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Studies of classical microbiology rely on the average behaviour of large cell populations without considering that clonal bacterial populations may bifurcate into phenotypic distinct sub-populations by random switching mechanisms.Listeria monocytogenes exposure to sublethal stresses may induce different physiological states that co-exist (i.e., sublethal injury or dormancy) and present variable resuscitation capacity. Exposures to peracetic acid (PAA; 10-30 ppm; for 3 h), acetic acid and hydrochloric acid (AA and HCl; pH 3.0-2.5; for 5 h) at 20 °C were used to induce different physiological states in L. monocytogenes, Scott A strain. After stress exposure, colony growth of single cells was monitored, on Tryptic Soy Agar supplemented with 0.6 % Yeast Extract, using time-lapse microscopy, at 37 °C. Images were acquired every 5 min and were analyzed using BaSCA framework. Most of the obtained growth curves of the colonies were fitted to the model of Baranyi and Roberts for the estimation of lag time (λ) and maximum specific growth rate (μ max ), except the ones obtained after exposure to AA pH 2.7 and 2.5 that were fitted to the Trilinear model. The data of λ and μ max that followed a multivariate normal distribution were used to predict growth variability using Monte Carlo simulations. Outgrowth kinetics after treatment with AA (pH 2.7 and 2.5; for 5 h at 20 °C), PAA (30 ppm; for 3 h at 20 °C) revealed that these stress conditions increase the skewness of the variability distributions to the right, meaning that the variability in lag times increases in favour of longer outgrowth. Exposures to AA pH 2.5 and 30 ppm PAA resulted in two distinct subpopulations per generation with different growth dynamics. This switching mechanism may have evolved as a survival strategy for L. monocytogenes cells, maximizing the chances of survival. Simulation of microbial growth showed that heterogeneity in growth dynamics is increased when cells are recovering from exposure to sublethal stresses (i.e. PAA and acidic conditions) that may induce injury or dormancy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Exposure to sublethal stresses related to food-processing may induce a heterogenous mixture of cells that co-exist, comprising healthy, sublethally injured, dormant and dead cells. Heterogeneity in survival capacity and dormancy of single cells may impede the detection of foodborne pathogens. In this study, we exposed Listeria monocytogenes Scott A strain, to peracetic acid (PAA; 20-40 ppm) and to acidic conditions (hydrochloric (HCl) and acetic (AA) acid, adjusted to pH 2.7-3.0, to evaluate the resuscitation capacity and outgrowth kinetics of metabolically active cells in two different media. Injury and the viable-but-non-culturable (VBNC) status of cells were assessed by flow cytometry using CFDA (metabolically active) and PI (dead) staining. Stressed CFDA + PI - cells were sorted on Tryptic Soy (TS) Agar or in TS broth, both supplemented with 0.6 % Yeast Extract (TSAYE or TSBYE), to evaluate culturability. Resuscitation capacity of CFDA + PI - sorted cells (10 events/well) was monitored by visual inspection on TSAYE and by optical density measurement in TSBYE for 5 days. Sorting of L. monocytogenes viable cells (CFDA + PI - ) in Ringer's solution on TSAYE and TSBYE showed 100 % recovery in both media (control condition), while the mean lag time in TSBYE was 9.6 h. Treatment with 20 ppm PAA for 90 and 180 min resulted in 74.79 % and 85.82 % of non-culturable cells in TSBYE and increased the average lag time to 41.7 h and 43.8 h, respectively, compared to the control (9.6 h). The longest average lag time (79.5 h) was detected after treatment with 30 ppm PAA for 90 min, while at the same condition sorting of CFDA + PI - cells resulted in 95.05 % and 93.94 % non-culturable cells on TSAYE and TSBYE, respectively. The highest percentage of wells with non-culturable cells (96.17 %) was detected on TSAYE after treatment with 40 ppm PAA for 30 min. Fractions of VBNC cells were detected in TSBYE after treatment with HCl pH 3.0 for 60 and 240 min, and in TSAYE and TSBYE after exposure to AA pH 2.7. Treatment with AA pH 2.7 for 150-300 min increased the range of recorded lag time values compared to 60 min, from 8.6 h up to 13.3 h, as well as the mean lag times in TSBYE. Modelling of the outgrowth kinetics comparing the two types of stress (oxidative vs acid) and the two systems of growth (colonial vs planktonic) revealed that low starting concentrations hindered the detection of viable L. monocytogenes cells, either due to VBNC induction or cell heterogeneity., Competing Interests: Declaration of competing interest This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The disinfectant peracetic acid (PAA) that is used in the food industry can cause sublethal injury in L. monocytogenes. The effect of preculture temperature on the inactivation and sublethal injury of L. monocytogenes cells due to PAA was evaluated by plating on non-selective and selective agar medium supplemented with 5 % (w/v) NaCl. L. monocytogenes cells were precultured at 30 °C, 20 °C or 4 °C, and the former was used as reference temperature. Preculture of cells at 20 °C or 4 °C and subsequent exposure to PAA at the respective growth temperatures caused higher injury compared to cells grown at 30 °C and exposed to PAA 20 °C and PAA 4 °C, respectively. Survival was also affected by the preculture temperature; 20 °C-grown cultures resulted in lower survival at PAA 20 °C. Nevertheless, preculture at 4 °C resulted in a similar number of surviving cells when exposed to PAA 4 °C compared to cells precultured at 30 °C and exposed to PAA at 4 °C. Flow cytometry was subsequently used to quantify outgrowth capacity of stressed and sublethal damaged populations following sorting of single cells in nutrient rich medium (Tryptone soy broth supplemented with yeast extract [TSBY]). PAA treatment affected the outgrowth of L. monocytogenes at single-cell level resulting in increased outgrowth-times reflecting higher single cell heterogeneity. To conclude, the response of L. monocytogenes when exposed to PAA depended on the preculture conditions, and the highly heterogeneous outgrowth potential of PAA-injured cells may affect their detection accuracy and pose a food safety risk., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The simultaneous presence of more than one strains of Listeria monocytogenes in the same food product may affect the growth capacity of each strain. The present study evaluated the metabolites composition that may potentially influence the growth of individual L. monocytogenes strains in a dual strain composite. Based on previous studies, L. monocytogenes strains, C5 (4b) and 6179 (1/2a) were selected due to the remarkable interaction, which was observed during their co-culture. The selected strains were inoculated (2.0 - 3.0 log CFU/mL) in Tryptic Soy Broth with 0.6% Yeast Extract (TSB-YE) in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic conditions (AC). Their resistance to different antibiotics enabled the selective enumeration of each strain in the co-culture. After reaching stationary phase, single and dual cultures were centrifuged and filtered. The cell-free spent medium (CFSM) was either characterized by Fourier transform infrared (FTIR-ATR) spectrometry or re-inoculated, after the addition of concentrated TSB-YE (for nutrient replenishment), with single and two-strain cultures for the evaluation of growth under the influence of metabolites produced from the same singly and co-cultured strains in the different combinations of strains and CFSM origin (7 °C/AC) (n = 2x3). By the end of storage, singly-cultured C5 and 6179 had reached 9.1 log CFU/mL, while in dual culture, 6179 was affected by the presence of C5 attaining only 6.4 ± 0.8 log CFU/mL. FTIR-ATR spectra of CFSM produced by singly-cultured 6179 and the co-culture were almost identical. Characteristic peaks in FTIR-ATR spectrum of CFSM of singly-cultured C5 at 1741, 1645 and 1223 cm -1 represent functional groups which were not present in the CFSM of the co-culture. These molecules may be located intracellularly or mounted on bacterial cell surface and removed from the supernatant during cell filtration of the co-culture. Both singly- and co-cultured 6179 managed to grow similarly regardless of CFSM origin. Contrarily, both singly- and co-cultured C5 managed to outgrow 6179 in CFSM which contained high concentration of C5 metabolites, while in CFSM produced by singly-cultured 6179, C5 did not grow, suggesting that the produced metabolites of strain 6179 appears to be harmful to strain C5. However, during co-culture, C5 may produce molecules that counteract the inhibitory effect of 6179. The findings shed more light on the mechanism behind the inter-strain interactions of L. monocytogenes indicating that both contact of cells and extracellular metabolites may influence the behavior of the different co-existing strains., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Temperature is a major determinant of Listeria (L.) monocytogenes adherence and biofilm formation on abiotic surfaces. However, its role on gene regulation of L. monocytogenes mature biofilms has not been investigated. In the present study, we aimed to evaluate the impact of temperature up- and down-shift on L. monocytogenes biofilms gene transcription. L. monocytogenes strain EGD-e biofilms were first developed on stainless steel surfaces in Brain Heart Infusion broth at 20 °C for 48 h. Then, nutrient broth was renewed, and mature biofilms were exposed to 10 °C, 20 °C or 37 °C for 24 h. Biofilm cells were harvested and RNA levels of plcA, prfA, hly, mpl, plcB, sigB, bapL, fbpA, fbpB, lmo2178, lmo0880, lmo0160, lmo1115, lmo 2089, lmo2576, lmo0159 and lmo0627 were evaluated by quantitative RT-PCR. The results revealed an over-expression of all genes tested in biofilm cells compared to planktonic cells. When biofilms were further allowed to proliferate at 20 °C for 24 h, the transcription levels of key virulence, stress response and putative binding proteins genes plcA, sigB, fbpA, fbpB, lmo1115, lmo0880 and lmo2089 decreased. A temperature-dependent transcription for sigB, plcA, hly, and lmo2089 genes was observed after biofilm proliferation at 10 °C or 37 °C. Our findings suggest that temperature differentially affects gene regulation of L. monocytogenes mature biofilms, thus modulating attributes such as virulence, stress response and pathogenesis., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The adaptive response of bacterial cells to changing environmental conditions depends on the behavior of single cells within the population. Exposure of Listeria monocytogenes to sublethal acidic conditions in foods or in the gastrointestinal track of the host may induce injuries relevant to difficult physiological states within the dormancy continuum. In this study, exposure to acidic conditions (acetic-AA and hydrochloric acid-HCl adjusted to pH 3.0, 2.7, 2.5 at 20 °C for 5 h) was used to evaluate injury of L. monocytogenes, Scott A strain. To differentiate the resistant sub-population from the total, Tryptic Soy Agar with 0.6 % Yeast Extract (TSAYE) supplemented or not with 5 % NaCl were comparatively used. Sublethally injured cells were detected by comparing plate counts with fluorescence microscopy, using combinations of CFDA (viability) and Propidium-Iodide (death). Effect of acid stress on the relative transcription of clpP, mazE, mazF, relA, gadC, gadD, gadB, sigB, inlA and prfA upon transition of total population into different physiological stages was evaluated through RT-qPCR. AA treated cells showed measurable logarithmic reduction at pH 2.7 and 2.5, while there was a significant percentage of CFDA - /PI + cells. Evaluation of the potentially culturable population on TSAYE, from the percentage of CFDA/PI-stained cells, revealed that unstained cells represented a non-culturable sub-population. Exposure to Ringer's solution pH 2.7, adjusted with AA, resulted in higher percentages of non-esterase active with membrane integrity cells (CFDA - /PI - ) compared to the percentages of the enumerated culturable cells on TSAYE after 4 and 5 h. Under the same conditions, after 1 h of exposure macroscopic observation revealed size colony variations (SCVs) of the total population (CFU on TSAYE). L. monocytogenes retained its culturability after hydrochloric acid exposure, while cells remained metabolically active (CFDA + ). However, a stochastic change in cell's shape, was detected after exposure to pH 3.0 and 2.5, adjusted with HCl, for 2 h at 20 °C. A pattern of gene up-regulation was observed during treatment with AA pH 2.7 and HCl pH 3.0 at the 3rd h of exposure. Deciphering L. monocytogenes sublethal injury sheds light into the physiological and molecular characteristics of this state and provides the food science community with quantitative data to improve risk assessment., Competing Interests: Declaration of competing interest None., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Due to the ubiquitous character of Listeria monocytogenes multiple strains of the pathogen may end up co-existing in/on the same final products and could potentially cause infection during consumption. Such multiple strain contamination may occur in different stages of the food supply chain. The present study evaluated the effect of oxygen availability and matrix structure on inter-strain interactions of L. monocytogenes that may occur at high population levels in/on different dairy model systems. L. monocytogenes strains C5 and ScottA (4b), 6179 (1/2a) and PL25 (1/2b) were selected as resistant to different antibiotics (enabling selective enumeration of each strain in co-culture) and inoculated (2.0-3.0 log CFU/mL, g or cm 2 ) in Ricotta and Camembert broth (1 dairy product: 2 ¼ Ringer solution) and in/on dairy-based structured media (dairy broth supplemented with 0.6 and 1.4% agar), in single and two-strain cultures (1:1 strain ratio). Bacterial growth was assessed during storage at 7 °C, under aerobic, hypoxic and anoxic conditions. Every experimental treatment was tested with three biological replicates and two technical repeats (n = 3 × 2). The simultaneously presence of different strains of the pathogen in/on the same substrate did not affect neither the duration of the lag phase nor the growth rate of the co-cultured strains. The observed inter-strain interactions were related with the final population reached/decrease during storage and occurred after the "critical" population density of ca. 6.0 log CFU/mL, g or cm 2 . The phenomenon was more pronounced in/on Ricotta than in/on Camembert-based substrates, indicating that the composition and the available nutrients of the substrate may affect the interactions that expressed as difference in the final population level between singly and co-cultured strains. Under aerobic and hypoxic conditions, most of the observed interactions were more pronounced in dairy-based broths and were mitigated with the addition of agar. The elimination of oxygen resulted in a prolonged lag time, which lasted at least 5 days and no observed interactions by the end of storage, due to low microbial counts. Investigating inter-strain interactions during growth in/on different substrates, which may have undergone temperature abuse during their transport along the supply chain or during storage in household refrigerators, could assist in explaining the mismatch between clinical and food samples during outbreak investigations., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The disinfectant peracetic acid (PAA) can cause high levels of sublethal injury to Listeria monocytogenes. This study aims to evaluate phenotypic and transcriptional characteristics concerning the surface attachment and virulence potential of sublethally injured L. monocytogenes ScottA and EGDe after exposure to 0.75 ppm PAA for 90 min at 4°C and subsequent incubation in tryptic soy broth supplemented with yeast extract (TSBY) at 4°C. The results showed that injured L. monocytogenes cells (99% of the total population) were able to attach (after 2 and 24 h) to stainless steel coupons at 4°C and 20°C. In vitro virulence assays using human intestinal epithelial Caco-2 cells showed that injured L. monocytogenes could invade host cells but could not proliferate intracellularly. The in vitro virulence response was strain dependent; injured ScottA was more invasive than EGDe. Assessment of PAA injury at the transcriptional level showed the upregulation of genes ( motB and flaA ) involved in flagellum motility and surface attachment. The transcriptional responses of L. monocytogenes EGDe and ScottA were different: only injured ScottA demonstrated upregulation of the virulence genes inlA and plcA . Downregulation of the stress-related genes fri and kat and upregulation of lmo0669 were observed in injured ScottA. The obtained results indicate that sublethally injured L. monocytogenes cells may retain part of their virulence properties as well as their ability to adhere to food-processing surfaces. Transmission to food products and the introduction of these cells into the food chain are therefore plausible scenarios that are worth taking into consideration in terms of risk assessment. IMPORTANCE L. monocytogenes is the causative agent of listeriosis, a serious foodborne illness. Antimicrobial practices such as disinfectants used for the elimination of this pathogen in the food industry can produce a sublethally injured population fraction. Injured cells of this pathogen that may survive antimicrobial treatment may pose a food safety risk. Nevertheless, knowledge regarding how sublethal injury may impact important cellular traits and phenotypic responses of this pathogen is limited. This work suggests that sublethally injured L. monocytogenes cells maintain virulence and surface attachment potential and highlights the importance of the occurrence of sublethally injured cells regarding food safety.

The dormancy continuum hypothesis states that in response to stress, cells enter different stages of dormancy ranging from unstressed living cells to cell death, in order to ensure their long-term survival under adverse conditions. Exposure of Listeria monocytogenes cells to sublethal stressors related to food processing may induce sublethal injury and the viable-but-nonculturable (VBNC) state. In this study, exposure to acetic acid (AA), hydrochloric acid (HCl), and two disinfectants, peracetic acid (PAA) and sodium hypochlorite (SH), at 20°C and 4°C was used to evaluate the potential induction of L. monocytogenes strain Scott A into different stages of dormancy. To differentiate the noninjured subpopulation from the total population, tryptic soy agar with 0.6% yeast extract (TSAYE), supplemented or not with 5% NaCl, was used. Sublethally injured and VBNC cells were detected by comparing plate counts obtained with fluorescence microscopy and by using combinations of carboxyfluorescein and propidium iodide (viable/dead cells). Induction of sublethal injury was more intense after PAA treatment. Two subpopulations were detected, with phenotypes of untreated cells and small colony variants (SCVs). SCVs appeared as smaller colonies of various sizes and were first observed after 5 min of exposure to 5 ppm PAA at 20°C. Increasing the stress intensity from 5 to 40 ppm PAA led to earlier detection of SCVs. L. monocytogenes remained culturable after exposure to 20 and 30 ppm PAA for 3 h. At 40 ppm, after 3 h of exposure, the whole population was considered nonculturable, while cells remained metabolically active. These results corroborate the induction of the VBNC state. IMPORTANCE Sublethally injured and VBNC cells may evade detection, resulting in underestimation of a food product's microbial load. Under favorable conditions, cells may regain their growth capacity and acquire new resistant characteristics, posing a major threat for public health. Induction of the VBNC state is crucial for foodborne pathogens, such as L. monocytogenes, the detection of which relies almost exclusively on the use of culture recovery techniques. In the present study, we confirmed that sublethal injury is an initial stage of dormancy in L. monocytogenes that is followed by the VBNC state. Our results showed that PAA induced SCVs (a phenomenon potentially triggered by external factors) and the VBNC state in L. monocytogenes, indicating that tests of lethality based only on culturability may provide false-positive results regarding the effectiveness of an inactivation treatment.

The aim of the present study was to evaluate the effect of oxygen availability (aerobic, hypoxic and anoxic conditions) and sub-optimal pH (6.2 and 5.5) in a structured medium (10% w/V gelatin) on the growth of two immobilized L. monocytogenes strains (C5, 6179) at 10 °C and their subsequent acid resistance (pH 2.0, e.g., gastric acidity). Anaerobic conditions resulted in lower bacterial population (P < 0.05) (7.8-8.2 log CFU/mL) at the end of storage than aerobic and hypoxic environment (8.5-9.0 log CFU/mL), a phenomenon that was intensified at lower pH (5.5), where no significant growth was observed for anaerobically grown cultures. Prolonged habituation of L. monocytogenes (15 days) at both pH increased its acid tolerance resulting in max. 10 times higher t 4D (appx. 60 min). The combined effect though of oxygen availability and suboptimal pH on L. monocytogenes acid resistance was found to vary with the strain. Anoxically grown cultures at pH 5.5 exhibited the lowest tolerance towards lethal acid stress, with countable survivors occurring only until 20 min of exposure at pH 2.0. Elucidating the role of oxygen limiting conditions, often encountered in structured foods, on acid resistance of L. monocytogenes, would assist in assessing the capacity of L. monocytogenes originated from different food-related niches to withstand gastric acidity and possibly initiate infection., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

The foodborne pathogen L. monocytogenes can be present in food processing environments where it is exposed to various stressors. These antimicrobial factors, which aim to eliminate the pathogen, can induce sub-lethal injury to the bacterial cells. In the present study, we investigated the efficacy of different treatments (stresses) relevant to food processing and preservation as well as sanitation methods, in generating sub-lethal injury at 4 °C and 20 °C to two L. monocytogenes strains, ScottA and EGDe. Additionally, we evaluated the survival and extent of L. monocytogenes injury after exposure to commonly used disinfectants (peracetic acid and benzalkonium chloride), following habituation in nutrient-deprived, high-salinity medium. Each stress had a different impact on the survival and injury kinetics of L. monocytogenes. The highest injury levels were caused by peracetic acid which, at 4 °C, generated high populations of injured cells without loss of viability. Other injury-inducing stresses were lactic acid and heating. Long-term habituation in nutrient-limited and high salinity medium (4 °C) and subsequent exposure to disinfectants resulted in higher survival and injury in benzalkonium chloride and increased survival, yet with lower injury levels, in peracetic acid at 20 °C. Taken together, these results highlight the potential food safety risk emerging from the occurrence of injured cells by commonly used food processing methods. Consequently, in order to accurately assess the impact of an antimicrobial method, its potential of inducing sublethal injury needs to be considered along with lethality., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The coexistence and interactions among Listeria monocytogenes strains in combination with the structural characteristics of foods, may influence their growth capacity and thus, the final levels at the time of consumption. In the present study, we aimed to evaluate the effect of oxygen availability in combination with substrate micro-structure on growth and inter-strain interactions of L. monocytogenes. L. monocytogenes strains, selected for resistance to different antibiotics (to enable distinct enumeration), belonging to serotypes 4b (C5, ScottA), 1/2a (6179) and 1/2b (PL25) and were inoculated in liquid (Tryptic Soy Broth supplemented with Yeast Extract - TSB-YE) and solid (TSB-YE supplemented with 0.6% and 1.2% agar) media (2-3 log CFU/mL, g or cm 2 ), single or as two-strain cultures (1:1 strain-ratio). Aerobic conditions (A) were achieved with constant shaking or surface inoculation for liquid and solid media respectively, while static incubation or pour plated media corresponded to hypoxic environment (H). Anoxic conditions (An) were attained by adding 0.1% w/v sodium thioglycolate and paraffin overlay (for solid media). Growth was assessed during storage at 7 °C (n = 3 × 2). Inter-strain interactions were manifested by the difference in the final population between singly and co-cultured strains. Τhe extent of suppression increased with reduction in agar concentration, while the impact of oxygen availability was dependent on strain combination. During co-culture, in liquid and solid media, 6179 was suppressed by C5 by 4.0 (in TSB-YE under H) to 1.8 log units (in solid medium under An), compared to the single culture, which attained population of ca. 9.4 log CFU/mL or g. The growth of 6179 was also inhibited by ScottA by 2.7 and 1.9 log units, in liquid culture under H and An, respectively. Interestingly, in liquid medium under A, H and An, ScottA was suppressed by C5, by 3.3, 2.4 and 2.3 log units, respectively, while in solid media, growth inhibition was less pronounced. Investigating growth interactions in different environments could assist in explaining the dominance of L. monocytogenes certain serotypes., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Biofilm-forming ability may vary significantly among different Listeria (L.) monocytogenes strains. This interstrain variation is also observed in L. monocytogenes biofilm resistance to antimicrobial compounds commonly used in the food-processing environment. The screening of a large set of L. monocytogenes strains with specific characteristics, such as serotype, MLST type, and other genetic characteristics under various environmental conditions, may lead to a better understanding of the mechanisms underlying the establishment of the pathogen on food contact surfaces. In this chapter, traditional methods for L. monocytogenes strains characterization with regard to biofilm formation and novel biofilm control methods will be described.

Abstract: The ability of the enterocin A-B-P-producing Enterococcus faecium KE82 adjunct strain to inactivate Listeria monocytogenes during protected designation of origin Galotyri processing was evaluated. Three trials were conducted with artisan cheeses made from traditionally "boiled" (85°C) ewe's milk. The milk was cooled at 42°C and divided in two treatments. A1 milk was inoculated with Streptococcus thermophilus ST1 and Lactococcus lactis subsp. cremoris M78, and A2 was inoculated with the basic starter ST1+M78 plus KE82 (step 1). All milks were fermented at 20 to 22°C for 24 h (step 2), and the curds were drained at 12°C for 72 h (step 3) and then salted with 1.5 to 1.8% salt to obtain the fresh Galotyri cheeses (step 4). These fresh cheeses were then ripened at 4°C for 30 days (step 5). Because artificial listerial contamination in the dairy plant was prohibited, samples of A1 and A2 cheese milk (200 mL) or curd (200 g) were collected after steps 1 through 5, inoculated with L. monocytogenes 10 (3 to 4 log CFU/mL or g), incubated at 37, 22, 12, and 4°C for predefined periods, and analyzed for microbial levels and pH. L. monocytogenes levels declined in all cheese curd portions contaminated after steps 2 through 5 (pH 4.36 to 4.84) when stored at 4 or 12°C for 15 days. The final net reductions in Listeria populations were 2.00-, 1.07-, 0.54-, and 0.61-log greater in the A2 than in the A1 curd portions after steps 2, 3, 4, and 5, respectively. In step 1, conducted to simulate the whole cheese milk fermentation process, L. monocytogenes levels declined by 1.47 log CFU/mL more in the A2 than in the A1 milk portions after 72 h at 22°C; however, slight growth (0.6 log CFU/mL) occurred during the first 6 h at 37°C. E. faecium KE82 was compatible with the starter culture and enhanced inactivation of L. monocytogenes during all steps of Galotyri cheese processing. The antilisterial effects of the combined acid and enterocin were the weakest in the fermenting milks, the strongest in the unsalted fermented curds, and declined again in the salted fresh cheeses., (Copyright ©, International Association for Food Protection.)

Interactions between Listeria monocytogenes and food-associated or environmental bacteria are critical not only for the growth but also for a number of key biological processes of the microorganism. In this regard, limited information exists on the impact of other microorganisms on the virulence of L. monocytogenes In this study, the growth of L. monocytogenes was evaluated in a single culture or in coculture with L. innocua , Bacillus subtilis , Lactobacillus plantarum , or Pseudomonas aeruginosa in tryptic soy broth (10°C/10 days and 37°C/24 h). Transcriptional levels of 9 key virulence genes ( inlA , inlB , inlC , inlJ , sigB , prfA , hly , plcA , and plcB ) and invasion efficiency and intracellular growth in Caco-2 cells were determined for L. monocytogenes following growth in mono- or coculture for 3 days at 10°C or 9 h at 37°C. The growth of L. monocytogenes was negatively affected by the presence of L. innocua and B. subtilis , while the effect of cell-to-cell contact on L. monocytogenes growth was dependent on the competing microorganism. Cocultivation affected the in vitro virulence properties of L. monocytogenes in a microorganism-specific manner, with L. innocua mainly enhancing and B. subtilis reducing the invasion of the pathogen in Caco-2 cells. Assessment of the mRNA levels of L. monocytogenes virulence genes in the presence of the four tested bacteria revealed a complex pattern in which the observed up- or downregulation was only partially correlated with growth or in vitro virulence and mainly suggested that L. monocytogenes may display a microorganism-specific transcriptional response. IMPORTANCE Listeria monocytogenes is the etiological agent of the severe foodborne disease listeriosis. Important insight regarding the physiology and the infection biology of this microorganism has been acquired in the past 20 years. However, despite the fact that L. monocytogenes coexists with various microorganisms throughout its life cycle and during transmission from the environment to foods and then to the host, there is still limited knowledge related to the impact of surrounding microorganisms on L. monocytogenes ' biological functions. In this study, we showed that L. monocytogenes modulates specific biological activities (i.e., growth and virulence potential) as a response to coexisting microorganisms and differentially alters the expression of virulence-associated genes when confronted with different bacterial genera and species. Our work suggests that the interaction with different bacteria plays a key role in the survival strategies of L. monocytogenes and supports the need to incorporate biotic factors into the research conducted to identify mechanisms deployed by this organism for establishment in different environments., (Copyright © 2020 American Society for Microbiology.)

Like with all food microorganisms, many basic aspects of L. monocytogenes life are likely to be influenced by its interactions with bacteria living in close proximity. This pathogenic bacterium is a major concern both for the food industry and health organizations since it is ubiquitous and able to withstand harsh environmental conditions. Due to the ubiquity of Listeria monocytogenes, various strains may contaminate foods at different stages of the supply chain. Consequently, simultaneous exposure of consumers to multiple strains is also possible. In this context even strain-to-strain interactions of L. monocytogenes play a significant role in fundamental processes for the life of the pathogen, such as growth or virulence, and subsequently compromise food safety, affect the evolution of a potential infection, or even introduce bias in the detection by classical enrichment techniques. This article summarizes the impact of microbial interactions on the growth and detection of L. monocytogenes primarily in foods and food-associated environments. Furthermore it provides an overview of L. monocytogenes virulence in the presence of other microorganisms., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The heat resistance of a cocktail of five Salmonella strains and five L. monocytogenes strains was determined in teriyaki-marinated chicken breasts. Inoculated meat, packaged in bags, were completely immersed in a circulating water bath and cooked to a final temperature of 55, 57.5 or 60°C in 1h, and then held for predetermined times. The surviving Salmonella and L. monocytogenes cells were enumerated by surface plating on XLD agar and Palcam agar, respectively. D-values, determined by linear regression, of Salmonella in chicken breast ranged from 47.65min at 55°C to 7.48min at 60°C; the values for L. monocytogenes ranged from 54.81min at 55°C to 10.39min at 60°C. Marination rendered the pathogen more sensitive to the lethal effect of heat. The results of this study will assist the food industry in ensuring microbiological safety of sous-vide processed marinated chicken breasts., (Published by Elsevier Ltd.)

Different physicochemical and microbiological characteristics of cheeses may affect Listeria monocytogenes potential to grow, survive, or exhibit an acid adaptive response during storage and digestion. The objectives of the present study were to assess: i) the survival or growth potential of L.monocytogenes on various cheeses during storage, ii) the effect of initial indigenous microbiota on pathogen growth in comparison to expected growth curves retrieved by existing predictive models, and iii) the impact of habituation on/in cheeses surfaces on the subsequent acid resistance during simulated gastric digestion. Portions of cream (Cottage and Mascarpone), soft (Anthotyros, Camembert, Mastelo®, Manouri, Mozzarella, Ricotta), and semi-hard (Edam, Halloumi, Gouda) cheeses were inoculated with ca. 100CFU/g or cm 2 of L.monocytogenes and stored under vacuum or aerobic conditions at 7°C (n=4). The impact of varying (initial) levels of starter culture or indigenous spoilage microbiota on pathogen growth was evaluated by purchasing cheese packages on different dates in relation to production and expiration date (subsequently reflecting to different batches) mimicking a potential situation of cheese contamination with L.monocytogenes during retail display. Values of pH and a w were also monitored and used to simulate growth of L. monocytogenes by existing models and compare it with the observed data of the study. Survival in simulated gastric fluid (SGF) (pH1.5; HCl; max. 120min) was assessed at three time points during storage. Mascarpone, Ricotta, Mozzarella, Camembert, and Halloumi supported L.monocytogenes growth by 0.5-0.8logCFU/g or cm 2 per day, since low initial levels of total viable counts (TVC) (1.8-3.8logCFU/g or cm 2 ) and high pH/a w values (ca. 6.23-6.64/0.965-0.993) were recorded. On Cottage, Anthotyros, Manouri, Mastelo®, Edam, and Gouda, the pathogen survived at populations similar or lower than the inoculation level due to the high reported competition and/or low pH/a w during storage. L. monocytogenes growth was significantly suppressed (p<0.05) on samples purchased close to expiration date (bearing high TVC), compared to those close to production date, regardless of cheese. Cheeses which supported growth of L.monocytogenes enabled higher survival in gastric acidity along their shelf-life compared to cheeses which did not support growth. However, even in the latter cheeses (i.e., Cottage, Mastelo®, Gouda), total elimination of a persisting low initial contamination was not always achieved. Such findings may provide useful evidence for assessing the risk posed by various cheeses types in relation to their compliance with food safety regulations., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Listeria monocytogenes is a foodborne pathogen able to tolerate adverse conditions by forming biofilms or by deploying stress resistant mechanisms, and thus manages to survive for long periods in food processing plants. This study sought to investigate the correlation between biofilm forming ability, tolerance to disinfectants and cell surface characteristics of twelve L. monocytogenes strains. The following attributes were evaluated: (i) biofilm formation by crystal violet staining method on polystyrene, and by standard cell enumeration on stainless steel and polystyrene; (ii) hydrophobicity assay using solvents; (iii) minimum inhibitory concentration (MIC) and biofilm eradication concentration (BEC) of peracetic acid (PAA) and quaternary ammonium compounds (QACs), and (iv) resistance to sanitizers (PAA 2000ppm; QACs 500ppm) of biofilms on polystyrene and stainless steel. After 72h of incubation, higher biofilm levels were formed in TSB at 20°C, followed by TSB at 37°C (P=0.087) and diluted TSB 1/10 at both 20 (P=0.005) and 37°C (P=0.004). Cells grown at 30°C to the stationary phase had significant electron donating nature and a low hydrophobicity, while no significant correlation of cell surface properties to biofilm formation was observed. Strains differed in MIC PAA and BEC PAA by 24- and 15-fold, respectively, while a positive correlation between MIC PAA and BEC PAA was observed (P=0.02). The MIC QACs was positively correlated with the biofilm-forming ability on stainless steel (P=0.03). Regarding the impact of surface type, higher biofilm populations were enumerated on polystyrene than on stainless steel, which were also more tolerant to disinfectants. Among all strains, the greatest biofilm producer was a persistent strain with significant tolerance to QACs. These results may contribute to better understanding of L. monocytogenes behavior and survival on food processing surfaces., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Pathogens found on fresh produce may encounter low temperatures, high acidity and limited nutrient availability. The aim of this study was to evaluate the effect of habituation of Listeria monocytogenes on cherry tomatoes or lettuce leaves on its subsequent response to inhibitory levels of acid, osmotic and heat stress. Habituation was performed by inoculating lettuce coupons, whole cherry tomatoes or tryptic soy broth (TSB) with a three-strains composite of L. monocytogenes, which were further incubated at 5°C for 24 hours or 5 days. Additionally, cells grown overnight in TSB supplemented with 0.6% yeast extract (TSBYE) at 30°C were used as control cells. Following habituation, L. monocytogenes cells were harvested and exposed to: (i) pH 3.5 adjusted with lactic acid, acetic acid or hydrochloric acid (HCl), and pH 1.5 (HCl) for 6 h; (ii) 20% NaCl and (iii) 60°C for 150 s. Results showed that tomato-habituated L. monocytogenes cells were more tolerant (P < 0.05) to acid or osmotic stress than those habituated on lettuce, and habituation on both foods resulted in more stress resistant cells than prior growth in TSB. On the contrary, the highest resistance to heat stress (P < 0.05) was exhibited by the lettuce-habituated L. monocytogenes cells followed by TSB-grown cells at 5°C for 24 h, whereas tomato-habituated cells were highly sensitized. Prolonged starvation on fresh produce (5 days vs. 24 h) increased resistance to osmotic and acid stress, but reduced thermotolerance, regardless of the pre-exposure environment (i.e., tomatoes, lettuce or TSB). These results indicate that L. monocytogenes cells habituated on fresh produce at low temperatures might acquire resistance to subsequent antimicrobial treatments raising important food safety implications., Competing Interests: The authors have declared that no competing interests exist.

Listeria monocytogenes strains are widespread in the environment where they live well mixed, often resulting in multiple strains contaminating a single food sample. The occurrence of different strains in the same food might trigger strain competition, contributing to uneven growth of strains in food and to bias during selective procedures. We tested the growth of seven L. monocytogenes strains (C5, 6179, ScottA, PL24, PL25, PL26, PL27) on ham slices and on nutrient-rich agar at 10°C, singly and in combinations. Strains were made resistant to different antibiotics for their selective enumeration. In addition, growth of single strains (axenic culture) and competition between strains in xenic cultures of two strains was evaluated in enrichment broth and on selective agar. According to ISO 11290-1:1996/Amd 1:2004 standard protocol for detection of L. monocytogenes, two enrichment steps both followed by streaking on ALOA were performed. Strain cultures were directly added in the enrichment broth or used to inoculate minced beef and sliced hams which were then mixed with enrichment broth. 180-360 colonies were used to determine the relative percentage of each strain recovered on plates per enrichment step. The data showed a significant impact of co-cultivation on the growth of six out of seven strains on ham and a bias towards certain strains during selective enrichment. Competition was manifested by: (i) cessation of growth for the outcompeted strain when the dominant strain reached stationary phase, (ii) reduction of growth rates or (iii) total suppression of growth (both on ham and in enrichment broth or ALOA). Outgrowth of strains by their competitors on ALOA resulted in limited to no recovery, with the outcompeting strain accounting for up to 100% of the total recovered colonies. The observed bias was associated with the enrichment conditions (i.e. food type added to the enrichment broth) and the strain-combination. The outcome of growth competition on food or nonselective agar surface did not necessarily coincide with the results of competition during enrichment. The results show that certain strains present in foods may be missed during classical detection due to strain competition and such likelihood should be taken into consideration when resolving a listeriosis outbreak., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Multiple Listeria monocytogenes strains can be present in the same food sample; moreover, infection with more than one L. monocytogenes strain can also occur. In this study we investigated the impact of strain competition on the growth and in vitro virulence potential of L. monocytogenes. We identified two strong competitor strains, whose growth was not (or only slightly) influenced by the presence of other strains and two weak competitor strains, which were outcompeted by other strains. Cell contact was essential for growth inhibition. In vitro virulence assays using human intestinal epithelial Caco2 cells showed a correlation between the invasion efficiency and growth inhibition: the strong growth competitor strains showed high invasiveness. Moreover, invasion efficiency of the highly invasive strain was further increased in certain combinations by the presence of a low invasive strain. In all tested combinations, the less invasive strain was outcompeted by the higher invasive strain. Studying the effect of cell contact on in vitro virulence competition revealed a complex pattern in which the observed effects depended only partially on cell-contact suggesting that competition occurs at two different levels: i) during co-cultivation prior to infection, which might influence the expression of virulence factors, and ii) during infection, when bacterial cells compete for the host cell. In conclusion, we show that growth of L. monocytogenes can be inhibited by strains of the same species leading potentially to biased recovery during enrichment procedures. Furthermore, the presence of more than one L. monocytogenes strain in food can lead to increased infection rates due to synergistic effects on the virulence potential.

Cutting and shredding of leafy vegetables increases the risk of cross contamination in household settings. The distribution of Escherichia coli O157:H7 and Listeria monocytogenes transfer rates (Tr) between cutting knives and lettuce leaves was investigated and a semi-mechanistic model describing the bacterial transfer during consecutive cuts of leafy vegetables was developed. For both pathogens the distribution of log10Trs from lettuce to knife was towards low values. Conversely log10Trs from knife to lettuce ranged from -2.1 to -0.1 for E. coli O157:H7 and -2.0 to 0 for L. monocytogenes, and indicated a more variable phenomenon. Regarding consecutive cuts, a rapid initial transfer was followed by an asymptotic tail at low populations moving to lettuce or residing on knife. E. coli O157:H7 was transferred at slower rates than L. monocytogenes. These trends were sufficiently described by the transfer-model, with RMSE values of 0.426-0.613 and 0.531-0.908 for L. monocytogenes and E. coli O157:H7, respectively. The model showed good performance in validation trials but underestimated bacterial transfer during extrapolation experiments. The results of the study can provide information regarding cross contamination events in a common household. The constructed model could be a useful tool for the risk-assessment during preparation of leafy-green salads., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Analysis for Listeria monocytogenes by ISO11290-1 is time-consuming, entailing two enrichment steps and subsequent plating on agar plates, taking five days without isolate confirmation. The aim of this study was to determine if a polymerase chain reaction (PCR) assay could be used for analysis of the first and second enrichment broths, saving four or two days, respectively. In a comprehensive approach involving six European laboratories, PCR and traditional plating of both enrichment broths from the ISO11290-1 method were compared for the detection of L. monocytogenes in 872 food, raw material and processing environment samples from 13 different dairy and meat food chains. After the first and second enrichments, total DNA was extracted from the enriched cultures and analysed for the presence of L. monocytogenes DNA by PCR. DNA extraction by chaotropic solid-phase extraction (spin column-based silica) combined with real-time PCR (RTi-PCR) was required as it was shown that crude DNA extraction applying sonication lysis and boiling followed by traditional gel-based PCR resulted in fewer positive results than plating. The RTi-PCR results were compared to plating, as defined by the ISO11290-1 method. For first and second enrichments, 90% of the samples gave the same results by RTi-PCR and plating, whatever the RTi-PCR method used. For the samples that gave different results, plating was significantly more accurate for detection of positive samples than RTi-PCR from the first enrichment, but RTi-PCR detected a greater number of positive samples than plating from the second enrichment, regardless of the RTi-PCR method used. RTi-PCR was more accurate for non-food contact surface and food contact surface samples than for food and raw material samples especially from the first enrichment, probably because of sample matrix interference. Even though RTi-PCR analysis of the first enrichment showed less positive results than plating, in outbreak scenarios where a rapid result is required, RTi-PCR could be an efficient way to get a preliminary result to be then confirmed by plating. Using DNA extraction from the second enrichment broth followed by RTi-PCR was reliable and a confirmed result could be obtained in three days, as against seven days by ISO11290-1., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Listeria monocytogenes can colonize in the food processing environment and thus pose a greater risk of cross-contamination to food. One of the proposed mechanisms that facilitates such colonization is biofilm formation. As part of a biofilm, it is hypothesized that L. monocytogenes can survive sanitization procedures. In addition, biofilms are difficult to remove and may require additional physical and chemical mechanisms to reduce their presence and occurrence. The initial stage of biofilm formation is attachment to surfaces, and therefore it is important to be able to determine the ability of L. monocytogenes strains to attach to various inert surfaces. In this chapter, methods to study bacterial attachment to surfaces are described. Attachment is commonly induced by bringing planktonic cells into contact with plastic, glass, or stainless steel surfaces with or without food residues ("soil") in batch or continuous (e.g., with constant flow of nutrients) culture. Measurement of biofilm formed is carried out by detaching cells (with various mechanical methods) and measuring the viable counts or by measuring the total attached biomass. Resistance of biofilms to sanitizers is commonly carried out by exposure of the whole model surface bearing the attached cells to a solution of sanitizer, followed by measuring the survivors as described above.

In this study, mathematical models were developed to predict the growth probability and kinetic behavior of Listeria monocytogenes on fresh pork skin during storage at different temperatures. A 10-strain mixture of L. monocytogenes was inoculated on fresh pork skin (3 by 5 cm) at 4 log CFU/cm(2). The inoculated samples were stored aerobically at 4, 7, and 10 °C for 240 h, at 15 and 20 °C for 96 h, and at 25 and 30 °C for 12 h. The Baranyi model was fitted to L. monocytogenes growth data on PALCAM agar to calculate the maximum specific growth rate, lag-phase duration, the lower asymptote, and the upper asymptote. The kinetic parameters were then further analyzed as a function of storage temperature. The model simulated growth of L. monocytogenes under constant and changing temperatures, and the performances of the models were evaluated by the root mean square error and bias factor (Bf). Of the 49 combinations (temperature × sampling time), the combinations with significant growth (P < 0.05) of L. monocytogenes were assigned a value of 1, and the combinations with nonsignificant growth (P > 0.05) were given a value of 0. These data were analyzed by logistic regression to develop a model predicting the probabilities of L. monocytogenes growth. At 4 to 10 °C, obvious L. monocytogenes growth was observable after 24 h of storage; but, at other temperatures, the pathogen had obvious growth after 12 h of storage. Because the root mean square error value (0.184) and Bf (1.01) were close to 0 and 1, respectively, the performance of the developed model was acceptable, and the probabilistic model also showed good performance. These results indicate that the developed model should be useful in predicting kinetic behavior and calculating growth probabilities of L. monocytogenes as a function of temperature and time.

The acid resistance of Listeria monocytogenes was evaluated: (i) after short (shock) or long-term (adaptation during growth) exposure to reduced (5.5) or neutral (7.2) pH in a liquid (broth) medium or on a solid surface (agar), and (ii) after growth on the surface of ham and turkey slices or in homogenates of these products. Three L. monocytogenes strains (serotypes 1/2a, 1/2b and 4b) were individually inoculated at: (i) 10(4)-10(5)CFU/ml in tryptic soy broth with 0.6% yeast extract (TSBYE) or on tryptic soy agar with 0.6% yeast extract (TSAYE) at pH 7.2 with 1% (+G) or without (-G) glucose of or TSBYE and TSAYE with 0.25% glucose at pH 5.5 (lactic acid) and incubated at 20°C, and (ii) 10(2)-10(3)CFU/cm(2) on ham and turkey slices (pH 6.39-6.42; formulated with potassium lactate and sodium diacetate) or in their homogenates (1:4 and 1:9; representing viscous [slurry] and liquid residues [purge], respectively), and stored at 10°C. The acid resistance of each strain was assessed in TSBYE of pH 3.5 (lactic acid) for strains growing in broth or on agar surfaces, and in TSBYE of pH 1.5 (HCl) for strains growing on ham and turkey slices or in their homogenates. Habituation at pH 5.5 for 3 or 24h at 20°C increased acid (pH 3.5) resistance of all strains compared to the control (pH 7.2). Cells grown on the surface of TSAYE-G (pH 7.2 or 5.5) showed higher resistance than cells grown in broth (TSBYE-G), whereas the opposite was observed for cells grown on TSAYE + G or in TSBYE + G. Growth of L. monocytogenes on meat product slices was markedly slower than in homogenates. Pathogen reductions following exposure to pH 1.5, after 10 and 27days of storage were strain-dependent and in the ranges of 0.5-2.5, 1.3-4.5 and 4.0-7.6 log units for cells grown on product slices in 1:4 and 1:9 homogenates, respectively. The results suggest that L. monocytogenes cells growing on food surfaces or in viscous matrices may show higher resistance to lethal acid conditions than cells growing in liquid substrates., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The purpose of this work was to estimate the prevalence and concentration of Listeria monocytogenes in minced pork meat by the application of a Bayesian modeling approach. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media. Presence of the pathogen was confirmed through biochemical and molecular tests. Independent experiments (n = 10) for validation purposes were performed. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. Sensitivity and specificity varied depending on the culture method. L. monocytogenes concentration was estimated at 14-17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = -2.17%). The use of at least two culture media in parallel enhanced the efficiency of L. monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L. monocytogenes presence and the uncertainty of the results obtained., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The present study aimed to describe the growth/no growth interface of Listeria monocytogenes at three potential states of growth in industrial environments, namely attached, (Att), detached (Det) from a biofilm, or in a planktonic state (suspended; Plan). A 3-strain composite of L. monocytogenes cells was left to colonize stainless steel (SS) surfaces in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) at 20 °C for 72 h and then transferred to TSBYE at 30 different pH and NaCl concentrations, which were renewed every two days during incubation at 10 °C. Survival of attached population was observed at optimal conditions (pH 7.2, a(w) 0.996), whereas at 4.5-8.0% salt and/or pH<6.0, reduction of attached population on SS surfaces was observed. PFGE patterns showed that 91% of the cells colonizing the SS coupons after 30 days, at any pH and a(w) conditions, belonged to a single strain. Furthermore, the change in the probability of a single cell to initiate growth (P(in)) over time, as well as the number of cells needed (CN) for growth initiation of planktonically growing Plan and Det L. monocytogenes cells were evaluated based on MPN Tables. An ordinary logistic regression model was also used to describe the growth/no growth interface of varying inoculation levels (from <10 to 10(4)CFU/ml) of Plan and Det cells in response to pH and a(w). Although both cell types demonstrated similar growth limits at populations of 10(2)-10(4)CFU/ml, higher numbers of Det than Plan cells were needed (CN) in order to initiate growth at low a(w) and pH. Individual Plan cells reached higher maximum levels of probability of growth initiation (P(max)) and had shorter times to reach P(max)/2 (t(au)), compared to their Det counterparts. Data on growth potential of cells in suspension, attached or detached status, may assist in ranking the risk from different sources of contamination. In addition, they may establish the link between the behavior of L. monocytogenes in foods and its origin from the processing plant. The latter link is important component of biotraceability., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The resistance of Listeria monocytogenes biofilms formed under food processing conditions, against various sanitizing agents and disinfection procedures was evaluated in the present study. The first sanitation procedure included biofilm formation on stainless steel coupons (SS) placed in tryptic soy broth supplemented with 0.6% yeast extract (TSBYE) of various concentrations of NaCl (0.5, 7.5 and 9.5%) at different temperatures (5 and 20 °C). The biofilms formed were exposed to warm (60 °C) water for 20 min, or to peroxyacetic acid (2% PAA) for 1, 2, 3 and 6 min. Treatment with warm water caused no significant (P ≥ 0.05) reductions in the attached populations. Conversely, surviving bacteria on SS coupons decreased as the exposure time to 2% PAA increased and could not be detected by culture after 6 min of exposure. Biofilms formed at 20°C were more resistant to PAA than biofilms formed at 5 °C. Salt concentration in the growth medium had no marked impact on the resistance to PAA. The second sanitation procedure included biofilm formation of nonadapted (NA) and acid-adapted (AA) cells in TSBYE of pH 5.0 and 7.0 (i.e., NA-5.0, NA-7.0 and AA-5.0, AA-7.0) at 4 °C. Coupons bearing attached cells of L. monocytogenes were periodically exposed to chlorine (0.465% Cl(-)), quaternary ammonium compound (1% QAC) and 2% PAA. The resistance of attached cells to QAC, PAA and Cl(-) followed the order: AA-5.0>NA-7.0 ≥ AA-7.0>NA-5.0. The most effective sanitizer was QAC followed by PAA and Cl(-). The results can lead to the development of efficient sanitation strategies in order to eliminate L. monocytogenes from the processing environment. Furthermore, such results may explain the presence of L. monocytogenes after sanitation as a result of cell attachment history., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The aim of this study was to investigate the potential effect of adaptive stationary phase acid tolerance response (ATR) of Listeria monocytogenes Scott A cells on their attachment to stainless steel (SS) under low pH or/and high salt conditions and on the subsequent resistance of sessile cells to strong acid challenge. Nonadapted or acid-adapted stationary-phase L. monocytogenes cells were used to inoculate (ca. 10⁸ CFU/ml) Brain Heart (BH) broth (pH 7.4, 0.5% w/v NaCl) in test tubes containing vertically placed SS coupons (used as abiotic substrates for bacterial attachment). Incubation was carried out at 16 °C for up to 15 days, without any nutrient refreshment. L. monocytogenes cells, prepared as described above, were also exposed to low pH (4.5; adjusted with HCl) or/and high salt (5.5% w/v NaCl) stresses, during attachment. On the 5th, 10th and 15th day of incubation, cells attached to SS coupons were detached (through bead vortexing) and enumerated (by agar plating). Results revealed that ATR significantly (p<0.05) affected bacterial attachment, when the latter took place under moderate acidic conditions (pH 4.5, 0.5 or 5.5% w/v NaCl), with the acid-adapted cells adhering slightly more than the nonadapted ones. Regardless of acidity/salinity conditions during attachment, ATR also enhanced the resistance of sessile cells to subsequent lethal acid challenge (exposure to pH 2 for 6 min; pH adjusted with either hydrochloric or lactic acid). The trend observed with viable count data agreed well with conductance measurements, used to indirectly quantify remaining attached bacteria (following the strong acid challenge) via their metabolic activity. To sum, this study demonstrates that acid adaptation of L. monocytogenes cells during their planktonic growth enhances their subsequent attachment to SS under extended exposure (at 16 °C for up to 15 days) to mild acidic conditions (pH 4.5), while it also improves the resistance of sessile cells to extreme acid treatment (pH 2). Therefore, the ATR of bacterial cells should be carefully considered when applying acidic decontamination strategies to eradicate L. monocytogenes attached to food processing equipment., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The presence of pathogens in dairy products is often associated with contamination via bacteria attached to food-processing equipment, especially from areas where cleaning/sanitation is difficult. In this study, the attachment of Listeria monocytogenes on stainless steel (SS), followed by detachment and growth in foods, was evaluated under conditions simulating a dairy processing environment. Initially, SS coupons were immersed in milk, vanilla custard, and yogurt inoculated with the pathogen (10(7) CFU/ml or CFU/g) and incubated at two temperatures (5 and 20 degrees C) for 7 days. By the end of incubation, cells were mechanically detached from coupons and used to inoculate freshly pasteurized milk which was subsequently stored at 5 degrees C for 20 days. The suspended cells in all three products in which SS coupons were immersed were also used to inoculate freshly pasteurized milk (5 degrees C for 20 days). When SS coupons were immersed in milk, shorter lag phases were obtained for detached than for planktonically grown cells, regardless of the preincubation temperature (5 or 20 degrees C). The opposite was observed when custard incubated at 20 degrees C was used to prepare the two types of inocula. However, in this case, a significant increase in growth rate was also evident when the inoculum was derived from detached cells. In another parallel study, while L. monocytogenes was not detectable on SS coupons after 7 days of incubation (at 5 degrees C) in inoculated yogurt, marked detachment and growth were observed when these coupons were subsequently transferred and incubated at 5 degrees C in fresh milk or/and custard. Overall, the results obtained extend our knowledge on the risk related to contamination of dairy products with detached L. monocytogenes cells.

During food processing, microorganisms are commonly exposed to multiple sublethal or lethal stresses (commonly a(w), pH) sequentially or simultaneously. The objectives of the present study were: (i) to comparatively evaluate the survival of Listeria monocytogenes and Salmonella Typhimurium in lethal acid (pH 4.0 and 4.5 with lactic acid) or osmotic conditions (15 and 20% NaCl), applied singly, sequentially (pH then NaCl or NaCl then pH), or simultaneously at 5 and 10 degrees C; and, (ii) to quantify the effect of osmotic shifts at pH 7.0, 6.0 or 5.0 (adjusted with lactic acid) on the lag phase and growth rate of L. monocytogenes at 10 degrees C. In sequential lethal stress applications, the second stress was applied 2 or 3 days after the first for Salmonella and L. monocytogenes, respectively. Acid tolerance of L. monocytogenes was higher than osmotic tolerance and the opposite was observed for Salmonella. Higher inactivation was observed after exposure to pH 4.0 compared to pH 4.5 as well as after exposure to 20% NaCl compared to 15% NaCl. Exposure to stresses sequentially resulted in faster (P<0.05) reductions than the exposure to single or double stresses applied simultaneously. The pH then NaCl sequence was more detrimental for pathogens than the reverse sequence. Incubation temperature (5 and 10 degrees C) did not show any profound (P<0.05) effect on microbial inactivation. When L. monocytogenes was incubated at a(w) 0.930 or 0.995 at 30 degrees C, then the lag phase increased both in subsequent osmotic downshift and upshift, respectively, at 10 degrees C. Shorter lag phase and higher ability to initiate growth at lower a(w) was observed after pre-adaptation at pH 6.0 or 5.0 compared to neutral pH. The results may contribute to the review of critical limits in low pH (with lactic acid) and water activity products, considering the risk of L. monocytogenes and Salmonella survival. In addition, the present indications may address the points in processing where stricter sanitation procedures should be applied in order to minimize the risk of survivors.

This study aimed to evaluate the effects of the level and sequence of hurdles, applied during growth, on the subsequent heat and acid tolerances of a 10-strain composite of Listeria monocytogenes. Individual strains were grown in glucose-free tryptic soy broth with 0.6% yeast extract (TSBYE-G). Then cultures were mixed and inoculated in fresh TSBYE-G (0.5% NaCl, pH 7.42; control), TSBYE-G that was supplemented with 3% NaCl (3.5% NaCl in total), or TSBYE-G with pH adjusted to 6.01 or 5.04 with lactic acid and incubated at 30 degrees C for 24 h. Furthermore, the culture composite was exposed to the following five combinations of double sequential hurdles (12 h in each at 30 degrees C): NaCl then pH 6.01, NaCl then pH 5.04, pH 7.42 then NaCl, pH 5.04 then NaCl, and pH 6.01 then NaCl. The heat and acid tolerances of the culture were assessed at 57 degrees C (for 2 h) and at pH 3.5 (for 7 h), respectively, in TSBYE-G. No significant (P > or = 0.05) differences in thermotolerance were observed among cultures exposed to various stresses. In contrast, the acid resistance followed the order: pH 6.01 = NaCl > NaCl then pH 5.04 > pH 6.01 then NaCl = pH 5.04 > pH 5.04 then NaCl > pH 7.42 then NaCl > control. The results suggest that exposure of L. monocytogenes to NaCl and low pH during growth may not affect its heat (57 degrees C) tolerance, but it may increase its acid (pH 3.5) resistance, depending on the sequence and intensity of the applied stresses.

The majority of published studies on the adaptive heat or acid tolerance response of Listeria monocytogenes have been performed with a single strain exposed to a single adaptation treatment; however, in food ecosystems, microorganisms commonly exist as multi-species communities and encounter multiple stresses, which may result in "stress hardening". Therefore, the present study evaluated the adaptive responses to heat (52, 57 and 63 degrees C) or lactic acid (pH 3.5) of a 10-strain composite of L. monocytogenes meat and human isolates at stationary phase, following exposure to combinations of osmotic (10% NaCl), acidic (pH 5.0 with HCl) and thermal (T; 46 degrees C) stresses, sequentially or simultaneously within 1.5h, in tryptic soy broth with 0.6% yeast extract (TSBYE). All treatments induced adaptive responses on L. monocytogenes at 57 degrees C, while no such cross-protection was observed at 52 and 63 degrees C. Survivor curves at 57 degrees C appeared convex with profound shoulders determined by a Weibull model. The highest thermotolerance was observed after combined exposure to acid and heat shock (pH-T), followed by exposure to osmotic shock, and by the combination of osmotic with heat shock (NaCl-T). Regarding acid tolerance, prior exposure to low pH, pH-T, or a combination of NaCl, pH and T resulted in a marked increase of resistance to pH 3.5, showing concave inactivation curves with tails at higher levels of survivors (log(10)CFU ml(-1)) than the control cultures. The sequence of exposure to sublethal stresses did not affect the thermotolerance of L. monocytogenes, whereas simultaneous exposure to most multiple stresses (e.g., NaCl-pH-T, NaCl-T and NaCl-pH) resulted in higher survivors of L. monocytogenes at pH 3.5 than exposure to the same stresses sequentially. The results indicate that combinations and sequences of sublethal hurdles may affect L. monocytogenes acid and heat tolerance, especially in acidic environments with mild heating or in low moisture environments.

The survival of postprocess Listeria monocytogenes contamination on sliced salami, stored under the temperatures associated with retail and domestic storage, was investigated. Sliced salami was inoculated with low and high concentrations of L. monocytogenes before being packaged under vacuum or air. Survival of L. monocytogenes was determined after storage of sausages for 45 or 90 days for low or high sample inocula, respectively, at 5, 15, and 25 degrees C. All survival curves of L. monocytogenes were characterized by an initial rapid inactivation within the first days of storage, followed by a second, slower inactivation phase or "tailing." Greater reduction of L. monocytogenes was observed at the high storage temperature (25 degrees C), followed by ambient (15 degrees C) and chill (5 degrees C) storage conditions. Moreover, vacuum packaging resulted in a slower destruction of L. monocytogenes than air packaging, and this effect increased as storage temperature decreased. Although L. monocytogenes numbers decreased to undetectable levels by the end of the storage period, the time (in days) needed for this reduction and for the total elimination of the pathogen decreased with high temperature, aerobic storage, and high inoculum. Results of this study clearly indicated that the kinetics of L. monocytogenes were highly dependent on the interaction of factors such as storage temperature, packaging conditions, and initial level of contamination (inoculum). These results may contribute to the exposure assessment of quantitative microbial risk assessment and to the establishment of storage-packaging recommendations of fermented sausages.

The effect of aerobic and anaerobic conditions on growth initiation by a 10-strain composite of Listeria monocytogenes (10(4) CFU/ml) was evaluated in tryptic soy broth with 0.6% yeast extract (TSBYE) as a function of 220 combinations of pH (3.82 to 7.42), sodium lactate (SL) (0 to 10%, vol/vol), and sodium diacetate (SD) (0 to 0.5%, wt/vol) at 10 or 30 degrees C (a slightly abusive and the optimal growth temperature, both above the growth limiting range of 0 to 3 degrees C for L. monocytogenes) in 96-well microplates. In addition, four probability-of-growth models were developed to quantify the effect of 346 aerobic and 346 anaerobic combinations of temperature (4 to 30 degrees C), SL (0 to 6%, vol/vol), and SD (0 to 0.5%, wt/vol) in the presence of NaCl (0.5 or 2.5%, wt/vol) on the growth-no growth responses of the same L. monocytogenes strain composite, with a microplate reader. Growth responses were evaluated turbidimetrically (620 nm) every 5 days for a total of 40 days. Data were modeled with logistic regression to determine the growth-no growth interfaces. The minimum pH values at which growth of L. monocytogenes occurred were higher under anaerobic than under aerobic conditions, and this difference was more evident at 10 degrees C or at higher SL and SD concentrations. The MIC of SD decreased with increasing SL levels. Anaerobic storage reduced the levels of SL-SD, allowing the growth of L. monocytogenes compared with aerobic storage, especially at low temperatures. In the presence of 2.5% NaCl, the MICs for SD were lower than those obtained with 0.5% NaCl, especially at 4 and 10 degrees C, or in the presence of 5 to 6% SL. The developed models for anaerobic incubation showed good performance (80% successful predictions; i.e., in 40 of 50 comparisons) with independent data from studies on survival-growth of L. monocytogenes on meat products. The study provides quantitative data on the antimicrobial activity of SL (0 to 10%) and SD (0 to 0.5%), temperature (4 to 30 degrees C), and pH (3.82 to 7.42) and on the probability of growth of L. monocytogenes under anaerobic or aerobic conditions in the presence of 0.5 or 2.5% NaCl, and hence, addresses important needs for risk assessment activities.

The fate of acid-adapted and nonadapted Listeria monocytogenes inoculated onto bologna slices (formulated with or without antimicrobials) was examined during storage and after exposure to in vitro gastric challenge. Bologna slices formulated with no antimicrobials (control), 3% sodium lactate (SL), or 1.8% SL plus 0.25% sodium diacetate (SD) were inoculated (2 log CFU/cm2) with a 10-strain composite of acid-adapted or nonadapted L. monocytogenes strains. Growth or survival of the two inocula on bologna was evaluated during vacuum-packaged storage (10 degrees C) for up to 36 days. Survival of previously acid-adapted or nonadapted L. monocytogenes on stored bologna exposed to simulated gastric fluid (adjusted to pH 1.0 with HCl) for 20, 40, and 60 min also was determined. As expected, inclusion of antimicrobials in the product formulation inhibited growth of L. monocytogenes during storage of vacuum-packaged bologna compared with growth on control samples. Acid adaptation of L. monocytogenes prior to product inoculation did not affect subsequent survival or growth on bologna or resistance to simulated gastric fluid (P > 0.05). Survival of L. monocytogenes exposed to simulated gastric fluid during storage increased with product age, growth phase of the cells, and possibly age of the cells, particularly for control samples (no antimicrobials), in which the pathogen grew uninhibited to approximately 6 log CFU/cm2 by day 8 of storage. Inhibition of L. monocytogenes growth on product formulated with antimicrobials was associated with only sporadic and small numbers of survivors following exposure of these samples to simulated gastric fluid, especially in samples stored longer. However, cell numbers in these treatment groups before the gastric challenge did not exceed 3.8 log CFU/cm2. Inhibition of growth on product with antimicrobials precluded detection of survivors resistant to the effects of simulated gastric fluid.

This study evaluated post-processing chemical solutions for their antilisterial effects on commercial smoked sausage formulated with or without 1.5% potassium lactate plus 0.05% sodium diacetate, and contaminated (approximately 3-4 log cfu/cm(2)) with 10-strain composite Listeria monocytogenes inocula prepared under various conditions. Inoculated samples were left untreated, or were immersed (2 min, 25 +/- 2 degrees C) in solutions of acetic acid (2.5%), lactic acid (2.5%), potassium benzoate (5%) or Nisaplin (0.5%, equivalent to 5000 IU/ml of nisin) alone, and in sequence (Nisaplin followed by acetic acid, lactic acid or potassium benzoate), before vacuum packaging and storage at 10 degrees C (48 days). Acetic acid, lactic acid or potassium benzoate applied alone reduced initial L. monocytogenes populations by 0.4-1.5 log cfu/cm(2), while treatments including Nisaplin caused reductions of 2.1-3.3 log cfu/cm(2). L. monocytogenes on untreated sausage formulated with antimicrobials had a lag phase duration of 10.2 days and maximum specific growth rate (mu(max)) of 0.089 per day, compared to no lag phase and mu(max) of 0.300 per day for L. monocytogenes on untreated product that did not contain antimicrobials in the formulation. The immersion treatments inhibited growth of the pathogen for 4.9-14.8 days on sausage formulated without potassium lactate-sodium diacetate; however, in all cases significant (P < 0.05) growth occurred by the end of storage. The antilisterial activity of chemical solutions was greatly enhanced when applied to product formulated with antimicrobials; growth was completely inhibited on sausage treated with acetic or lactic acid alone, and in sequence with Nisaplin. In general, habituation (15 degrees C, 7 days) of L. monocytogenes cells, planktonically or as attached cells to stainless-steel coupons in sausage homogenate prior to contamination of product, resulted in shorter lag phase durations compared with cells cultivated planktonically in a broth medium. Furthermore, when present, high levels of spoilage flora were found to suppress growth of the pathogen. Findings of this study could be useful to US meat processors in their efforts to select required regulatory alternatives for control of post-processing contamination in meat products.

The antilisterial effect of postprocess antimicrobial treatments on commercially manufactured frankfurters formulated with and without a 1.5% potassium lactate-0.05% sodium diacetate combination was evaluated. Frankfurters were inoculated (ca. 3 to 4 log CFU/cm2) with 10-strain composite Listeria monocytogenes cultures originating from different sources. The inocula evaluated were cells grown planktonically in tryptic soy broth plus 0.6% yeast extract (30 degrees C, 24 h) or in a smoked sausage homogenate (15 degrees C, 7 days) and cells that had been removed from stainless steel coupons immersed in an inoculated smoked sausage homogenate (15 degrees C, 7 days). Inoculated frankfurters were dipped (2 min, 25 +/- 2 degrees C) in acetic acid (AA; 2.5%), lactic acid (LA; 2.5%), potassium benzoate (PB; 5%), or Nisaplin (commercial form of nisin; 0.5%, equivalent to 5,000 IU/ml of nisin) solutions, or in Nisaplin followed by AA, LA, or PB, and were subsequently vacuum packaged and stored for 48 days at 10 degrees C. In addition to microbiological analyses, sensory evaluations were performed with uninoculated samples that had been treated with AA, LA, or PB for 2 min. Initial L. monocytogenes populations were reduced by 1.0 to 1.8 log CFU/cm2 following treatment with AA, LA, or PB solutions, and treatments that included Nisaplin reduced initial levels by 2.4 to >3.8 log CFU/ cm2. All postprocessing treatments resulted in some inhibition of L. monocytogenes during the initial stages of storage of frankfurters that were not formulated with potassium lactate-sodium diacetate; however, in all cases, significant (P < 0.05) growth occurred by the end of storage. The dipping of products formulated with potassium lactate-sodium diacetate in AA or LA alone--or in Nisaplin followed by AA, LA, or PB-increased lag-phase durations and lowered the maximum specific growth rates of the pathogen. Moreover, depending on the origin of the inoculum, this dipping of products led to listericidal effects. In general, differences in growth kinetics were obtained for the three inocula that were used to contaminate the frankfurters. Possible reasons for these differences include the presence of stress-adapted subpopulations and the inhibition of the growth of the pathogen due to high levels of spoilage microflora. The dipping of frankfurters in AA, LA, or PB did not (P > 0.05) affect the sensory attributes of the product when compared to the control samples. The data generated in this study may be useful to U.S. ready-to-eat meat processors in their efforts to comply with regulatory requirements.

The objective of this study was to model the effect of drying temperatures (52, 57, and 63 degrees C) and predrying treatments on the inactivation of Listeria monocytogenes on beef jerky. Before drying, beef slices were inoculated with a 10-strain composite of L. monocytogenes and then treated with the following: (i) nothing (C), (ii) traditional marinade (M), or (iii) dipping in 5% acetic acid solution for 10 min, followed by M (AM). In addition, sequential stresses (exposure to 10% NaCl, followed by an adjustment of the pH to 5.0 and, subsequently, a water bath at 45 degrees C) were applied to the inocula before beef contamination and drying at 63 degrees C. Surviving L. monocytogenes were determined on tryptic soy agar plus 0.6% yeast extract (TSAYE) and on PALCAM agar at 0, 2, 4, 6, 8, and 10 h during drying. Data were modeled by a linear regression (treatment AM) and a logistic-based equation capable of fitting biphasic inactivation curves without initial shoulder (treatments C and M). The total log reductions expressed as the CFU per square centimeter of L. monocytogenes (3.9 to 5.1) for the samples treated with M (3.5 to 5.4) when compared with C were similar, whereas AM-treated samples had higher (6.1 to 6.8) reductions. All survival curves were characterized by an initial rapid decrease in populations within the first 2 h, which was followed by a secondary death phase at a lower rate. No significant (P > or = 0.05) differences in inactivation were observed due to drying temperatures in the range (52 to 63 degrees C) tested. Inactivation differences between recovered counts of stressed and unstressed cells were significant (P < 0.05) in PALCAM but not in TSAYE. The acidified predrying treatment (AM) had higher pathogen inactivation during drying than other treatments, regardless of drying temperature. The models developed may be useful in designing effective drying processes for beef jerky.

The effect of gamma irradiation on the natural microflora of whole salted vacuum-packaged trout at 4 and 10 degrees C was studied. In addition, the effectiveness of gamma irradiation in controlling Listeria monocytogenes inoculated into trout was investigated. Irradiation at doses of 0.5 and 2 kGy affected populations of bacteria, namely, Pseudomonas spp., Brochothrix thermosfacta, lactic acid bacteria, H2S-producing bacteria typical of Shewanella putrefaciens, and Enterobacteriaceae, at both 4 and 10 degrees C. This effect was more pronounced at the higher dose (2 kGy) and the lower temperature (4 degrees C). Pseudomonads, H2S-producing bacteria typical of S. putrefaciens, and Enterobacteriaceae showed higher sensitivity to gamma irradiation than did the rest of the microbial species. Sensory evaluation did not show a good correlation with bacterial populations. On the basis of sensory odor scores, a shelf life of 28 days (2 kGy, 4 degrees C) was obtained for salted vacuum-packaged freshwater trout, compared with a shelf life of 7 days for the unirradiated sample. Under the same conditions, the growth of L. monocytogenes inoculated into the samples was suppressed by 2 log cycles after irradiation (2 kGy) and storage for up to 18 days at 4 degrees C.

Lately, the inclusion of additional lactic acid bacteria (LAB) strains to cheeses is becoming more popular since they can affect cheese's nutritional, technological, and sensory properties, as well as increase the product's safety. This work studied the effect of Lactiplantibacillus pentosus L33 and Lactiplantibacillus plantarum L125 free cells and supernatants on feta cheese quality and Listeria monocytogenes fate. In addition, rapid and non-invasive techniques such as Fourier transform infrared (FTIR) and multispectral imaging (MSI) analysis were used to classify the cheese samples based on their sensory attributes. Slices of feta cheese were contaminated with 3 log CFU/g of L. monocytogenes, and then the cheese slices were sprayed with (i) free cells of the two strains of the lactic acid bacteria (LAB) in co-culture (F, ~5 log CFU/g), (ii) supernatant of the LAB co-culture (S) and control (C, UHT milk) or wrapped with Na-alginate edible films containing the pellet (cells, FF) or the supernatant (SF) of the LAB strains. Subsequently, samples were stored in air, in brine, or in vacuum at 4 and 10 °C. During storage, microbiological counts, pH, and water activity (aw) were monitored while sensory assessment was conducted. Also, in every sampling point, spectral data were acquired by means of FTIR and MSI techniques. Results showed that the initial microbial population of Feta was ca. 7.6 log CFU/g and consisted of LAB (>7 log CFU/g) and yeast molds in lower levels, while no Enterobacteriaceae were detected. During aerobic, brine, and vacuum storage for both temperatures, pathogen population was slightly postponed for S and F samples and reached lower levels compared to the C ones. The yeast mold population was slightly delayed in brine and vacuum packaging. For aerobic storage at 4 °C, an elongation in the shelf life of F samples by 4 days was observed compared to C and S samples. At 10 °C, the shelf life of both F and S samples was extended by 13 days compared to C samples. FTIR and MSI analyses provided reliable estimations of feta quality using the PLS-DA method, with total accuracy (%) ranging from 65.26 to 84.31 and 60.43 to 89.12, respectively. In conclusion, the application of bioprotective LAB strains can result in the extension of feta's shelf life and provide a mild antimicrobial action against L. monocytogenes and spoilage microbiota. Furthermore, the findings of this study validate the effectiveness of FTIR and MSI techniques, in tandem with data analytics, for the rapid assessment of the quality of feta samples. [ABSTRACT FROM AUTHOR]

Globally, fresh vegetables or minimally processed salads have been implicated in several foodborne disease outbreaks. This work studied the effect of Lactiplantibacillus pentosus FMCC-B281 cells (F) and its supernatant (S) on spoilage and on the fate of Listeria monocytogenes and Escherichia coli O157:H7 on fresh-cut ready-to-eat (RTE) salads during storage. Also, Fourier transform infrared (FTIR) and multispectral imaging (MSI) analysis were used as rapid and non-destructive techniques to estimate the microbiological status of the samples. Fresh romaine lettuce, rocket cabbage, and white cabbage were used in the present study and were inoculated with L. pentosus and the two pathogens. The strains were grown at 37 °C for 24 h in MRS and BHI broths, respectively, and then were centrifuged to collect the supernatant and the pellet (cells). Cells (F, ~5 log CFU/g), the supernatant (S), and a control (C, broth) were used to spray the leaves of each fresh vegetable that had been previously contaminated (sprayed) with the pathogen (3 log CFU/g). Subsequently, the salads were packed under modified atmosphere packaging (10%CO2/10%O2/80%N2) and stored at 4 and 10 °C until spoilage. During storage, microbiological counts and pH were monitored in parallel with FTIR and MSI analyses. The results showed that during storage, the population of the pathogens increased for lettuce and rocket independent of the treatment. For cabbage, pathogen populations remained stable throughout storage. Regarding the spoilage microbiota, the Pseudomonas population was lower in the F samples, while no differences in the populations of Enterobacteriaceae and yeasts/molds were observed for the C, F, and S samples stored at 4 °C. According to sensory evaluation, the shelf-life was shorter for the control samples in contrast to the S and F samples, where their shelf-life was elongated by 1–2 days. Initial pH values were ca. 6.0 for the three leafy vegetables. An increase in the pH of ca. 0.5 values was recorded until the end of storage at both temperatures for all cases of leafy vegetables. FTIR and MSI analyses did not satisfactorily lead to the estimation of the microbiological quality of salads. In conclusion, the applied bioprotective strain (L. pentosus) can elongate the shelf-life of the RTE salads without an effect on pathogen growth. [ABSTRACT FROM AUTHOR]

Listeria monocytogenes (in the meat, fish and seafood, dairy and fruit and vegetable sectors), Salmonella enterica (in the feed, meat, egg and low moisture food sectors) and Cronobacter sakazakii (in the low moisture food sector) were identified as the bacterial food safety hazards most relevant to public health that are associated with persistence in the food and feed processing environment (FFPE). There is a wide range of subtypes of these hazards involved in persistence in the FFPE. While some specific subtypes are more commonly reported as persistent, it is currently not possible to identify universal markers (i.e. genetic determinants) for this trait. Common risk factors for persistence in the FFPE are inadequate zoning and hygiene barriers; lack of hygienic design of equipment and machines; and inadequate cleaning and disinfection. A well‐designed environmental sampling and testing programme is the most effective strategy to identify contamination sources and detect potentially persistent hazards. The establishment of hygienic barriers and measures within the food safety management system, during implementation of hazard analysis and critical control points, is key to prevent and/or control bacterial persistence in the FFPE. Once persistence is suspected in a plant, a 'seek‐and‐destroy' approach is frequently recommended, including intensified monitoring, the introduction of control measures and the continuation of the intensified monitoring. Successful actions triggered by persistence of L. monocytogenes are described, as well as interventions with direct bactericidal activity. These interventions could be efficient if properly validated, correctly applied and verified under industrial conditions. Perspectives are provided for performing a risk assessment for relevant combinations of hazard and food sector to assess the relative public health risk that can be associated with persistence, based on bottom‐up and top‐down approaches. Knowledge gaps related to bacterial food safety hazards associated with persistence in the FFPE and priorities for future research are provided. [ABSTRACT FROM AUTHOR]

The aim of the present study was to assess the effect of sourdough related parameters on the growth and plantaricin activity of six Lactiplantibacillus plantarum strains against a mixture of 5 Listeria monocytogenes strains and to analyze the transcriptomic response of their pln genes. Parameters included 3 substrates (MRS broth, mMRS broth, WFE), 3 temperatures (20, 30, 37 °C), 2 initial pH values (5.0, 6.0), 2 NaCl concentrations (0.0, 1.8%) and 12 time points (ranging from 0 to 33 h). The transcriptomic response of the plantaricin genes to the aforementioned parameters was assessed after 21 h of growth. In general, plantaricin activity was strain dependent with that of Lp. plantarum strains LQC 2422, 2441, 2485 and 2516, harboring four pln genes, namely, pln423 (plαA), plαB, plαC and plαD, reaching 2560 AU/mL. However, strains LQC 2320 and 2520, in which 18 pln genes were detected, namely, plNC8a, plNC8b, plNC8c, plnL, plnR, plnJ, plnK, plnE, plnF, plnH, plnS, plnY, plNC8-IF, plNC8-HK, plnD, plnI, plnM and plnG, exhibited plantaricin activity barely reaching 160 AU/mL. Substrate, temperature, initial pH value and strains significantly affected plantaricin activity, while NaCl had only a marginal effect. Similarly, growth substrate and temperature had a more pronounced effect than initial pH value on gene transcription. A strong correlation between the transcription of the genes belonging to the same locus was observed; however, only a weak correlation, if any, was observed between plantaricin activity and the transcription of the genes assessed. [ABSTRACT FROM AUTHOR]

A multi‐country outbreak of Listeria monocytogenes ST6 linked to blanched frozen vegetables (bfV) took place in the EU (2015–2018). Evidence of food‐borne outbreaks shows that L. monocytogenes is the most relevant pathogen associated with bfV. The probability of illness per serving of uncooked bfV, for the elderly (65–74 years old) population, is up to 3,600 times greater than cooked bfV and very likely lower than any of the evaluated ready‐to‐eat food categories. The main factors affecting contamination and growth of L. monocytogenes in bfV during processing are the hygiene of the raw materials and process water; the hygienic conditions of the food processing environment (FPE); and the time/Temperature (t/T) combinations used for storage and processing (e.g. blanching, cooling). Relevant factors after processing are the intrinsic characteristics of the bfV, the t/T combinations used for thawing and storage and subsequent cooking conditions, unless eaten uncooked. Analysis of the possible control options suggests that application of a complete HACCP plan is either not possible or would not further enhance food safety. Instead, specific prerequisite programmes (PRP) and operational PRP activities should be applied such as cleaning and disinfection of the FPE, water control, t/T control and product information and consumer awareness. The occurrence of low levels of L. monocytogenes at the end of the production process (e.g. < 10 CFU/g) would be compatible with the limit of 100 CFU/g at the moment of consumption if any labelling recommendations are strictly followed (i.e. 24 h at 5°C). Under reasonably foreseeable conditions of use (i.e. 48 h at 12°C), L. monocytogenes levels need to be considerably lower (not detected in 25 g). Routine monitoring programmes for L. monocytogenes should be designed following a risk‐based approach and regularly revised based on trend analysis, being FPE monitoring a key activity in the frozen vegetable industry. [ABSTRACT FROM AUTHOR]