Guidance on validation of lethal control measures for foodborne pathogens in foods.

Food manufacturers are required to obtain scientific and technical evidence that a control measure or combination of control measures is capable of reducing a significant hazard to an acceptable level that does not pose a public health risk under normal conditions of distribution and storage. A validation study provides evidence that a control measure is capable of controlling the identified hazard under a worst-case scenario for process and product parameters tested. It also defines the critical parameters that must be controlled, monitored, and verified during processing. This review document is intended as guidance for the food industry to support appropriate validation studies, and aims to limit methodological discrepancies in validation studies that can occur among food safety professionals, consultants, and third-party laboratories. The document describes product and process factors that are essential when designing a validation study, and gives selection criteria for identifying an appropriate target pathogen or surrogate organism for a food product and process validation. Guidance is provided for approaches to evaluate available microbiological data for the target pathogen or surrogate organism in the product type of interest that can serve as part of the weight of evidence to support a validation study. The document intends to help food manufacturers, processors, and food safety professionals to better understand, plan, and perform validation studies by offering an overview of the choices and key technical elements of a validation plan, the necessary preparations including assembling the validation team and establishing prerequisite programs, and the elements of a validation report.

Modeling and Validation of the Ecological Behavior of Wild-Type Listeria monocytogenes and Stress-Resistant Variants.

UNLABELLED: Listeria monocytogenes exhibits a heterogeneous response upon stress exposure which can be partially attributed to the presence of stable stress-resistant variants. This study aimed to evaluate the impact of the presence of stress-resistant variants of Listeria monocytogenes and their corresponding trade-offs on population composition under different environmental conditions. A set of stress robustness and growth parameters of the wild type (WT) and an rpsU deletion variant was obtained and used to model their growth behavior under combined mild stress conditions and to model their kinetics under single- and mixed-strain conditions in a simulated food chain. Growth predictions for the WT and the rpsU deletion variant matched the experimental data generally well, although some deviations from the predictions were observed. The data highlighted the influence of the environmental conditions on the ratio between the WT and variant. Prediction of performance in the simulated food chain proved to be challenging. The trend of faster growth and lower stress robustness for the WT than for the rpsU variant in the different steps of the chain was confirmed, but especially for the inactivation steps and the time needed to resume growth after an inactivation step, the experimental data deviated from the model predictions. This report provides insights into the conditions which can select for stress-resistant variants in industrial settings and discusses their potential persistence in food processing environments. IMPORTANCE: Listeria monocytogenes exhibits a heterogeneous stress response which can partially be attributed to the presence of genetic variants. These stress-resistant variants survive better under severe conditions but have, on the other hand, a reduced growth rate. To date, the ecological behavior and potential impact of the presence of stress-resistant variants is not fully understood. In this study, we quantitatively assessed growth and inactivation behavior of wild-type L. monocytogenes and its stress-resistant variants. Predictions were validated under different conditions, as well as along a model food chain. This work illustrates the effects of environmental factors on population dynamics of L. monocytogenes and is a first step in evaluating the impact of population diversity on food safety.

Spoilage evaluation, shelf-life prediction, and potential spoilage organisms of tropical brackish water shrimp (Penaeus notialis) at different storage temperatures.

Maintaining the freshness of shrimp is a concern to shrimp stakeholders. To improve shrimp quality management, it is of importance to evaluate shrimp spoilage characteristics. Therefore, microbiological, sensory, and chemical changes of naturally contaminated tropical brackish water shrimp (Penaeus notialis) during storage at 28 °C, 7 °C and 0 °C were assessed. H2S-producing bacteria were the dominant group of microorganisms at 28 °C and 7 °C whereas Pseudomonas spp. were dominant at 0 °C. Total volatile basic nitrogen and trimethylamine correlated well (R(2) > 0.90) with the sensory scores. An empirical model to predict the shelf-life of naturally contaminated tropical shrimp as a function of storage temperature was developed. Specific groups of organisms were isolated at the sensory rejection times and assessed for spoilage potential in shrimps of which the endogenous flora was heat inactivated. Isolates capable of producing strong off-odor identified by 16S rRNA sequencing were mainly lactic acid bacteria (LAB) and Enterobacteriaceae at 28 °C or 7 °C and Pseudomonas spp. and LAB at 0 °C. The study contributes to the knowledge about tropical shrimp spoilage and provides a basis for the development of methods and tools to improve shrimp quality management.

Effects of the antimicrobial glabridin on membrane integrity and stress response activation in Listeria monocytogenes.

Glabridin is a prenylated isoflavan which can be extracted from liquorice roots and has shown antimicrobial activity against foodborne pathogens and spoilage microorganisms. However, its application may be hindered due to limited information about its mode of action. In this study, we aimed to investigate the mode of action of glabridin using a combined phenotypic and proteomic approach on Listeria monocytogenes. Fluorescence and transmission electron microscopy of cells exposed to glabridin showed membrane permeabilization upon treatment with lethal concentrations of glabridin. Comparative proteomics analysis of control cells and cells exposed to sub-lethal concentrations of glabridin showed upregulation of proteins related to the two-component systems LiaSR and VirRS, confirming cell envelope damage during glabridin treatment. Additional upregulation of SigmaB regulon members signified activation of the general stress response in L. monocytogenes during this treatment. In line with the observed upregulation of cell envelope and general stress response proteins, sub-lethal treatment of glabridin induced (cross)protection against lethal heat and low pH stress and against antimicrobials such as nisin and glabridin itself. Overall, this study sheds light on the mode of action of glabridin and activation of the main stress responses to this antimicrobial isoflavan and highlights possible implications of its use as a naturally derived antimicrobial compound.

A web-based microbiological hazard identification tool for infant foods.

An integrated approach to identify and assess Microbiological Hazards (MHs) and mitigate risks in infant food chains is crucial to ensure safe foods for infants and young children. A systematic procedure was developed to identify MHs in specific infant foods. This includes five major steps: 1) relevant hazard-food pairing, 2) process inactivation efficiency, 3) recontamination possibility after processing, 4) MHs growth opportunity, and 5) MHs-food association level. These steps were integrated into an online tool called the Microbiological Hazards IDentification (MiID) decision support system (DSS), targeting food companies, governmental agencies and academia users, and is accessible at https://foodmicrobiologywur.shinyapps.io/Microbial_hazards_ID/. The MiID DSS was validated in four case studies, focussing on infant formula, fruit puree, cereal-based meals, and fresh fruits, each representing distinct products and processing characteristics. The results obtained through the application of the MiID DSS, compared with identification by food safety experts, consistently identified the top MHs in these food products. This process affirms its effectiveness in systematic hazard identification. The introduction of the MiID DSS helps to structure the first steps in HACCP (hazard analysis) and in risk assessment (hazard identification) to follow a structured and well-documented procedure, balancing the risk of overlooking relevant MHs or including too many irrelevant MHs. It is a valuable addition to risk analysis/assessment in infant food chains and has the potential for future extension. This includes the incorporation of newly acquired data related to infant foods via a semi-publicly hosted platform, or it can be adapted for hazard identification in general food products using a similar framework.

Quantitative assessment of food safety interventions for Campylobacter spp. and Salmonella spp. along the chicken meat supply chain in Burkina Faso and Ethiopia.

Rural and small-scale chicken farming is a major source of income in most African countries, and chicken meat is an important source of nutrients. However, chicken meat can be contaminated with Campylobacter spp. and Salmonella spp., pathogens with a high reported burden of foodborne illnesses. Therefore, it is essential to control these pathogens in chicken meat. Quantitative microbial risk assessments (QMRA) can aid the development of effective food safety control measures and are currently lacking in chicken meat supply chains in the African context. In this study, we developed stochastic QMRA models for Salmonella spp. and Campylobacter spp. in the chicken meat supply chain in Burkina Faso and Ethiopia employing the modular process risk model in @Risk software. The study scope covered chicken farming, transport, slaughtering, consumer handling, and consumption. Effectiveness of candidate interventions was assessed against baseline models' outputs, which showed that the mean annual Campylobacter spp. risk estimates were 6482 cases of illness per 100,000 persons and 164 disability adjusted life years (DALYs) per 100,000 persons in Burkina Faso, and 12,145 cases and 272 DALYs per 100,000 persons in Ethiopia. For Salmonella spp., mean annual estimates were 2713 cases and 1212 DALYs per 100,000 persons in Burkina Faso, and 4745 cases and 432 DALYs per 100,000 persons in Ethiopia. Combining interventions (improved hand washing plus designated kitchen utensils plus improved cooking) resulted in 75 % risk reduction in Burkina Faso at restaurants and 93 to 94 % in Ethiopia at homes for both Salmonella spp. and Campylobacter spp. For Burkina Faso, adding good hygienic slaughter practices at the market to these combined interventions led to over 91 % microbial risk reduction. Interventions that involved multiple food safety actions in a particular step of the supply chain or combining different interventions from different steps of the supply chain resulted in more risk reduction than individual action interventions. Overall, this study demonstrates how diverse and scanty food supply chain information can be applied in QMRA to provide estimates that can be used to stimulate risk-based food safety action in African countries.

Temperature status of domestic refrigerators and its effect on the risk of listeriosis from ready-to-eat (RTE) cooked meat products.

Inadequate domestic refrigeration is frequently cited as a factor that contributes to foodborne poisoning and infection, and consumer behaviour in this regard can vary largely. This study provides insight into the temperature profiles of domestic refrigerators in the Netherlands and the impact on the number of listeriosis cases related to ready-to-eat (RTE) cooked meat products. A survey was conducted among Dutch consumers (n = 1020) to assess their knowledge and behaviour related to refrigerators. Out of these participants, 534 measured their refrigerator's temperature, revealing an average temperature of 5.7 °C (standard deviation (SD) of 2.2 °C) with a maximum of 17 °C. Elderly people (65 years and older) had refrigerators with temperatures that were on average 0.6 °C higher than those of younger people (35 years or younger). The 24-hour temperature profiles of an additional set of actively surveyed refrigerators (n = 50) showed that the temperature measured on the upper shelf was significantly higher (mean 7.7 °C, SD 2.7 °C) than the temperature measured on the bottom shelf (5.7 °C, SD 2.1 °C). Quantitative Microbiological Risk Assessment (QMRA) predicted that the primary factors contributing to the risk of listeriosis were the initial concentration and the time and temperature during household storage. Scenario analysis revealed that storing opened RTE cooked meat products at home for either <7 days or at temperatures <7 °C resulted in a significant reduction of over 80 % in predicted illness cases. Among all illness cases, the elderly represented nearly 90 %. When assessing the impact of the disease in terms of Years of Life Lost (YLL), the contribution of the elderly was 59 %. Targeted communication, particularly directed towards the elderly, on the importance of storing RTE cooked meat products at the recommended temperature on the bottom or middle shelf as well as consuming within two to three days after opening, holds the potential to significantly reduce the number of cases.

Insight in lag phase of Listeria monocytogenes during enrichment through proteomic and transcriptomic responses.

The dynamics of the enrichment-based detection procedure of the foodborne pathogen Listeria monocytogenes from food still remains poorly understood. This enrichment is crucial in the reliable detection of this pathogen and more insight into the recovery mechanism during this step is important to advance our understanding of lag phase behaviour during enrichment. In this study we combined transcriptomic and proteomic analyses to better understand the physiological processes within the lag phase of L. monocytogenes during enrichment. Upon transfer of BHI-cultured stationary phase L. monocytogenes cells to half-Fraser enrichment broth (HFB), motility-associated genes and proteins were downregulated, while expression of metal uptake transporters, resuscitation-promoting factors that stimulate growth from dormancy, antibiotic efflux pumps and oxidative stress proteins were upregulated. Next to this, when cells with a heat stress history were cultured in enrichment broth, proteins necessary for recovery were upregulated with functions in DNA-damage repair, protein refolding, cell-wall repair, and zinc transport. Proteomic results pointed to possible factors that support shortening the lag duration, including the addition of 10 µM zinc and the addition of spent HFB containing presumed concentrations of resuscitation-promoting factors. However, these interventions did not lead to biologically relevant reduction of lag phase. Also, when cells were enriched in spent HFB, final cell concentrations were similar to enrichments in fresh HFB, indicating that the enrichment broth seems not to lack critical substrates. Concludingly, this study gives insight into the proteomic changes in the lag phase during enrichment and shows that supplementation of HFB is not the best strategy to optimize the current enrichment method.

Pulsed electric field treatment for preservation of Chlorella suspensions and retention of gelling capacity.

Pulsed electric field (PEF) processing has emerged as an alternative to thermal pasteurization for the shelf-life extension of heat-sensitive liquids at industrial scale. It offers the advantage of minimal alteration in physicochemical characteristics and functional properties. In this study, a pilot-scale continuous PEF processing (Toutlet < 55 °C) was applied to microalgae Chlorella vulgaris (Cv) suspensions (pH = 6.5), which was proposed as a functional ingredient for plant-based foods. Cv suspensions were inoculated with three distinct food spoilage microorganisms (Pseudomonas guariconensis, Enterobacter soli and Lactococcus lactis), isolated from the Cv biomass. PEF treatments were applied with varying electric field strength Eel of 16 to 28 kV/cm, pulse repetition rate f of 100 to 140 Hz, with a pulse width τ of 20 µs and an inlet product temperature Tin of 30 °C. The aim was to evaluate the PEF-induced microbial reduction and monitor the microbial outgrowth during a 10-day cold storage period (10 °C). Maximum inactivation of 4.1, 3.7 and 3.6 logs was achieved (28 kV/cm and 120 Hz) for the investigated isolates, respectively. Under these conditions, the critical electric field strengths Ecrit, above which inactivation was observed, ranged from 22.6 to 24.6 kV/cm. Moreover, repeated PEF treatment resulted in similar inactivation efficiency, indicating its potential to enhance shelf-life further.

Stress resistant rpsU variants of Listeria monocytogenes can become underrepresented due to enrichment bias.

Population heterogeneity is an important component of the survival mechanism of Listeria monocytogenes, leading to cells in a population with diverse stress resistance levels. We previously demonstrated that several ribosomal gene rpsU mutations enhanced the stress resistance of L. monocytogenes and lowered the growth rate at 30 °C and lower temperatures. This study investigated whether these switches in phenotypes could result in a bias in strain detection when standard enrichment-based procedures are applied to a variety of strains. Detailed growth kinetics analysis of L. monocytogenes strains were performed, including the LO28 wild type (WT) and rpsU variants V14 and V15, during two commonly used enrichment-based procedures described in the ISO 11290-1:2017 and the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM). WT had a higher growth rate than the variants during the enrichment processes. Co-culture growth kinetics predictions for WT and rpsU variants showed that the detection chances of the rpsU mutants were reduced from ∼52 % to less than ∼13 % and âˆ¼ 3 % during ISO and BAM enrichment, respectively, which were further validated through subsequent qPCR experiments. Higher heat stress resistance of rpsU variants did not lead to faster recovery during enrichment after heat treatment, and different pre-culturing temperatures before heat treatment did not significantly affect the growth kinetics of the WT and rpsU variants. Additionally, post-enrichment isolation procedures involving streaking on selective agar plates did not show preferences for isolating WT or rpsU variants nor affect the detection chance of rpsU variants. The difference in detection chance suggests that the selective enrichment procedures inadequately represent the genotypic diversity present in a sample. Hence, the enrichment bias during the L. monocytogenes isolation procedure may contribute to the observed underrepresentation of the rpsU mutation among L. monocytogenes isolates deposited in publicly available genome databases. The underrepresentation of rpsU mutants in our findings suggests that biases introduced by standard isolation and enrichment procedures could inadvertently skew our understanding of genetic diversity when relying on public databases.

A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV.

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function.

Ribosomal mutations enable a switch between high fitness and high stress resistance in Listeria monocytogenes.

Multiple stress resistant variants of Listeria monocytogenes with mutations in rpsU encoding ribosomal protein RpsU have previously been isolated after a single exposure to acid stress. These variants, including L. monocytogenes LO28 variant V14 with a complete deletion of the rpsU gene, showed upregulation of the general stress sigma factor Sigma B-mediated stress resistance genes and had a lower maximum specific growth rate than the LO28 WT, signifying a trade-off between stress resistance and fitness. In the current work V14 has been subjected to an experimental evolution regime, selecting for higher fitness in two parallel evolving cultures. This resulted in two evolved variants with WT-like fitness: 14EV1 and 14EV2. Comparative analysis of growth performance, acid and heat stress resistance, in combination with proteomics and RNA-sequencing, indicated that in both lines reversion to WT-like fitness also resulted in WT-like stress sensitivity, due to lack of Sigma B-activated stress defense. Notably, genotyping of 14EV1 and 14EV2 provided evidence for unique point-mutations in the ribosomal rpsB gene causing amino acid substitutions at the same position in RpsB, resulting in RpsB22Arg-His and RpsB22Arg-Ser, respectively. Combined with data obtained with constructed RpsB22Arg-His and RpsB22Arg-Ser mutants in the V14 background, we provide evidence that loss of function of RpsU resulting in the multiple stress resistant and reduced fitness phenotype, can be reversed by single point mutations in rpsB leading to arginine substitutions in RpsB at position 22 into histidine or serine, resulting in a WT-like high fitness and low stress resistance phenotype. This demonstrates the impact of genetic changes in L. monocytogenes' ribosomes on fitness and stress resistance.

Activation of a silent lactose utilization pathway in an evolved Listeria monocytogenes F2365 outbreak isolate.

Listeria monocytogenes, a widespread food-borne pathogen, utilizes diverse growth substrates including mono- and di-saccharides via PEP-phosphotransferase (PTS) systems. We evaluated a collection of L. monocytogenes isolates of different origins for their ability to utilize lactose, a disaccharide composed of galactose and glucose and the main carbon source in milk and dairy products. Notably, the dairy-associated outbreak strain F2365 could not utilize lactose efficiently, conceivably due to a frameshift mutation (lacR887del) resulting in a truncated LacR. Transcriptional activator LacR is involved in the expression of two PTS systems, encoded by the lpo operon lmo1718-1720 in combination with lmo2708 and the lmo2683-2685 operon, and linked to lactose and/or cellobiose metabolism in L. monocytogenes. Via experimental evolution of the ancestral strain F2365, an evolved isolate F2365 EV was obtained which showed enhanced growth and metabolism of lactose. Using the lactose-positive model strain L. monocytogenes EGDe as a control, HPLC experiments showed that EGDe and F2365 EV could consume lactose and utilize the glucose moiety, while the galactose moiety was exported from the cells. Genome sequencing of F2365 EV found the original lacR887del mutation was still present but an additional point mutation lmo2766C415T had occurred, resulting in an amino acid substitution in the putative regulator Lmo2766. The lmo2766 gene is located next to operon lmo2761-2765 with putative PTS genes in the genome. Notably, comparative RNAseq analysis confirmed that the lmo2761-2765 operon was strongly upregulated in F2365 EV in the presence of lactose but not in EGDe and F2365. Conversely, the LacR-regulated lpo operon, lmo2708, and lmo2683-2685 operon were only upregulated in EGDe. Additional growth and HPLC experiments, using mutants constructed in lactose-positive L. monocytogenes EGDe, showed reduced growth of the EGDe lacR887del mutant with no utilization of lactose, while the double mutant EGDe lacR887dellmo2766C415T showed enhanced growth and lactose utilization. Hence, these results demonstrate that an amino acid substitution in the Lmo2766 regulator activates a previously silent lactose utilization pathway encoded by PTS operon lmo2761-2765, facilitating the growth and metabolism of L. monocytogenes with lactose as a substrate. This finding enhances our understanding of the metabolic capabilities and adaptability of L. monocytogenes, offering a broader view of the lactose utilization capacity of this pathogen.

Biofilm formation and desiccation survival of Listeria monocytogenes with microbiota on mushroom processing surfaces and the effect of cleaning and disinfection.

Microbial multispecies communities consisting of background microbiota and Listeria monocytogenes could be established on materials used in food processing environments. The presence, abundance and diversity of the strains within these microbial multispecies communities may be affected by mutual interactions and differences in resistance towards regular cleaning and disinfection (C&D) procedures. Therefore, this study aimed to characterize the growth and diversity of a L. monocytogenes strain cocktail (n = 6) during biofilm formation on polyvinyl chloride (PVC) and stainless steel (SS) without and with the presence of a diverse set of background microbiota (n = 18). L. monocytogenes and background microbiota strains were isolated from mushroom processing environments and experiments were conducted in simulated mushroom processing environmental conditions using mushroom extract as growth medium and ambient temperature (20 °C) as culturing temperature. The L. monocytogenes strains applied during monospecies biofilm incubation formed biofilms on both PVC and SS coupons, and four cycles of C&D treatment were applied with a chlorinated alkaline cleaning agent and a disinfection agent based on peracetic acid and hydrogen peroxide. After each C&D treatment, the coupons were re-incubated for two days during an incubation period for 8 days in total, and C&D resulted in effective removal of biofilms from SS (reduction of 4.5 log CFU/cm2 or less, resulting in counts below detection limit of 1.5 log CFU/cm2 after every C&D treatment), while C&D treatments on biofilms formed on PVC resulted in limited reductions (reductions between 1.2 and 2.4 log CFU/cm2, which equals a reduction of 93.7 % and 99.6 %, respectively). Incubation of the L. monocytogenes strains with the microbiota during multispecies biofilm incubation led to the establishment of L. monocytogenes in the biofilm after 48 h incubation with corresponding high L. monocytogenes strain diversity in the multispecies biofilm on SS and PVC. C&D treatments removed L. monocytogenes from multispecies biofilm communities on SS (reduction of 3.5 log CFU/cm2 or less, resulting in counts below detection limit of 1.5 log CFU/cm2 after every C&D treatment), with varying dominance of microbiota species during different C&D cycles. However, C&D treatments of multispecies biofilm on PVC resulted in lower reductions of L. monocytogenes (between 0.2 and 2.4 log CFU/cm2) compared to single species biofilm, and subsequent regrowth of L. monocytogenes and stable dominance of Enterobacteriaceae and Pseudomonas. In addition, planktonic cultures of L. monocytogenes were deposited and desiccated on dry surfaces without and with the presence of planktonic background microbiota cultures. The observed decline of desiccated cell counts over time was faster on SS compared to PVC. However, the application of C&D resulted in counts below the detection limit of 1.7 log CFU/coupon on both surfaces (reduction of 5.9 log CFU/coupon or less). This study shows that L. monocytogenes is able to form single and multispecies biofilms on PVC with high strain diversity following C&D treatments. This highlights the need to apply more stringent C&D regime treatments for especially PVC and similar surfaces to efficiently remove biofilm cells from food processing surfaces.

Catalase activity as a biomarker for mild-stress-induced robustness in Bacillus weihenstephanensis.

Microorganisms are able to survive and grow in changing environments by activating stress adaptation mechanisms which may enhance bacterial robustness. Stress-induced enhanced robustness complicates the predictability of microbial inactivation. Using psychrotolerant Bacillus weihenstephanensis strain KBAB4 as a model, we investigated the impact of the culturing temperature on mild-oxidative-stress-induced (cross-)protection toward multiple stresses, including severe oxidative, heat, and acid stresses. Culturing at a refrigeration temperature (7°C) compared to the optimal growth temperature (30°C) affected both the robustness level of B. weihenstephanensis and the oxidative stress adaptive response. Scavengers of reactive oxygen species have a crucial role in adaptation to oxidative stresses, and this points to a possible predictive role in mild-oxidative-stress-induced robustness. Therefore, the catalase activity was determined upon mild oxidative stress treatment and was demonstrated to be significantly correlated with the robustness level of mild-stress-treated cells toward severe oxidative and heat stresses but not toward severe acid stress for cells grown at both refrigeration and optimal temperatures. The quantified correlations supported the predictive quality of catalase activity as a biomarker and also underlined that the predictive quality is stress specific. Biomarkers that are able to predict stress-induced enhanced robustness can be used to better understand stress adaptation mechanisms and might allow the design of effective combinations of hurdles to control microbial behavior.

The importance of what we cannot observe: Experimental limitations as a source of bias for meta-regression models in predictive microbiology.

Meta-regression models have gained in popularity during the last years as a way to create more generic models for Microbial Risk Assessments that also include variability. However, as with most meta-analyses and empirical models, systematic biases in the data can result in inaccurate models. In this article, we define experimental bias as a type of selection bias due to the practical limitations of microbial inactivation experiments. Conditions with extremely high D-values (i.e. slow inactivation) need very long experimental runs to cause significant reductions. On the other hand, when the D-value is extremely low, not enough data points can be gathered before the microbial population is below the detection limit. Consequently, experimental designs favour conditions within a practical experimental range, introducing a selection bias in the D-values. We demonstrate the impact of experimental bias in meta-regression models using numerical simulations. Models fitted to data with experimental bias overestimated the z-value and underestimated variability. We propose a rapid heuristic method to identify experimental bias in datasets, and we propose truncated regression to mitigate its impact in meta-regression models. Both methods were validated using simulated data. Thereafter the procedures were tested by building a meta-regression model for actual data for the inactivation of Bacillus cereus spores. We concluded that the dataset included experimental bias, and that it would cause an overestimation of the microbial resistance at high temperatures (>120 °C) for classical meta-regression models. This effect was mitigated when the model was built using truncated regression. In conclusion, we demonstrate that experimental bias could potentially result in inaccurate models for predictive microbiology. Therefore, checking for experimental bias should be a routine step in meta-regression modelling, and be included in guidelines on data analysis for meta-regression.

Impact of food-relevant conditions and food matrix on the efficacy of prenylated isoflavonoids glabridin and 6,8-diprenylgenistein as potential natural preservatives against Listeria monocytogenes.

Prenylated isoflavonoids can be extracted from plants of the Leguminosae/Fabaceae family and have shown remarkable antimicrobial activity against Gram-positive food-borne pathogens, such as Listeria monocytogenes. Promising candidates from this class of compounds are glabridin and 6,8-diprenylgenistein. This research aimed to investigate the potential of glabridin and 6,8-diprenylgenistein as food preservatives against L. monocytogenes. Their antimicrobial activity was tested in vitro at various conditions relevant for food application, such as different temperatures (from 10 °C to 37 °C), pH (5 and 7.2), and in the presence or absence of oxygen. The minimum inhibitory concentrations of glabridin and 6,8-diprenylgenistein in vitro were between 0.8 and 12.5 µg/mL in all tested conditions. Growth inhibitory activities were similar at 10 °C compared to higher temperatures, although bactericidal activities decreased when the temperature decreased. Notably, lower pH (pH 5) increased the growth inhibitory and bactericidal activity of the compounds, especially for 6,8-diprenylgenistein. Furthermore, similar antimicrobial efficacies were shown anaerobically compared to aerobically at the tested conditions. Glabridin showed a more stable inhibitory and bactericidal activity when the temperature decreased compared to 6,8-diprenylgenistein. Therefore, we further determined the antimicrobial efficacy of glabridin against L. monocytogenes growth on fresh-cut cantaloupe at 10 °C. In these conditions, concentrations of glabridin of 50, 100 and 250 µg/g significantly reduced the growth of L. monocytogenes compared to the control, resulting on average in >1 Log CFU/g difference after 4 days compared to the control. Our results further underscored the importance of considering the food matrix when assessing the activity of novel antimicrobials. Overall, this study highlights the potential of prenylated isoflavonoids as naturally derived food preservatives.

Impact of preculture temperature on peracetic acid-induced inactivation and sublethal injury of L. monocytogenes and subsequent growth potential of single cells.

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.

Variability in growth and biofilm formation of Listeria monocytogenes in Agaricus bisporus mushroom products.

Foods and food production environments can be contaminated with Listeria monocytogenes and may support growth of this foodborne pathogen. This study aims to characterize the growth and biofilm formation of sixteen L. monocytogenes strains, isolated from mushroom production and processing environments, in filter-sterilized mushroom medium. Strain performance was compared to twelve L. monocytogenes strains isolated from other sources including food and human isolates. All twenty-eight L. monocytogenes strains showed rather similar growth performance at 20 °C in mushroom medium, and also significant biofilm formation was observed for all strains. HPLC analysis revealed the presence of mannitol, trehalose, glucose, fructose and glycerol, that were all metabolized by L. monocytogenes, except mannitol, in line with the inability of L. monocytogenes to metabolize this carbohydrate. Additionally, the growing behavior of L. monocytogenes was tested on whole, sliced and smashed mushroom products to quantify performance in the presence of product-associated microbiota. A significant increase of L. monocytogenes was observed with higher increase of counts when the mushroom products were more damaged, even with the presence of high background microbiota counts. This study demonstrated that L. monocytogenes grows well in mushroom products, even when the background microbiota is high, highlighting the importance to control (re)contamination of mushrooms.

Growth performance of Listeria monocytogenes and background microbiota from mushroom processing environments.

Interaction between Listeria monocytogenes and resident background microbiota may occur in food processing environments and may influence the survival of this pathogen in a factory environment. Therefore the aim of this study was to characterize the growth performance of microbiota isolated from the processing environments of frozen sliced mushrooms, and to investigate the competitive performance of L. monocytogenes when co-cultured with accompanying environmental microbiota. Acinetobacter, Enterobacteriaceae, Lactococcus and Pseudomonas were the most prominent background microbiota isolated from the processing environment of frozen sliced mushrooms. All individual microbiota strains were able to grow and form biofilm in filter-sterilized mushroom medium, with the mannitol-consumers Raoultella and Ewingella as top performers, reaching up to 9.6 and 9.8 log CFU/mL after 48 h incubation at room temperature. When L. monocytogenes mushroom isolates were co-cultured with the microbiota strains, L. monocytogenes counts ranged from 7.6 to 8.9 log CFU/mL after 24 h of incubation, while counts of the microbiota strains ranged from 5.5 to 9.0 log CFU/mL. Prolonged incubation up to 48 h resulted in further increase of L. monocytogenes counts when co-cultured with non-acidifying species Pseudomonas and Acinetobacter reaching 9.1 to 9.2 log CFU/mL, while a decrease of L. monocytogenes counts reaching 5.8 to 7.7 log CFU/mL was observed in co-culture with Enterobacteriaceae and acidifying Lactococcus representatives. In addition, L. monocytogenes grew also in spent mushroom media of the microbiota strains, except in acidified spent media of Lactococcus strains. These results highlight the competitive ability of L. monocytogenes during co-incubation with microbiota in fresh and in spent mushroom medium, indicative of its invasion and persistence capacity in food processing factory environments.