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The deviations from log-linearity that are often observed in bacterial survivor curves can be explained using different arguments, both biological and experimental. In this study, we used Bacillus subtilis as a model organism to demonstrate that the generally accepted vitalistic arguments (initial heterogeneities in the stress resistance of the cells in the population) may fail to describe microbial inactivation in some situations. In this sense, we showed how dynamic stress acclimation during an isothermal treatment provides an alternative explanation for survivor curves with an upwards curvature. We also provided an innovative experimental approach based on preadaptation experiments to evaluate which hypothesis is more suitable for the bacterial response. Furthermore, we used our experimental results to define bounds for the possible stress acclimation that may take place during dynamic treatments, concluding that the magnitude of stress acclimation may be larger for dynamic treatments than for isothermal experiments. We also evaluated the contribution of the SigB general stress response system to heat resistance by comparing the heat survival of wt and the ΔsigB mutant. Both strains survived better in 51, 52.5 and 55 °C when cells were pre-adapted at 48 °C than non-pre-adapted cells. However, ΔsigB was less resistant to heat than wt due to the missing SigB general stress system. Although these conclusions were based on B. subtilis as a model organism, this study can be the first step towards the development of a novel methodology able to estimate dynamic effects using only isothermal experiments. This would improve the models developed within the predictive microbiology community, improving our ability to predict microbial inactivation during industrial treatments, which are most often dynamic., 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 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We developed a quantitative microbiological risk assessment (QMRA) of haemolytic uremic syndrome (HUS) associated with Shiga toxin-producing Escherichia coli (STEC)-contaminated beef (intact beef cuts, ground beef and commercial hamburgers) in children under 15 years of age from Argentina. The QMRA was used to characterize STEC prevalence and concentration levels in each product through the Argentinean beef supply chain, including cattle primary production, cattle transport, processing and storage in the abattoir, retail and home preparation, and consumption. Median HUS probability from beef cut, ground beef and commercial hamburger consumption was <10-15, 5.4x10-8 and 3.5x10-8, respectively. The expected average annual number of HUS cases was 0, 28 and 4, respectively. Risk of infection and HUS probability were sensitive to the type of abattoir, the application or not of Hazard Analysis and Critical Control Points (HACCP) for STEC (HACCP-STEC), stx prevalence in carcasses and trimmings, storage conditions from the abattoir to retailers and home, the joint consumption of salads and beef products, and cooking preference. The QMRA results showed that the probability of HUS was higher if beef cuts (1.7x) and ground beef (1.2x) were from carcasses provided by abattoirs not applying HACCP-STEC. Thus, the use of a single sanitary standard that included the application of HACCP-STEC in all Argentinean abattoirs would greatly reduce HUS incidence. The average number of annual HUS cases estimated by the QMRA (n = 32) would explain about 10.0% of cases in children under 15 years per year in Argentina. Since other routes of contamination can be involved, including those not related to food, further research on the beef production chain, other food chains, person-to-person transmission and outbreak studies should be conducted to reduce the impact of HUS on the child population of Argentina., Competing Interests: The authors have declared that no competing interests exist.

Food is traded across the global markets to satisfy consumer demands, mainly from developed countries, for year-round access to a wide range of foods. This has resulted in an increasingly complex network of food trade and has made importing countries vulnerable to the spread of foodborne disease outbreaks originating from "foreign" food networks. Analysis of these networks can provide information on potential food safety risks and also on the potential spread of these risks through the food network in question. In this study, network theory has been used to analyse global trade. A mathematical model was developed enabling a simulation of the distribution of food products based on the publicly available data on international imports, exports and production provided by the Food and Agriculture Organization of the United Nations. Through numerical simulations we demonstrate, for the first time, the impact that the network structure has on the distribution of food products in terms of food safety risks. As a case study, a recent trans-national food safety incident was analysed, illustrating the potential application of the model in a foodborne pathogen outbreak. Using only the type of contaminated food and the countries where the outbreak was reported, the model was used to identify the most likely origin of the contaminated eggs, narrowing down the options to three countries (including the actual origin). Furthermore, it is used to identify those countries with significant food safety risks, due to imports of food produced in these three countries. The approach can help regulatory agencies and the food industry to design improved surveillance and risk mitigation actions against transnational food safety risks., (Copyright © 2019. Published by Elsevier Ltd.)

The mathematical models used in predictive microbiology contain parameters that must be estimated based on experimental data. Due to experimental uncertainty and variability, they cannot be known exactly and must be reported with a measure of uncertainty (usually a standard deviation). In order to increase precision (i.e. reduce the standard deviation), it is usual to add extra sampling points. However, recent studies have shown that precision can also be increased without adding extra sampling points by using Optimal Experiment Design, which applies optimization and information theory to identify the most informative experiment under a set of constraints. Nevertheless, to date, there has been scarce contributions to know a priori whether an experimental design is likely to provide the desired precision in the parameter estimates. In this article, two complementary methodologies to predict the parameter precision for a given experimental design are proposed. Both approaches are based on in silico simulations, so they can be performed before any experimental work. The first one applies Monte Carlo simulations to estimate the standard deviation of the model parameters, whereas the second one applies the properties of the Fisher Information Matrix to estimate the volume of the confidence ellipsoids. The application of these methods to a case study of dynamic microbial inactivation, showing how they can be used to compare experimental designs and assess their precision, is illustrated. The results show that, as expected, the optimal experimental design is more accurate than the uniform design with the same number of data points. Furthermore, it is demonstrated that, for some heating profiles, the uniform design does not ensure that a higher number of sampling points increases precision. Therefore, optimal experimental designs are highly recommended in predictive microbiology., Competing Interests: The authors have declared that no competing interests exist.

The estimation of the concentration of microorganisms in a sample is crucial for food microbiology. For instance, it is essential for prevalence studies, challenge tests (growth and/or inactivation studies) or microbial risk assessment. The application of serial dilutions followed by viable counts in Petri dishes is probably the most extended experimental methodology for this purpose. However, this enumeration technique is also a source of uncertainty. In this article, the uncertainty of the serial dilution and viable count methodology related to the sampling error is analyzed, as well as the approximation of the microbial concentration by the number of colonies in a Petri dish. We analyze from a theoretical point of view (statistical analysis) the application of the binomial and Poisson models, demonstrating that the Poisson distribution increases the variance when used to model individual serial dilutions. On the other hand, the binomial model produces unbiased results. Therefore, the Poisson distribution is only applicable when it is a good approximation of the binomial distribution, so the use of the latter is recommended. The relevance of this uncertainty is demonstrated by Monte Carlo simulations of a generic microbial inactivation experiment, where the only source of uncertainty/variability considered is the one generated by serial plating and viable cell enumeration. Due to both the uncertainty of the methodology and the omission of zero-count plates because of the log-transformation, the simulated survival curve can have a tail. Therefore, this phenomenon, which is usually attributed to biological variability, can be to some extent an artefact of the experimental design and/or methodology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Decisions regarding microbial risk assessment usually have to be carried out with incomplete information. This is due to the large number of possible scenarios and the lack of specific data for the problem considered. Consequently, risk assessment studies are based on the information obtained with a small number of bacterial cells which are considered the most heat resistant and/or more capable of multiplying during storage. The identification of the most resistant strains is usually based on D and z-values, normally estimated from isothermal experiments. This procedure omits the potential effect that the shape of the dynamic thermal profile applied in industry has on the microbial inactivation. One example of such effects is stress acclimation, which is related to a physiological response of the cells during sub-lethal treatments that increases their resistance. In this article, we use a recently published mathematical model to compare the development of thermal resistance for Escherichia coli K12 MG1655 and E. coli CECT 515 using inactivation data already published for these strains. Based only on the isothermal experiments, E. coli K12 MG1655 would be identified as more resistant to the thermal treatment than the CECT 515 strain in the 50-65°C temperature range. However, we conclude that stress acclimation is strain (and/or media)-dependent; the CECT 515 strain has a higher capacity for developing a stress acclimation than K12 MG1655 (300% increase of the D-value for CECT 515, 50% for K12 MG1655). It, thus, has the potential to be more resistant to the thermal treatment than the K12 MG1655 strain for some conditions allowing acclimation. A methodology is proposed to identify for which conditions this may be the case. After calibrating the model parameters representing acclimation using real experimental data, the applicability of the proposed approach is demonstrated using numerical simulations, showing how the CECT 515 strain can be more resistant for some heating profiles. Consequently, the most resistant bacterial strain to a dynamic heating profile should not be identified based only on isothermal experiments (D- and z-value). The relevance of stress acclimation for the treatment studied should also be evaluated.

Bacillus sporothermodurans produces highly heat-resistant endospores, that can survive under ultra-high temperature. High heat-resistant sporeforming bacteria are one of the main causes for spoilage and safety of low-acid foods. They can be used as indicators or surrogates to establish the minimum requirements for heat processes, but it is necessary to understand their thermal inactivation kinetics. The aim of the present work was to study the inactivation kinetics under both static and dynamic conditions in a vegetable soup. Ordinary least squares one-step regression and sequential procedures were applied for estimating these parameters. Results showed that multiple dynamic heating profiles, when analyzed simultaneously, can be used to accurately estimate the kinetic parameters while significantly reducing estimation errors and data collection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In 1999 it has been recognized that 3 BRCA1 abnormalities - 5382insC, C61G and 4153delA - constitute almost 90% of all germline mutations of this gene in Poland. Due to the above findings we started performing the cheap and quick large scale testing for BRCA1 mutations and, these days, we have almost 4,000 carriers diagnosed and under direct or indirect supervision what is probably the largest number in the world. Additionally, the above results pushed us to hypothesize that genetic homogeneity will be seen in Poland in studies of other genes. Actually, the next studies allowed us to identify genes / changes associated with moderate / low breast cancer risk and showed, similarly to BRCA1, high level of genetic homogeneity. This series included BRCA2, C5972T, CHEK2 del5395; 1100delC, I157T or IVS2 + 1G > A, CDKN2A (p16) A148T, XPD Asp312Asn and Lys751Gln, CYP1B1 R48G, A119S and L43V. The results of the above studies led us in 2004 already to hypothesize that >90% of all cancers have genetic (constitutional) background. Two years later we were able to show a panel of markers covering 92% of consecutive breast cancers in Poland, and we formulated the hypothesis that all cancers have a genetic background. These days we are demonstrating for the first time that genetic components to malignancy play a role in all cancers. We are presenting it on examples of late-onset breast cancers from Poland, but it seems to be justified to expect that similar results can be achieved from other malignancies.

The isothermal survival curves of the heat resistant spores of Bacillus sporothermodurans IC4, in the range of 117-125 degrees C, were determined in chicken, mushroom and pea soups by the capillary method. They were all non-linear with a noticeable upper concavity and could be described by the equation log(10) [N(t)/N(0)]=-b(T)t(n) with a fixed power, n, of the order of 0.7-0.8. The temperature dependence of b(T) could be described by the equation b(T)=log(e)[1+exp[k(T-T(c))]], where T(c) is the temperature where intensive inactivation starts and k is the slope of b(T) at temperatures well above T(c). They were 121-123 degrees C and 0.2-0.4 degrees C(-1), respectively, depending on the soup. These parameters were used to estimate the survival curves of the spores in two non-isothermal heat treatments using the procedure originally proposed by Peleg and Penchina [Crit. Rev. Food Sci. Nutr. 40 (2000) 159]. The results were compared with experimental survival curves, determined by the direct injection method, in another laboratory. There was a general agreement, although not perfect, between the predicted and experimentally observed survival ratios. Also, the isothermal survival parameters, estimated directly from the non-isothermal inactivation data using the model, were in general agreement with those calculated from the isothermal data. This suggests that the heat inactivation patterns of B. sporothermodurans IC4 spores in soups can be at least roughly estimated using the same general survival model, which has until now only been experimentally validated for vegetative bacterial cells.

Positron emission tomography (PET) using F-18-fluoro-2-deoxy-d-glucose(F-18-FDG) is gaining acceptance as a useful imaging method for head and neck tumors. Results of 59 PET scans done on 45 patients with head and neck tumors were evaluated retrospectively. Thirty-six patients had prior treatment consisting of surgery, radiation therapy, chemotherapy, or a combination of these modalities. PET results were compared to computed tomography and magnetic resonance imaging results and validated by histologic findings or survival. PET identified 36 out of 37 tumors (sensitivity 97%) and 18 of 22 benign processes (specificity 82%). Tumor was ruled out in 18 out of 19 patients. For CT, the sensitivity was 80% (20/25) and specificity was 31% (4/13). FDG-PET facilitates differentiation of recurrent head and neck tumors from treatment related changes sometimes difficult to characterize by CT or MRI and may have a significant impact on the management of patients reducing morbidity and costs.

A risk analysis was applied to experimental heat resistance data. This analysis is an approach for processing experimental thermobacteriological data in order to study the variability of D and z values of target microorganisms depending on the deviations range of environmental factors, to determine the critical factors and to specify their critical tolerance. This analysis is based on sets of sensitivity functions applied to a specific case of experimental data related to the thermoresistance of Clostridium sporogenes and Bacillus stearothermophilus spores. The effect of the following factors was analyzed: the type of target microorganism; nature of the heating substrate; pH, temperature; type of acid employed and NaCl concentration. The type of target microorganism to be inactivated, the nature of the substrate (reference or real food) and the heating temperature were identified as critical factors, determining about 90% of the alteration of the microbiological risk. The effect of the type of acid used for the acidification of products and the concentration of NaCl can be assumed to be negligible factors for the purposes of engineering calculations. The critical non-uniformity in temperature during thermobacteriological studies was set as 0.5% and the critical tolerances of pH value and NaCl concentration were 5%. These results are related to a specific case study, for that reason their direct generalization is not correct.

The effect of growth temperature and growth rate on the susceptibility to heat and pH stress were investigated in Listeria monocytogenes grown in continuous culture where these two growth variables could be varied independently of each other, and in batch culture. After growth at 30 degrees C or 10 degrees C at constant growth rate, or at 30 degrees C at different growth rates, cells did not differ in their resistance to heat at 55 degrees C. Cells grown at 30 degrees C were more resistant to acid stress at pH 2.5 than cells grown at the same growth rates at 10 degrees C. Cells grown at low growth rate at 30 degrees C gave greater resistance to acid stress than those grown at high growth rate. Growth temperature and growth rate had independent effects on the susceptibility of L. monocytogenes to acid stress conditions. This may have implications for the survival of L. monocytogenes in acidic foods.

The heat treatment necessary to inactivate spores of non-proteolytic Clostridium botulinum in refrigerated, processed foods may be influenced by the occurrence of lysozyme in these foods. Spores of six strains of non-proteolytic Cl. botulinum were inoculated into tubes of an anaerobic meat medium, to give 10(6) spores per tube. Hen egg white lysozyme (0-50 micrograms ml-1 was added, and the tubes were given a heat treatment equivalent to 19.8 min at 90 degrees C, cooled, and incubated at 8 degrees, 12 degrees, 16 degrees and 25 degrees C for up to 93 d. In the absence of added lysozyme, neither growth nor toxin formation were observed. A 6-D inactivation was therefore achieved. In tubes to which lysozyme (5-50 micrograms ml-1 had been added prior to heating, growth and toxin formation were observed. With lysozyme added at 50 micrograms ml-1, growth was first observed after 68 d at 8 degrees C, 31 d at 12 degrees C, 24 d at 16 degrees C, and 9 d at 25 degrees C. Thus, in these circumstances, a heat treatment equivalent to 19.8 min at 90 degrees C was not sufficient, on its own, to give a 6-D inactivation. A combination of the heat treatment, maintenance at less than 12 degrees C, and a shelf-life not more than 4 weeks reduced the risk of growth of non-proteolytic Cl. botulinum by a factor of 10(6).

The influence of mushroom extract, acidification and type of acidulant (citric acid or glucono-delta-lactone) on the thermal resistance of PA 3679 spores has been studied. The pH values were 7 (phosphate buffer), 6.7 (non acidified mushroom extract), 6.22, 5.34 and 4.65 (acidified mushroom extract). Spores were less heat resistant in the non-acidified mushroom extract than in phosphate buffer. However, acidification of the extract had an effect on D values which was dependent on the specific pH. At pH 6.22, D values were higher than those in non-acidified extract, for both type of acidulants, but the difference between D values in both substrates was affected by temperature, the higher the temperature, the less the difference. Acidification at other pH levels did not significantly affect D values obtained, neither did the type of acidulant. The z value ranged from 9.79 to 12.09 degrees C for citric acid and from 8.18 to 10.18 degrees C for glucono-delta-lactone. When pH was decreased the z value increased for both acidulants.

The thermal resistance of Bacillus stearothermophilus spores was studied in bidistilled water as the reference medium, mushroom extract and acidified mushroom extract. Citric acid and glucono-δ-lactone were used as acidulants. Results indicated that mushroom extract affects the heat resistance of spores; D values were lower than for those in bidistilled water. The pH effect was lower with higher treatment temperatures. Acidification reduced the thermal resistance of spores, the reduction being similar for both types of acidulants, and in general it also had the effect of increasing the z values. It has been confirmed that acidification of the canned mushrooms could actually help to control the thermophilic spoilage. This acidification could also be obtained by the use of glucono-δ-lactone, which has been shown to be as effective as citric acid in reducing the heat resistance of spores.