms and planktonic multi-family assemblages.These findings suggest that L. monocytogenes control strategies should focus not only on assessing the efficacy of sanitizers against L. monocytogenes, but also against biofilm-forming microorganisms that reside in the food processing built environment, such as Pseudomonadaceae or Xanthomonadaceae.

In this study, isolate Bacillus velezensis1-3 was selected out for its anti- Listeria potency, from which a novel circular bacteriocin, velezin, was purified out of the fermentate, and then characterized. Facilitated with a broad antibacterial spectrum, velezin has demonstrated decent inhibitive activity against of foodborne pathogen L. monocytogenes ATCC 19115. It exerted the antibacterial activity through damaging the membrane integrity of targeted cell and causing leakage of vital elements, including K+ ion. It was noteworthy that velezin also inhibited the biofilm formation by L. monocytogenes ATCC 19115. At the challenge of velezin, L. monocytogenes ATCC 19115 up-regulated expression of genes associated with membrane, ion transporters, stressing-related proteins as well as the genes responsible for the synthesis of small molecule. Taken together, velezin may have potential to be a candidate as natural additive used in food/feed in the future.

The Contamination Sanitization Inspection and Disinfection (CSI-D) device is a handheld fluorescence-based imaging system designed to disinfect food contact surfaces using ultraviolet-C (UVC) illumination. This study aimed to determine the optimal CSI-D parameters (i.e., UVC exposure time and intensity) for the inactivation of the following foodborne bacteria plated on non-selective media: generic Escherichia coli (indicator organism) and the pathogens enterohemorrhagic E. coli, enterotoxigenic E. coli, Salmonella enterica, and Listeria monocytogenes. Each bacterial strain was spread-plated on non-selective agar and exposed to high-intensity (10 mW/cm2) or low-intensity (5 mW/cm2) UVC for 1–5 s. Control plates were not exposed to UVC. The plates were incubated overnight at 37 °C and then enumerated. Three trials for each bacterial strain were conducted. Statistical analysis was carried out to determine if there were significant differences in bacterial growth between UVC intensities and exposure times. Overall, exposure to low or high intensity for 3–5 s resulted in consistent inhibition of bacterial growth, with reductions of 99.9–100 % for E. coli, 96.8–100 % for S. enterica, and 99.2–100 % for L. monocytogenes. The 1 s exposure time showed inconsistent results, with a 66.0–100 % reduction in growth depending on the intensity and bacterial strain. When the results for all strains within each species were combined, the 3–5 s exposure times showed significantly greater (p 0.05) in growth inhibition between the high and low UVC intensities. The results of this study show that, in pure culture conditions, exposure to UVC with the CSI-D device for ≥3 s is required to achieve consistent reduction of E. coli, S. enterica, and L. monocytogenes.

Listeriosis is a disease caused by L. monocytogenes, a relevant microorganism as a causative agent of foodborne diseases - FBD. This study aimed to evaluate the distribution of Listeria spp., and L. monocytogenes in different production areas in two small plants (A and B) and two micro-food processing plants (C and D) producing meat derivatives, located in different cities of Colombia. The methodology implemented was i. The analysis of sampling points is based on a harmonised tool. ii. Four samplings in each production plant between 2019 and 2020. iii. Isolation and identification of microorganisms through conventional microbiology, a semi-automated system, molecular serotyping and clonal characterisation by ERIC-PCR. L. monocytogenes frequency in the production plants belonging to the study ranged between 5.9 and 28.6 %; for Listeria spp., plants A and D had isolated, plant A had the highest proportion, while for L. monocytogenes geno-serotypes found were: 1/2a, 1/2c, 4a-4c, 4b, 4d - 4e, with geno-serotype 4b as the most frequent. Furthermore, possible persistent isolates were detected in plant C as the feasible sources of contamination, based on failures in flow management, raw material contaminated with L. monocytogenes, lack of standardised cooking processes and transfer of the microorganism through equipment and surfaces. Finally, in three of the four production plants assayed, L. monocytogenes or Listeria spp. were present in the packaging area in some of the samples taken during the study, which calls for increased and frequent monitoring, as well as constant technical support for the control of L. monocytogenes in micro and small-scale production plants.

After finishing waxes are applied, citrus fruits are typically dried at 32–60°C for 2–3 min before final packing. The survival of Listeria monocytogenes, Salmonella, and Enterococcus faecium NRRL B-2354 was evaluated under laboratory conditions on lemons after applying one of four finishing waxes (F4, F6, F8, and F15) followed by an ambient hold or heated (50 or 60°C) drying step. The reduction of inoculated microorganisms during drying was significantly influenced by wax type and temperature, with greater reductions at higher temperatures. Greater reductions after waxing and drying at 60°C were observed with L. monocytogenes (2.84–4.44 log) than with Salmonella (1.65–3.67 log), and with Salmonella than with E. faecium (0.99–2.93 log). The survival of Salmonella inoculated at 5.8–5.9 log/fruit on lemons and oranges after applying wax F6 and drying at 60°C was evaluated during storage at 4 and 22°C. The reductions of Salmonella after waxing and drying were 1.7 log; additional reductions during storage at 4 or 22°C were 1.40–1.43 or 0.18–0.29 log, respectively, on waxed lemons, and 0.56–1.02 or 0.54–0.57 log, respectively, on waxed oranges. Under pilot-scale packinghouse conditions with wax F4, mean and minimum reductions of E. faecium ranged from 2.15 to 2.89 and 1.64 to 2.12 log, respectively. However, E. faecium was recovered by whole-fruit enrichment (limit of detection: 0.60 log CFU/lemon) but not by plating (LOD: 1.3 log CFU/lemon) from uninoculated lemons run with or after the inoculated lemons. The findings should provide useful information to establish and implement packinghouse food safety plans.

Small- and medium-sized dairy processing facilities (SMDFs) may face unique challenges with respect to controlling Listeria in their processing environments, e.g., due to limited resources. The aim of this study was to implement and evaluate environmental monitoring programs (EMPs) for Listeria control in eight SMDFs in a ∼1-year longitudinal study; this included a comparison of pre-operation (i.e., after cleaning and sanitation and prior to production) and mid-operation (i.e., at least 4 h into production) sampling strategies. Among 2,072 environmental sponge samples collected across all facilities, 272 (13%) were positive for Listeria. Listeria prevalence among pre- and mid-operation samples (15% and 17%, respectively), was not significantly different. Whole genome sequencing (WGS) performed on select isolates to characterize Listeria persistence patterns revealed repeated isolation of closely related Listeria isolates (i.e., ≤20 high-quality single nucleotide polymorphism [hqSNP] differences) in 5/8 facilities over >6 months, suggesting Listeria persistence and/or reintroduction was relatively common among the SMDFs evaluated here. WGS furthermore showed that for 41 sites where samples collected pre- and mid-operation were positive for Listeria, Listeria isolates obtained were highly related (i.e., ≤10 hqSNP differences), suggesting that pre-operation sampling alone may be sufficient and more effective for detecting sites of Listeria persistence. Importantly, our data also showed that only 1/8 of facilities showed a significant decrease in Listeria prevalence over 1 year, indicating continued challenges with Listeria control in at least some SMDFs. We conclude that options for simplified Listeria EMPs (e.g., with a focus on pre-operation sampling, which allows for more rapid identification of likely persistence sites) may be valuable for improved Listeria control in SMDFs.

Thermal inactivation studies were undertaken on Listeria monocytogenes and Salmonella spp. inoculated on the surface of country ham. Hams (average = ca. 3.4 ± 0.5 kg each; average = ca. ≥18% shrinkage) were used as provided by the processor (i.e., “salted hams”), desalted in tap water (i.e., “desalted hams”), or dried for an additional period (i.e., “extra-dried hams”). Hams were surface inoculated (ca. 9.5 log CFU/ham) with a multistrain cocktail of L. monocytogenes or Salmonella spp. and cooked within a bag in a circulating water bath to an internal temperature of 130°F (54.4°C) instantaneous, 145°F (62.8°C) and held for 4 min, 153°F (67.2°C) and held for 34 s, or 160°F (71.1°C) instantaneous. Regardless of ham type, all four time and temperature combinations tested herein delivered a ≥6.7-log reduction of cells of L. monocytogenes or Salmonella spp. Differences in product pH, moisture content, or aw did not have an appreciable impact on the thermal inactivation of L. monocytogenes or Salmonella spp. on country ham. In addition, shelf-life studies were undertaken using slices of “salted” country ham that were surface inoculated (ca. 5.5 log CFU/slice) with a multistrain cocktail of L. monocytogenes or Staphylococcus aureus and then stored at 20°C. Levels of S. aureus increased by ca. ≤1.4 log CFU/slice during storage for 90 days, whereas levels of L. monocytogenes remained relatively unchanged (≤0.2 log CFU/slice increase). Our data validated that cooking parameters elaborated in the U.S. Department of Agriculture’s Food Safety and Inspection Service Cooking Guideline for Meat and Poultry Products (Revised Appendix A) are sufficient to deliver significant reductions (ca. ≥6.8 log CFU/ham) in levels of L. monocytogenes and Salmonella spp. on country ham. In addition, in the event of postprocessing contamination, country ham may support the outgrowth of S. aureus or survival of L. monocytogenes during storage at 20°C for 90 days.

Sous-vide is a cooking technique which is considered safe for food preparation, however, the recommended temperatures and times for preparing seafood and fish may not be sufficient to ensure safety, and additionally, thermal stress may cause changes in virulence and resistance. The objective of the present investigation was to determine the reduction of Listeria monocytogenes isolated from fish after sous-vide preparation. For this purpose flow cytometry was used. The impact of stress during the preparation of the fish-based dish using the sous-vide technique on the virulence potential and resistance profile was determined geno- and phenotypically. The study was conducted at four different temperatures, that are typical for fish preparation (42, 52, 57 and 60 °C) showing that none of them could be effective against L. monocytogenes. In addition, the investigation of changes in the virulence, phenotypic antibiotic resistance and gene expression showed that the stress during the preparation of sous-vide decrease virulence potential of the L. monocytogenes strains. It was observed that the greatest changes in antibiotic resistance profile were noted for tetracycline and notable changes were also observed for some strains in the case of fosfomycin and ciprofloxacin. Therefore it is important not to consider the sous-vide method as ensuring the microbiological quality of products, especially those with high contamination levels, and choose products of the best quality for processing.

Listeria monocytogenes (Lm), a foodborne bacterium, can infect people and has a high fatality rate in immunocompromised individuals. Listeriolysin O (LLO), the primary virulence factor of Lm, is critical in regulating the pathogenicity of Lm. This review concludes that LLO may either directly or indirectly activate a number of host cell viral pathophysiology processes, such as apoptosis, pyroptosis, autophagy, necrosis and necroptosis. We describe the invasion of host cells by Lm and the subsequent removal of Lm by CD8 T cells and CD4 T cells upon receipt of the LLO epitopes from major histocompatibility complex class I (MHC-I) and major histocompatibility complex class II (MHC-II). The development of several LLO-based vaccines that make use of the pore-forming capabilities of LLO and the immune response of the host cells is then described. Finally, we conclude by outlining the several natural substances that have been shown to alter the three-dimensional conformation of LLO by binding to particular amino acid residues of LLO, which reduces LLO pathogenicity and may be a possible pharmacological treatment for Lm.

Validated alternative test methodologies may be used in place of culture-based methods recommended for environmental monitoring programs (EMPs) for Listeria in food production facilities. In order to help guide decisions on which testing method to use to simplify Listeria EMP implementation in food production facilities, alternative methods were compared to the culture-based method in actual EMPs for Listeria. Seventy-two samples collected from two facilities of souzai production businesses that use meat and meat products as ingredients, one facility of processed meat product production business, and one facility of processed meat product and souzai production business were applied to EMPs for Listeria using the culture-based method, 3MTM Molecular Detection System (MDS), and InSite L. mono Glo (InSite). The kappa coefficient in MDS was 0.65 for Listeria monocytogenes and 0.74 for Listeria spp., both of which were deemed substantial compared with the culture-based method. The kappa coefficient in InSite was −0.01 for L. monocytogenes and 0.50 for Listeria spp., which indicated poor and moderate reproducibility, respectively. When the medium of InSite was smeared on agar medium, 7 of the 19 samples tested positive only for Listeria spp. (negative for L. monocytogenes) but L. monocytogenes was cultured, indicating that the sensitivity of detecting L. monocytogenes via fluorescence may be low. MDS was considered a useful alternative for both L. monocytogenes and Listeria spp. as targets, and InSite was not possible as a substitute for detecting L. monocytogenes; however, it is considered a helpful alternative method for detecting Listeria spp. EMPs for Listeria often target Listeria spp. as an indicator of L. monocytogenes. The alternative methods studied in this study are rapid, simple, and useful in EMPs for Listeria. However, the data in this study were a comparatively small sample set and impacted by variability, so more robust comparisons are desirable in the future.

Background: Listeria monocytogenes, a Gram-positive bacillus, primarily affects immunocompromised individuals. Endocarditis is a rare but severe complication of L. monocytogenes bacteremia, irrespective of native or prosthetic valves. While there is no standardized treatment, the use of ampicillin proves effective in most cases. Surgical intervention is reserved for cases involving valve dehiscence, heart failure, or myocardial abscess. Case presentation: A 54-year-old female, with mitral valve replacement, presented with fever, chest pain and dyspnea at rest. Patient was initially diagnosed with bacterial pneumonia; however, subsequent evaluation revealed L. monocytogenes bacteremia, resulting in endocarditis. Surgical management was contraindicated due to multiple prior valve replacement surgeries. Symptoms resolution, along with improvements in echocardiographic and clinical parameters, was achieved through extended antibiotic treatment only with no surgical intervention. Conclusion - key takeaways: This case underscores the critical importance of individualized treatment approaches in endocarditis, particularly in patients with surgery approach contraindication, and emphasized the success achieved through ampicillin-based management.

This study investigated the antibacterial effects of ultrasound (US), β-citronellol (CT), and a combination of the two treatments on Listeria monocytogenes. Results showed that US or CT alone did not show apparent antibacterial effect (0.02–0.76 log CFU/mL reduction). The combined treatment showed obviously inactivate effect of L. monocytogenes, the populations of L. monocytogenes decreased by 8.93 log CFU/mL after US (253 W/cm2, 20 kHz) + 0.8 mg/mL CT treatment. US + CT treatment also had a significant (P 0.05) change the surface color of carrots but significantly (P

Commercial cheese brines are used repeatedly over extended periods, potentially for years, and can be a reservoir for salt-tolerant pathogens, such as Listeria monocytogenes. The objective of this study was to determine the inactivation of L. monocytogenes in cheese brines treated with hydrogen peroxide (H2O2) (0, 50, and 100 ppm) at holding temperatures representing manufacturing conditions. In experiment one, four fresh cheese brines were prepared with 10 or 20% salt and pH 4.6 or 5.4 (2x2 design; duplicate trials). Brines were inoculated with L. monocytogenes, treated with H2O2, and stored at 10 and 15.6°C. For experiment two, seven used commercial brines (representing five cheese types, 15–30% NaCl, pH 4.5–5.5; three seasonal trials) were inoculated with L. monocytogenes or S. aureus, treated with H2O2, and stored at 12.8°C (both L. monocytogenes and S. aureus), 7.2 and 0°C (L. monocytogenes only). Each treatment was assayed on Days 0, 1, and 7 for microbial populations and residual H2O2. Data revealed that pathogen populations decreased ≤1 log in cheese brines with no hydrogen peroxide stored for 7 days, regardless of the storage temperature. In fresh brine treated with 50 or 100 ppm of H2O2, populations of L. monocytogenes were reduced to less than the detectable limit by 7 days at 10 and 15.6°C (>4 log reduction). For unfiltered used brines, H2O2 had no effect on L. monocytogenes populations in Brick J (pH 5.4, 15% NaCl) due to rapid inactivation of H2O2, likely by indigenous yeasts (∼3-log CFU/ml). For the remaining brines, the addition of 100 ppm H2O2 killed >4 log L. monocytogenes when stored at 7.2 or 12.8°C for 1 week, but only 3–4 log reduction when stored at 0°C. The addition of 50 ppm H2O2 had similar lethal effects at 12.8°C but was less effective at 7.2 or 0°C. Inactivation rates of S. aureus were similar to that of L. monocytogenes. This study confirmed that high salt, warmer temperature, and 100-ppm H2O2 accelerated the inactivation of L. monocytogenes in cheese brines. Data also suggest that the presence of catalase-positive indigenous microorganisms may neutralize the effect of H2O2.

Listeria monocytogenes, is a Gram-positive facultative intracellular bacterium without spores. It can cause invasive diseases such as septicemia, meningitis, and encephalitis, and has a high mortality rate. This is a report on a recent case of neurolisteriosis isolated from cerebrospinal fluid sample of a patient with diabetes and chronic heart failure in our hospital. The patient initially received the combined treatment of cephalosporin and meropenem (both 1.0 g every 8 hours). We identified the pathogenic organism as L. monocytogenes using three identification methods: mass spectrometry, biochemical assays, and molecular techniques. After determining the pathogenic bacteria, we quickly informed the clinician and suggesting a change in antibiotic treatment and immediately discontinued cephalosporin and meropenem. The patient's symptoms were significantly improved after 9 days of penicillin G treatment, and the patient chose to be discharged for personal reasons. In conclusion, certain strains of wild-type Listeria monocytogenes can lead to identification errors that occur across platforms and methods.

The objectives of the present study were to isolate and identify Listeria monocytogenes in salad vegetables and milk products sold in different markets of Mymensingh district in Bangladesh, and to characterize this organism (serogroups, virulence genes) along with antibiogram study. A total of 85 samples of salad vegetables (tomato, carrot and cucumber) were collected from six different markets of five upazilas of Mymensingh district, and 83 milk products such as cheese, yogurt and icecream of two available brands were collected from Mymensingh sadar upazila. Isolation and identification of Listeria monocytogenes was done by cultural, biochemical, PCR and MALDI-TOF assays. The presence of serogroups (lineage I, lineage II, lineage III) and virulence-associated genes (inlA, actA, prfA, and hlyA) were determined by multiplex PCR. The antimicrobial susceptibility test of the isolates was done with 22 antimicrobial agents using disk diffusion assay. The overall prevalence of L. monocytogenes was 7.1%. Among salad vegetables, contamination of cucumber was highest (19.2%), and among milk products, the highest contamination was in icecream (7.1%). Five isolates (41.7%) belonged to Lineage II and seven isolates (58.3%) to Lineage III. Only two isolates (16.67%) harbored one or more virulence-associated genes. The highest resistance was observed to oxacillin and nalidixic acid followed by erythromycin and meropenem. Findings of the present study would be useful to evaluate the risk posed by salad vegetables and milk products, and to the concerned authorities for eventual future food regulations.

Background: Relatively few studies have focused on mortality risk factors for listeriosis in the literature. Information on the mortality of populations with listeriosis is needed. We aimed to explore the mortality risk factors related to listeriosis in this meta-analysis. Methods: The studies were considered eligible for inclusion only if they directly compared the mortality risk factors for listeriosis between dead patients and surviving patients. The mortality risk factors included clinical predisposing factors and predisposing comorbidities. Results: Thirteen studies were included in this study. There were 12,265 listeriosis patients, including 2863 (23.3%) dead patients. The meta-analysis provided evidence that the mortality risk factors related to listeriosis were as follows: 1. Clinical predisposing factors included age ≥ 60 years, primary bacteremia and central nervous system involvement; 2. Predisposing comorbidities included non-hematological malignancies, alcoholism, chronic kidney disease, cardiovascular disease, and pulmonary disease. In addition, autoimmune disease comorbidity had a protective effect against listeriosis. Conclusion: The presence of older patients, primary bacteremia, central nervous system involvement, non-hematological malignancies, alcoholism, chronic kidney disease, cardiovascular disease, and pulmonary disease should alert physicians to the higher risk of mortality.

ABSTRACT: The gram-positive bacterium Listeria monocytogenes is an important foodborne pathogen contaminating dairy products. Closely related to L. monocytogenes saprophytic Listeria spp. are also frequent contaminators of food and, particularly, dairy products. To distinguish L. monocytogenes from nonpathogenic Listeria spp. and other bacteria, a dot-immunoassay was developed. The immunoassay is based on the polyclonal antibody to the secreted form of the surface virulence-associated L. monocytogenes-specific InlB protein. To increase InlB production, bacteria were grown on the brain-heart infusion agar supplemented with 0.2% activated charcoal (BHIC agar). Direct plating of artificially contaminated raw milk samples on the BHIC agar followed by the dot-immunoassay allowed a rapid identification of L. monocytogenes in concentrations as little as 10 cfu/mL. Using the developed approach, preliminary results were obtained within 14 h, and the final results were obtained after 26 h. The dot-immunoassay was tested on L. monocytogenes strains belonging to different clonal complexes and phylogenetic lineages, Listeria spp., and other bacterial species. Results showed the exceptional specificity of the developed dot-immunoassay for the rapid identification of L. monocytogenes.

The present study evaluates the efficacy of a batch wash ozone sanitation system (BWOSS) and spray wash ozone sanitation system (SWOSS) against Listeria monocytogenes (two strains) and Salmonella enterica subsp. enterica (three serovars) inoculated on the surface of carrots, sweet potatoes, and butternut squash, commonly used in raw meat-based diets (RMBDs) marketed for companion animals such as dogs and cats. Produce either remained at room temperature for 2 h or were frozen at −20°C and then tempered overnight at 4°C to mimic the preprocessing steps of a raw pet food processing operation (‘freeze-temper’) prior to ozone treatment. Two ozone concentrations (0 and 5 ppm) were applied for either 20 s or 60 s for BWOSS and 20 s for SWOSS. Based on an ANOVA, BWOSS data showed no significant difference (P > 0.05) in microbial reduction between 0 and 5 ppm ozone concentration across all treatment durations for each produce type. BWOSS resulted in mean microbial reductions of up to 1.56 log CFU/mL depending on the treatment time and produce type. SWOSS data were analyzed using a generalized linear model with Quasipoisson errors. Freeze-tempered produce treated with SWOSS had a higher bacterial log reduction at 5 ppm ozone compared to 0 ppm ozone (P = 0.0013) whereas room temperature produce treated with SWOSS did not show any significant difference in microbial reduction between ozone concentrations. The potential to mitigate microbial cross-contamination was also investigated during SWOSS treatment. The results indicate that 5 ppm ozone decreased pathogens in the rinsate and proximal surfaces by 0.63–1.66 log CFU/mL greater than no ozone depending on the pathogen and sample. Overall, data from this study indicate that SWOSS would be more effective compared to BWOSS in reducing the microbial load present on the surface of root tubers and squash subjected to freezing and thawing and has the potential to mitigate cross-contamination within RMDB manufacturing environments.

Listeria monocytogenes (Lm) is a Gram-positive bacterium that causes invasive listeriosis, an illness with high mortality and hospitalization rates. Due to the severity of illness associated with Lm, rapid identification and characterization of isolates from foods and the food-processing environment are critical to properly identify and track the pathogen and quickly remove adulterated foods from the market. Prior methods can rely on time-consuming biochemical or sera-agglutination assays to perform these tasks. Development of a high-throughput method that would rapidly perform these tasks is critical to improve response to contamination events. Previously, a single laboratory validation of a qPCR-based method was presented that could rapidly verify Lm isolates and characterize them into six molecular serogroups. In the current study, a multi-laboratory validation (MLV) was performed to evaluate the reliability of the qPCR method for identification and serogrouping of Lm isolates. Sixteen collaborating laboratories independently analyzed a panel of 43 blinded isolates plus three control strains using the qPCR method. This panel was comprised of representatives for non-Listeria (n = 7), Listeria sp. (n = 8), and Lm (n = 28) strains. The Lm isolates contained representatives of the six serogroups: 2A, 2B, 2C, 4B, NT, and 4bV/IVb-v1, with five strains for each serogroup except 4bV/IVb-v1 (n = 3). The results generated by 16 laboratories showed high sensitivity, specificity, and accuracy, generally ≥97%, for both the genus-species and serogrouping qPCRs. Results from one laboratory lowered the sensitivity of the non-Listeria group to 93%. These results indicated the method was highly reliable. However, only the previously evaluated serogroups were tested within the MLV panel, though there is the potential for other serogroup results. Sequence Read Archive (SRA) files for Lm isolates were evaluated to determine the frequency of other potential serogroup profiles. This effort identified a low percentage of isolates with atypical qPCR serogroups (0.30%) that are consistent with Lm and were generally associated with lineage II and the natural environment. In summary, the results indicate that the proposed qPCR method is reliable and has a high degree of sensitivity, accuracy, and specificity, while also decreasing hands-on analysis time and increasing throughput of the analysis.

Environmental monitoring programs (EMPs) for food production facilities are useful for verifying general sanitation controls and are recommended as verification measures to ensure that the Hazard Analysis Critical Control Point plan is working effectively. In this study, EMPs for Listeria were conducted at three food production facilities to assess the efficacy of sanitation control and establish effective sanitation control methods. In Facility A, L. monocytogenes was detected in the clean area although in Zone 3, non–food-contact surfaces. To prevent contamination from dirty areas, the cleaning practices in the preparation room were investigated. Normal cleaning combined with disinfection with carbonated hypochlorite water (chlorine concentration, 150 ppm) proved effective. At Facility B, a salad product and its ingredients (pastrami and salami) were positive for L. monocytogenes serotype 3b. The bacterial count was

Pet food formulated with raw meat can pose health risks to pets and humans. High-pressure processing (HPP) was evaluated to achieve a 5-log reduction of Salmonella, E. coli STEC, and L. monocytogenes in commercial raw pet foods and maintain a 5-log reduction throughout post-HPP storage. Three formulation types that varied in the amounts of striated meat, organ meat, bone, seeds, and other ingredients (fruits, vegetables, and minor ingredients) designated as A-, S-, and R-formulations were used.Eight raw diet pet foods, consisting of three beef formulations (A-, S- and R-Beef), three chicken formulations (A-, S-, and R-Chicken), and two lamb formulations (A- and S-Lamb), were inoculated with 7 log CFU/g cocktails of Salmonella, E. coli STEC or L. monocytogenes, HPP at 586 MPa for 1–4 min, and stored refrigerated (4°C) or frozen (−10 to −18°C) for 21 days with microbiological analyses at various time intervals.A- formulations (20–46% meat, 42–68% organs, 0.9–1.3% seeds, and 10.7–11.1% fruits, vegetables, and minor ingredients) inoculated with Salmonella and treated at 586 MPa for at least 2 min achieved a 5-log reduction 1 day post-HPP and maintained that inactivation level during frozen storage. A- and S-formulations inoculated with E. coli STEC and treated at 586 MPa for at least 2 min achieved a 5-log reduction from day 6 of frozen storage.L. monocytogenes was more HPP resistant than Salmonella and E. coli STEC. S-formulations containing chicken or beef and stored frozen post-HPP had lower inactivation of L. monocytogenes compared to A-formulations containing chicken or beef. S-Lamb had higher frozen storage inactivation (5.95 ± 0.20 log CFU/g) compared to chicken (2.52 ± 0.38 log CFU/g) or beef (2.36 ± 0.48 log CFU/g). HPP coupled with frozen storage time was effective in achieving and maintaining a 5-log reduction of Salmonella and E. coli STEC while L. monocytogenes was more resistant and requires further optimization to achieve a 5-log reduction.

ABSTRACT: Foodborne pathogens detection is important to ensure food safety and human health. In this study, we designed a comet structure to rapidly and sensitively detect foodborne Listeria monocytogenes. This method combined isothermal sequence exchange amplification (SEA) and surface-enhanced Raman spectroscopy. Listeria monocytogenes DNA could be rapidly amplified at a constant temperature via SEA with a pair of modified primers, which rendered the precise thermal control instrumentation unnecessary. Efficient SEA amplification generated a large number of DNA duplexes that could be easily captured by streptavidin-modified magnetic bead and AuMB@Ag-isothiocyanate fluorescein antibody (anti-FITC). AuMB@Ag-anti-FITC was used as a signal probe, which generated a significant excitation signal at 1,616 cm−1 for quantitative detection and analysis. The results displayed sensitive detection of L. monocytogenes in cheese from 2.0 × 101 cfu/mL to 2.0 × 106 cfu/mL within 1.0 h with a detection limit of 7.8 cfu/mL. Furthermore, this comet structure displayed the desirable specificity as its specific primers and amplified DNA ends were attached to streptavidin-modified magnetic beads and AuMB@Ag-anti-FITC, respectively. We expected that the method devised would provide a promising new approach to screening for L. monocytogenes and guarantee the microbiological safety of dairy products.

In 2016, the U.S. Food and Drug Administration (FDA), the Centers for Disease Control and Prevention (CDC), and state partners investigated nine Listeria monocytogenes infections linked to frozen vegetables. The investigation began with two environmental L. monocytogenes isolates recovered from Manufacturer A, primarily a processor of frozen onions, that were a match by whole genome sequencing (WGS) to eight clinical isolates and historical onion isolates with limited collection details. Epidemiologic information, product distribution, and laboratory evidence linked suspect food items, including products sourced from Manufacturer B, also a manufacturer of frozen vegetable/fruit products, with an additional illness. The environmental isolates were obtained during investigations at Manufacturers A and B. State and federal partners interviewed ill people, analyzed shopper card data, and collected household and retail samples. Nine ill persons between 2013 and 2016 were reported in four states. Of four ill people with information available, frozen vegetable consumption was reported by three, with shopper cards confirming purchases of Manufacturer B brands. Two identified outbreak strains of L. monocytogenes (Outbreak Strain 1 and Outbreak Strain 2) were a match to environmental isolates from Manufacturer A and/or isolates from frozen vegetables recovered from open and unopened product samples sourced from Manufacturer B; the investigation resulted in extensive voluntary recalls. The close genetic relationship between isolates helped investigators determine the source of the outbreak and take steps to protect public health. This is the first known multistate outbreak of listeriosis in the United States linked to frozen vegetables and highlights the significance of sampling and WGS analyses when there is limited epidemiologic information. Additionally, this investigation emphasizes the need for further research regarding food safety risks associated with frozen foods.

Smoked salmon and smoked trout are ready-to-eat and potentially contaminated with the pathogenic bacterium Listeria monocytogenes making them high risk for the consumer. This raises questions about the presence of hypervirulent or persistent strains in the salmon and trout industries. Knowledge of the genetic diversity of circulating strains in these sectors is essential to evaluate the risk associated with this pathogen and improve food safety. We analyzed the genetic structure of 698 strains of L. monocytogenes isolated from 2006 to 2017 in France, based on their serogroup, lineage and clonal complexes (CCs) determined by Multilocus sequence typing (MLST). Most of the CCs were identified by mapping the strains PFGE profiles and a novel high-throughput real-time PCR method for CC identification. We identified thirteen CCs and one sequence type (ST) with variable distribution in salmon and trout samples (food, environment). The three most prevalent CCs were CC121, CC26 and CC204. Strains from ST191 and CC54 were detected for the first time in these sectors, while less than 0.6% of the isolates belonged to the hyper-virulent CC1, CC6 and CC20. No CC was exclusively associated with the salmon sector. This project allowed us to assess the population diversity of CCs of L. monocytogenes in the salmon and trout industries.

There is a growing interest in using models to predict foodborne pathogen inactivation as a way to validate or verify preventive controls. Unlike liquid foods, solid, low water activity foods (LWAF) are heterogenous in composition and structure and do not transfer heat uniformly. Using models constructed from one food to predict pathogen inactivation on another LWAF is complex and may not always be possible, even if the foods have similar composition. Using models constructed from inactivation kinetics of three foodborne pathogens and a surrogate from vacuum-steam-pasteurized (72 and 82 °C) whole macadamia nuts and dried apricot halves, 3-log reductions were predicted for the same pathogens and foods of reduced size. Model fits (First-order, Weibull, and Gompertz) were significantly impacted by the food type regardless of particle size. Despite the foods being identical in composition with particle size as the only altered characteristic, best-fit models accurately predicted the 3-log reductions only 50% of the time, but the surrogate inactivation models provided conservative predictions for pathogen reductions, highlighting that a surrogate's model may be a suitable tool for predicting pathogen reduction on LWAFs.

Keeping the global food supply safe necessitates international collaborations between countries. Health and regulatory agencies routinely communicate during foodborne illness outbreaks, allowing partners to share investigational evidence. A 2016–2020 outbreak of Listeria monocytogenes infections linked to imported enoki mushrooms required a multinational collaborative investigation among the United States, Canada, Australia, and France. Ultimately, this outbreak included 48 ill people, 36 in the United States and 12 in Canada, and was linked to enoki mushrooms sourced from one manufacturer located in the Republic of Korea. Epidemiologic, laboratory, and traceback evidence led to multiple regulatory actions, including extensive voluntary recalls by three firms in the United States and one firm in Canada. In the United States and Canada, the Korean manufacturer was placed on import alert while other international partners provided information about their respective investigations and advised the public not to eat the recalled enoki mushrooms. The breadth of the geographic distribution of this outbreak emphasizes the global reach of the food industry. This investigation provides a powerful example of the impact of national and international coordination of efforts to respond to foodborne illness outbreaks and protect consumers. It also demonstrates the importance of fast international data sharing and collaboration in identifying and stopping foodborne outbreaks in the global community. Additionally, it is a meaningful example of the importance of food sampling, testing, and integration of sequencing results into surveillance databases.

Testing of foods for low levels of the human pathogen, Listeria monocytogenes (Lm), involves a selective enrichment procedure. A nonpathogenic species of Listeria, L. innocua (Li), is often present in foods and food-manufacturing environments and is an interference organism for Lm detection due to competition during enrichment. The present study investigated whether a novel enrichment strategy incorporating the sugar allose into the secondary enrichment broth (allose method) could improve the detection of Lm from foods when Li is present. First, Canadian food isolates of Listeria spp. were tested to confirm recent reports that lineage II Lm (LII-Lm), but not Li, could metabolize allose. All LII-Lm isolates (n = 81), but not Li (n = 36), possessed the allose genes lmo0734-lmo0739, and could efficiently metabolize allose. Next, smoked salmon was contaminated with mixtures of LII-Lm and Li and tested using different enrichment procedures to compare the ability to recover Lm. Allose broth was more effective than Fraser Broth, with Lm detected in 87% (74 of 85) compared to 59% (50 of 85) of the samples (P

The foodborne pathogen Listeria monocytogenes generally infects immunocompromised individuals, such as cancer patients, more frequently and with higher morbidity and mortality than the general population. Because of the anticipated risk associated with L. monocytogenes and other pathogens in produce, immunocompromised individuals are often placed on neutropenic diets that exclude fresh produce, though these risks have not been quantified. Therefore, this study developed a data-driven risk model for listeriosis in cancer patients who consume ready-to-eat (RTE) salads, consisting of leafy greens, cucumbers, and tomatoes, as influenced by kitchen-scale treatments and storage practices. Monte Carlo simulations were used to model the risk of invasive listeriosis during one chemotherapy cycle. Refrigerating all salad components decreased the median risk by approximately one-half log. For refrigerated salads with no treatment, the predicted median risk was ≤ 4.3 × 10−08. When salad ingredients were surface blanched with greens rinsed, the predicted risk decreased to 5.4 × 10−10. Predicted risk was lowest (1.4 × 10−13) for a blanched “salad” consisting of solely cucumbers and tomatoes. Interestingly, rinsing, as recommended by FDA, only decreased the median risk by 1 log. A sensitivity analysis revealed that the highly variable dose-response parameter k strongly influenced risk, indicating that reducing uncertainty in this variable may improve model accuracy. Overall, this study demonstrates that kitchen-scale pathogen reduction approaches have high risk reduction efficacy and could be considered as an alternative to diets that exclude produce when making risk management decisions.

Nut, seed, and legume butters have become increasingly popular with consumers. Listeria monocytogenes contamination of a variety of butters has resulted in several recalls, although no known outbreaks have been identified. L. monocytogenes has been shown to survive on a variety of seeds for up to 6 months, legumes and nuts for over 12 months, and in peanut butter and peanut-chocolate spreads for 21 to 60 weeks depending on formulation; however, long-term survival in other butters has not yet been characterized. In this study, the survival of L. monocytogenes in various nut, seed, legume, and chocolate-containing butters (n = 10) based on inoculation level, storage temperature, and the pH, aw, and nutrient contents of the butters was examined. First, butters were inoculated with L. monocytogenes at 4 log CFU/g and stored at either 5 or 25°C with enumeration and/or enrichment at intervals over 12 months. L. monocytogenes survived in all butters examined with no significant change in population after storage at 5°C, whereas the population was reduced to

Fresh-cut apples, being rich in antioxidants and other nutrients, have emerged as popular snacks in restaurants, at home, and in school lunch programs, partially due to freshness, convenience, and portion size availability. Two major challenges in processing fresh-cut apples are the browning of cut surfaces and contamination with human pathogens. Regarding human pathogens, contamination by Listeria monocytogenes is a major concern, as evidenced by two outbreaks of whole apples and numerous recalls of fresh-cut apples. Antibrowning agents currently used by the industry have little to no antimicrobial properties. The present review discusses the possible origins of L. monocytogenes in fresh-cut apples, including contaminated whole apples, and contamination via the processing environment and the equipment in fresh-cut facilities. Treatment with antibrowning solutions could possibly be an opportunity for Listeria contamination and represents the last chance to inactivate pathogens. The discussion is focused on the antibrowning treatments where formulations and coatings with antibrowning and antimicrobial properties have been developed and evaluated against Listeria and other microorganisms. In addition, several research needs and considerations are discussed to further reduce the chance of pathogen contamination on fresh-cut apples.

Comparisons among a qPCR assay, VIDAS® assays and a conventional agar streaking method following the same enrichment for the detection of Listeria monocytogenes were performed under two challenging conditions. In the first comparison, L. innocua and L. monocytogenes were coinoculated into sausages at ratios (L. innocua-to-L. monocytogenes) of 10, 100, 1000, and 10 000. qPCR provided the most sensitive detection at all ratios after both 24-h and 48-h enrichments. A modified VIDAS® LMO2 assay (i.e., replacement of the kit-specified enrichment scheme with the enrichment scheme used in this study) and agar streaking yielded equivalent results when the ratio was 10 and 100; agar streaking was more sensitive when the ratio was 1000; neither method could detect L. monocytogenes at the ratio of 10 000. Enrichment duration of 48 h was needed for modified VIDAS® to detect L. monocytogenes when the ratio was 1000. Agar streaking after 24-h enrichment isolated L. monocytogenes better than after 48-h enrichment when the ratio was 100 and 1000. In the second comparison, we followed the validation guidelines of AOAC International and inoculated L. monocytogenes, without any L. innocua, onto lettuce and stainless-steel surfaces at low levels. The numbers of positive samples detected by qPCR, VIDAS® LIS assay, modified VIDAS® LMO2 assay, and agar streaking after 48-h enrichment were not statistically different. Our data showed that qPCR was the most sensitive method, while agar streaking and VIDAS® performed reasonably well. Streaking after 24-h enrichment was needed when background flora could overgrow L. monocytogenes during prolonged enrichment, and this is critical for confirming rapid screening assays. Appropriate selection of enrichment duration and rapid assays will enhance the testing of L. monocytogenes in food and environmental samples.

Mild cooking thermal treatments, like sous-vide, can compromise ground meat entrees such as meatballs with chipotle sauce, especially when salt levels are reduced during its preparation. Listeria monocytogenes is a thermoresistant pathogen that can be in ready-to-eat food. On the other hand, nisin, due to its thermal stability, can be a good alternative to aid on the thermal inactivation of L. monocytogenes and ensure meat safety. The objective was to optimize the amount of nisin and salt concentrations to thermally inactivate L. monocytogenes during the sous-vide cooking of ground beef marinated in chipotle sauce, and to generate a predictive model. A four-strain cocktail was prepared and inoculated in ground beef in combination (3:2) with chipotle sauce added with nisin (0–150 IU) and salt (0–2%). After that, meat samples were sous-vide cooked at different temperatures, nisin, and salt concentrations, established by a central composite design. Depending on the levels of these factors, D-values ranged from 49.71 to 0.27 min. A predictive model (p

Formulating ready-to-eat (RTE) products with growth inhibitors minimizes the risk of listeriosis. In part I, RTE egg products formulated with 6.25 ppm nisin were evaluated to control Listeria monocytogenes. Individual experimental units were surface inoculated with 2.5-log CFU/g of L. monocytogenes, packaged in pouches with a headspace gas of 20:80 CO2:NO2, and stored at 4.4°C for 8 weeks. Formulations with finished product pH of 6.29 ± 0.07 limited growth to 0.05) from the control without nisin at pH 7.34 ± 0.13, all supported 4-log growth by 4 weeks. In part II, a nisin bioassay test was performed to evaluate the stability of nisin in eggs as affected by the product pH (6.00 ± 0.03, 7.00 ± 0.00, 7.50 ± 0.03, and 8.00 ± 0.02) and cooking to an internal temperature of 73.9 or 85°C for 90 s. The nisin activity loss increased as the product pH or the cooking temperature increased (p 0.05) in the CO2 and O2 headspace gas composition, generally no detection or growth of background microbes, and no changes (p > 0.05) in the pH of the formulations during storage, all assuring absence of uncontrolled interferences for the growth of L. monocytogenes.