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.

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.

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.

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.

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.

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.

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.

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.

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.

Shelf life estimation of refrigerated vacuum packed beef accounting for uncertainty.

This study estimates the shelf life of vacuum packed beef meat (three muscles: striploin (longissimus thoracis et lumborum, LTL), tenderloin (psoas major, PM) and outside chuck (trapezius thoracis, TT)) at refrigeration temperatures (0 °C-10 °C) based on modelling the growth of two relevant groups of spoilage microorganisms: lactic acid bacteria (LAB) and Enterobacteriaceae. The growth models were developed combining a two-step and a one-step approach. The primary modelling was used to identify the parameters affecting the growth kinetics, guiding the definition of secondary growth models. For LAB, the secondary model included the effect of temperature and initial pH on the specific growth rate. On the other hand, the model for Enterobacteriaceae incorporated the effect of temperature on the specific growth rate and the lag phase; as well as the effect of the initial pH on the specific growth rate, the lag phase and the initial microbial count. We did not observe any significant effect of the type of muscle on the growth kinetics. Once the equations were defined, the models were fitted to the complete dataset using a one-step approach. Model validation was carried out by cross-validation, mitigating the impact of an arbitrary division between training and validation sets. The models were used to estimate the shelf life of the product, based on the maximum admissible microbial concentration (7 log CFU/g for LAB, 5 log CFU/g for Enterobacteriaceae). Although LAB was the dominant microbiota, in several cases, both LAB and Enterobacteriaceae reached the critical concentration practically at the same time. Furthermore, in some scenarios, the end of shelf life would be determined by Enterobacteriaceae, pointing at the potential importance of non-dominant microorganisms for product spoilage. These results can aid in the implementation of effective control measures in the meat processing industry.

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.

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.

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.

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.

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.

Critical comparison of statistical methods for quantifying variability and uncertainty of microbial responses from experimental data.

Variability and uncertainty are important factors for quantitative microbiological risk assessment (QMRA). In this context, variability refers to inherent sources of variation, whereas uncertainty refers to imprecise knowledge or lack of it. In this work we compare three statistical methods to estimate variability in the kinetic parameters of microbial populations: mixed-effect models, multilevel Bayesian models, and a simplified algebraic method previously suggested. We use two case studies that analyse the influence of three levels of variability: (1) between-strain variability (different strains of the same species), (2) within-strain variability (biologically independent reproductions of the same strain) and, at the most nested level, (3) experimental variability (species independent technical lab variability resulting in uncertainty about the population characteristic of interest) on the growth and inactivation of Listeria monocytogenes. We demonstrate that the algebraic method, although relatively easy to use, overestimates the contribution of between-strain and within-strain variability due to the propagation of experimental variability in the nested experimental design. The magnitude of the bias is proportional to the variance of the lower levels and inversely proportional to the number of repetitions. This bias was very relevant in the case study related to growth, whereas for the case study on inactivation the resulting insights in variability were practically independent of the method used. The mixed-effects model and the multilevel Bayesian models calculate unbiased estimates for all levels of variability in all the cases tested. Consequently, we recommend using the algebraic method for initial screenings due to its simplicity. However, to obtain parameter estimates for QMRA, the more complex methods should generally be used to obtain unbiased estimates.

Intraspecific variability in heat resistance of fungal conidia.

Microbial species are inherently variable, which is reflected in intraspecies genotypic and phenotypic differences. Strain-to-strain variation gives rise to variability in stress resistance and plays a crucial role in food safety and food quality. Here, strain variability in heat resistance of asexual spores (conidia) of the fungal species Aspergillus niger, Penicillium roqueforti and Paecilomyces variotii was quantified and compared to bacterial variability found in the literature. After heat treatment, a 5.4- to 8.6-fold difference in inactivation rate was found between individual strains within each species, while the strain variability of the three fungal species was not statistically different. We evaluated whether the degree of intraspecies variability is uniform, not only within the fungal kingdom, but also amongst different bacterial species. Comparison with three spore-forming bacteria and two non-spore-forming bacteria revealed that the variability of the different species was indeed in the same order of magnitude, which hints to a microbial signature of variation that exceeds kingdom boundaries.

Level of Detection (LOD50) of Campylobacter Is Strongly Dependent on Strain, Enrichment Broth, and Food Matrix.

The detection of thermotolerant Campylobacter in food may be difficult due to the growth of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae during enrichment, resulting in false-negative samples. Therefore, the ISO protocol (ISO 10272-1:2017) suggests that, next to Bolton broth (BB), Preston broth (PB) is used as an enrichment broth to inhibit competitive flora in samples with suspected high levels of background microorganisms, such as ESBL-producing bacteria. However, the application of the strains used for validation of this ISO was not clearly characterized. This study examined the LOD50 (level of detection, the concentration where the probability of detection is 50%) of the validation strains (three C. jejuni and two C. coli strains) in BB and PB using different food matrices, namely, raw milk, chicken skin, frozen minced meat, and frozen spinach. The LOD50 was calculated by inoculating multiple portions with at least two inoculum levels. For each reproduction, eight test portions were used for each inoculum level and the test portion size was 10 g (chicken skin, frozen minced meat, and frozen spinach) or 10 mL (raw milk). Furthermore, the effect of artificially inoculated ESBL-producing E. coli on the LOD50 was examined to mimic the presence of ESBL-producing background microorganisms in the food matrices, namely, raw milk and chicken skin. In BB, the LOD50 of all strains tested in raw milk, chicken skin, and frozen spinach was rather low (0.4-37 CFU/test portion), while the LOD50 in frozen minced meat was higher and much more variable (1-1,500 CFU/test portion), depending on the strain. Generally, enrichment in PB resulted in higher LOD50 than in BB, especially for C. coli. Co-inoculation with ESBL-producing E. coli increased the LOD50 in BB, while PB successfully inhibited the growth of this competitive microorganism. In conclusion, food matrix and enrichment broth may have a large influence on the LOD50 of different Campylobacter strains. Therefore, it is not possible to give an unequivocal advice on when to use which enrichment broth, and this advocates the use of both methods in case of doubt. Furthermore, this study indicates specific strains that would be a good choice to use for Campylobacter method verification as described in ISO 16140-3:2021.

Comparative Analysis of L-Fucose Utilization and Its Impact on Growth and Survival of Campylobacter Isolates.

Campylobacter jejuni and Campylobacter coli were previously considered asaccharolytic, but are now known to possess specific saccharide metabolization pathways, including L-fucose. To investigate the influence of the L-fucose utilization cluster on Campylobacter growth, survival and metabolism, we performed comparative genotyping and phenotyping of the C. jejuni reference isolate NCTC11168 (human isolate), C. jejuni Ca1352 (chicken meat isolate), C. jejuni Ca2426 (sheep manure isolate), and C. coli Ca0121 (pig manure isolate), that all possess the L-fucose utilization cluster. All isolates showed enhanced survival and prolonged spiral cell morphology in aging cultures up to day seven in L-fucose-enriched MEMα medium (MEMαF) compared to MEMα. HPLC analysis indicated L-fucose utilization linked to acetate, lactate, pyruvate and succinate production, confirming the activation of the L-fucose pathway in these isolates and its impact on general metabolism. Highest consumption of L-fucose by C. coli Ca0121 is conceivably linked to its enhanced growth performance up to day 7, reaching 9.3 log CFU/ml compared to approximately 8.3 log CFU/ml for the C. jejuni isolates. Genetic analysis of the respective L-fucose clusters revealed several differences, including a 1 bp deletion in the Cj0489 gene of C. jejuni NCTC11168, causing a frameshift in this isolate resulting in two separate genes, Cj0489 and Cj0490, while no apparent phenotype could be linked to the presumed frameshift in this isolate. Additionally, we found that the L-fucose cluster of C. coli Ca0121 was most distant from C. jejuni NCTC11168, but confirmation of links to L-fucose metabolism associated phenotypic traits in C. coli versus C. jejuni isolates requires further studies.