A comprehensive investigation of protein expression profiles in L. monocytogenes exposed to thermal abuse, mild acid, and salt stress conditions.

Preventing L. monocytogenes infection is crucial for food safety, considering its widespread presence in the environment and its association with contaminated RTE foods. The pathogen's ability to persist under adverse conditions, for example, in food processing facilities, is linked to virulence and resistance mechanisms, including biofilm formation. In this study, the protein expression patterns of two L. monocytogenes 1/2a strains, grown under environmental stressors (mild acidic pH, thermal abuse, and high concentration of NaCl), were investigated. Protein identification and prediction were performed by nLC-ESI-MS/MS and nine different bioinformatic software programs, respectively. Gene enrichment analysis was carried out by STRING v11.05. A total of 1,215 proteins were identified, of which 335 were non-cytosolic proteins and 265 were immunogenic proteins. Proteomic analysis revealed differences in protein expression between L. monocytogenes strains in stressful conditions. The two strains exhibited unique protein expression profiles linked to stress response, virulence, and pathogenesis. Studying the proteomic profiles of such microorganisms provides information about adaptation and potential treatments, highlighting their genetic diversity and demonstrating the utility of bioinformatics and proteomics for a broader analysis of pathogens.

Stress Adaptation Responses of a Listeria monocytogenes 1/2a Strain via Proteome Profiling.

Listeria monocytogenes is a foodborne pathogen that is ubiquitous and largely distributed in food manufacturing environments. It is responsible for listeriosis, a disease that can lead to significant morbidity and fatality in immunocompromised patients, pregnant women, and newborns. Few reports have been published about proteome adaptation when L. monocytogenes is cultivated in stress conditions. In this study, we applied one-dimensional electrophoresis and 2D-PAGE combined with tandem mass spectrometry to evaluate proteome profiling in the following conditions: mild acid, low temperature, and high NaCl concentration. The total proteome was analyzed, also considering the case of normal growth-supporting conditions. A total of 1,160 proteins were identified and those related to pathogenesis and stress response pathways were analyzed. The proteins involved in the expression of virulent pathways when L. monocytogenes ST7 strain was grown under different stress conditions were described. Certain proteins, particularly those involved in the pathogenesis pathway, such as Listeriolysin regulatory protein and Internalin A, were only found when the strain was grown under specific stress conditions. Studying how L. monocytogenes adapts to stress can help to control its growth in food, reducing the risk for consumers.

The Slaughterhouse as Hotspot of CC1 and CC6 Listeria monocytogenes Strains with Hypervirulent Profiles in an Integrated Poultry Chain of Italy.

In Europe, very few studies are available regarding the diversity of Listeria monocytogenes (L. monocytogenes) clonal complexes (CCs) and sequence types (ST) in poultry and on the related typing of isolates using whole genome sequencing (WGS). In this study, we used a WGS approach to type 122 L. monocytogenes strains isolated from chicken neck skin samples collected in two different slaughterhouses of an integrated Italian poultry company. The studied strains were classified into five CCs: CC1-ST1 (21.3%), CC6-ST6 (22.9%), CC9-ST9 (44.2%), CC121-ST121 (10.6%) and CC193-ST193 (0.8%). CC1 and CC6 strains presented a virulence gene profile composed of 60 virulence genes and including the Listeria Pathogenicity Island 3, aut_IVb, gltA and gltB. According to cgMLST and SNPs analysis, long-term persistent clusters belonging to CC1 and CC6 were found in one of the two slaughterhouses. The reasons mediating the persistence of these CCs (up to 20 months) remain to be elucidated, and may involve the presence and the expression of stress response and environmental adaptation genes including heavy metals resistance genes (cadAC, arsBC, CsoR-copA-copZ), multidrug efflux pumps (mrpABCEF, EmrB, mepA, bmrA, bmr3, norm), cold-shock tolerance (cspD) and biofilm-formation determinants (lmo0673, lmo2504, luxS, recO). These findings indicated a serious risk of poultry finished products contamination with hypervirulent L. monocytogenes clones and raised concern for the consumer health. In addition to the AMR genes norB, mprF, lin and fosX, ubiquitous in L. monocytogenes strains, we also identified parC for quinolones, msrA for macrolides and tetA for tetracyclines. Although the phenotypical expression of these AMR genes was not tested, none of them is known to confer resistance to the primary antibiotics used to treat listeriosis The obtained results increase the data on the L. monocytogenes clones circulating in Italy and in particular in the poultry chain.

Catering of high-risk foods and potential of stored food menu data for timely outbreak investigations in healthcare facilities, Italy and Germany.

Healthcare-associated foodborne outbreaks (HA-FBOs) can cause significant morbidity and mortality, affecting particularly vulnerable hospital populations. Electronic records of food served in healthcare facilities (HCFs) could be useful for timely investigations of HA-FBOs. We explored the availability and usability of electronic food menu data to support investigations of HA-FBOs through a survey among 35 HCFs in Germany (n = 13) and in Italy (n = 22). Large variability was reported in the storage time of menu data (from no storage up to 10 years) and their formats, including paper, electronic (PDF, Word, Excel), or fully searchable databases (15/22 in Italian HCFs, 3/13 in German HCFs). Food products that may present a risk to vulnerable persons - including deli salads, raw/fermented sausage products, soft cheese, smoked fish or frozen berries - were offered on the menu of all HCFs in Germany, and one-third of the Italian HCFs. The usability of electronic food menu data for the prevention or investigation of HA-FBOs may be suboptimal in a large number of HCFs in Germany, as well as in some HCFs in Italy. Standardised collection for use of electronic food menu data might help discover the association between illnesses and food eaten during outbreak investigations. Hospital hygienists, food safety and public health authorities should collaborate to increase implementation of food safety guidelines.

Immunoproteome profiling of Listeria monocytogenes under mild acid and salt stress conditions.

Listeria monocytogenes is one of the main foodborne pathogens worldwide. Although its response to stress conditions has been extensively studied, it is still present in the food processing environments and is a concern for consumers. To investigate how this microorganism adapts its proteome in mild stress conditions, a combined proteomics and bioinformatics approach was used to characterize the immunogenic protein profile of a sequence type 7 (ST7) strain that caused severe listeriosis outbreaks in central Italy. Extracted proteins were analyzed by immunoblotting using positive sera against L. monocytogenes and nLC-ESI-MS/MS, and all data were examined by five software to predict subcellular localization (SCL). A total of 226 proteins were extracted from the bands of interest, 58 of which were classified as potential immunogenic antigens. Compared to control cells grown under optimal conditions, six proteins, some of which under-described, were expressed under mild acid and salt stress conditions and/or at 12°C. In particular, adaptation and shaping of the proteome mainly involved cell motility at 12°C without acid and salt stress, whereas the combination of the same temperature with mild acid and salt stress induced a response concerning carbohydrate metabolism, oxidative stress, and DNA repair. Raw data are available via ProteomeXchange with identifier PXD033519.

Investigating the cecal microbiota in broiler poultry farms and its potential relationships with animal welfare.

The present study assessed the modulation of cecal microbiota and correlations with Campylobacter colonization and animal welfare status. For these purposes, we conducted a cross sectional study of the cecal microbiota from 187 broilers reared in 13 batches from 10 poultry farms by performing 16S rRNA sequencing (regions V3-4). The welfare of each batch was assessed using a simplified Welfare Quality® protocol, scoring higher in organic batches, compared to both antibiotic-free and conventional batches. The bioinformatics analyses were conducted in QIIME 2 and a linear discriminant analysis determined the association between microbiota and animals with different Campylobacter carriage status and welfare levels. In the microbiota from the subjects negative for Campylobacter or with high welfare scores, Bacteroidetes was the predominant phylum with the genus Megamonas significantly increased in abundance. A greater abundance of Parabacteroides, Phascolarctobacterium, Helicobacter in poultry negative for Campylobacter was also found at the genus level. Animals with the lowest welfare scores showed an increased abundance of Proteobacteria. The results suggested a different microbial composition and diversity in the analyzed groups.

Healthcare-associated foodborne outbreaks in high-income countries: a literature review and surveillance study, 16 OECD countries, 2001 to 2019.

BackgroundHealthcare-associated foodborne outbreaks (HA-FBO) may have severe consequences, especially in vulnerable groups.AimThe aim was to describe the current state of HA-FBO and propose public health recommendations for prevention.MethodsWe searched PubMed, the Outbreak Database (Charité, University Medicine Berlin), and hand-searched reference lists for HA-FBO with outbreak onset between 2001 and 2018 from Organisation for Economic Co-operation and Development (OECD) countries and HA-FBO (2012-2018) from the German surveillance system. Additionally, data from the European Food Safety Authority were analysed.ResultsThe literature search retrieved 57 HA-FBO from 16 OECD countries, primarily in the US (n = 11), Germany (n = 11) and the United Kingdom (n = 9). In addition, 28 HA-FBO were retrieved from the German surveillance system. Based on the number of outbreaks, the top three pathogens associated with the overall 85 HA-FBO were Salmonella (n = 24), norovirus (n = 22) and Listeria monocytogenes (n = 19). Based on the number of deaths, L. monocytogenes was the main pathogen causing HA-FBO. Frequently reported implicated foods were 'mixed foods' (n = 16), 'vegetables and fruits' (n = 15) and 'meat and meat products' (n = 10). Consumption of high-risk food by vulnerable patients, inadequate time-temperature control, insufficient kitchen hygiene and food hygiene and carriers of pathogens among food handlers were reported as reasons for HA-FBO.ConclusionTo prevent HA-FBO, the supply of high-risk food to vulnerable people should be avoided. Well working outbreak surveillance facilitates early detection and requires close interdisciplinary collaboration and exchange of information between hospitals, food safety and public health authorities.

Animal Welfare Assessment in Antibiotic-Free and Conventional Broiler Chicken.

The poultry sector is moving towards antibiotic-free production, both to challenge the increasing spread of the antibiotic resistance phenomenon and to meet market demands. This could negatively impact the health and welfare of the animals. In this study, the welfare of 14 batches of 41-47-day-old broilers raised by the same integrated company with and without antibiotics was assessed using the Welfare Quality® protocol. The total welfare score did not significantly differ between the two systems: the good-feeding principle was, on average, higher in the conventional batches, with statistical significance (t = -2.45; p = 0.024), while the other welfare principles (good housing, good health and appropriate behaviour) were slightly better in the antibiotic-free batches. Despite stocking densities averagely higher in the antibiotic-free batches, the absence of antibiotics did not seem to impact the good-health principle; in particular, hock burns, foot pad dermatitis and lameness were significantly less severe in the antibiotic-free batches (p < 0.0001, p = 0.018, p < 0.0001, respectively), which showed also a lower death rate (2.34% vs. 2.50%). Better management of antibiotic-free batches was reported, particularly concerning litter conditions. Further studies would be required to identify and standardise a set of managerial methodologies in order to improve the health of broilers raised without antibiotics.

Survival rate of Escherichia coli O157 in artificially contaminated raw and thermized ewe milk in different Pecorino cheese production processes.

Pecorino is a typical Italian cheese, mostly produced in central and southern Italy regions using ewe raw milk and following traditional procedures. The use of raw milk constitutes a risk linked to the potential survival or multiplication of pathogenic microorganisms, as Shiga toxin-producing Escherichia coli (STEC). The aim of this study was to compare different Italian traditional Pecorino production methods to determine if there were any phases that could influence the Escherichia coli O157 survival rate, but also if they could negatively influence lactic acid bacteria survival rate, during the phases of production and ripening. Therefore batches of Pecorino cheese were prepared using different production methods, representing the real and typical cheese production in southern and central Italy regions: 1) heating the milk at 37 °C for about 40 min before curding, 2) heating the milk at 60 °C (thermization) for 13 min, so that the alkaline phosphatase reaction is still positive before curding, 3) cooking curd at 41 °C and 4) at 45 °C, both for 5 min. Our results demonstrated that traditional milk treatments different from pasteurization can help but do not eliminate serious microbiological treats, as E. coli O157, especially if the raw milk is heavily contaminated. The heat treatment at 60 °C applied to raw milk was able to decrease the concentration of E. coli O157 of 1.7 log10CFU/ml and, according to the inactivation slope, it would be further reduced prolonging the heating treatment. The results obtained also showed that, during the Pecorino cheese ripening, E. coli O157 was always enumerable for 60 days, remaining detectable after 90 days of ripening.

Humoral immune response and delayed-type hypersensitivity in rabbits infected with Trypanosoma equiperdum.

Trypanosoma equiperdum is the causative agent of dourine, a parasitic venereal disease of equids. In this work, rabbits were infected with T. equiperdum strain OVI; serological tests (complement fixation test, ELISA and immunoblotting), used for the diagnosis of dourine in horses, were applied to study rabbit humoral immune response and to characterise T. equiperdum antigen pattern recognised by antibodies from infected rabbits. Moreover a protein extract of T. equiperdum strain OVI was produced and tested in skin tests on infected rabbits to detect the cell-mediated response induced by T. equiperdum, in order to evaluate its use in the field diagnosis of dourine. Sera of infected rabbits recognized in immunoblotting Trypanosoma protein bands with molecular weight below 37 kDa, providing a serological response comparable with that already observed in dourine infected horses. Moreover the trypanosome protein extract was capable to produce in vivo delayed-type hypersensitivity (DHT Type IV) in rabbits and proved itself to be non-toxic and non-sensitizing.

Validation via challenge test of a dynamic growth-death model for the prediction of Listeria monocytogenes kinetics in Pecorino di Farindola cheese.

Pecorino di Farindola is a typical cheese produced in the area surrounding the village of Farindola, located in the Abruzzo Region (central Italy), unique among Italian cheese because only raw ewe milk and pig rennet are used for its production. In the literature it is well documented that raw milk is able to support the growth of pathogenic microorganisms such as Listeria monocytogenes. Predictive microbiology can be useful in order to predict growth-death kinetics of pathogenic bacteria, on the basis of known environmental conditions. Aim of this study was to compare predictions obtained from a model, originally designed to predict the kinetics of L. monocytogenes in the dynamic growth-death environment of drying fresh sausage, with the results of challenge tests performed during the ripening of Pecorino di Farindola produced from artificially contaminated raw ewe milk. A challenge test was carried out using ewe raw milk inoculated with L. monocytogenes, in order to produce Pecorino di Farindola cheese stored at 18 °C for 149 days of ripening. During the ripening period, pH and aw values decreased in all samples analysed; lactic acid bacteria become the prevailing microbial population, while for L. monocytogenes a period of stability (neither growth nor death) followed the initial situation. The growth inhibition and the following inactivation may mostly be due to competition with the autochthonous microbiota and to the reduction of water activity. Mathematical modelling was used in order to predict microbial kinetics in the dynamic ripening environment, joining growth and death patterns in a continuous way, and including the highly uncertain growth/no growth range separating the two regions. The effect of lactic acid bacteria on the growth of pathogens was also included. Predicted microbial kinetics were satisfactory, as confirmed by the absence of statistically significant difference between observed and predicted values (p > 0.05). The present study proved, via challenge tests, that a dynamic growth/death model, previously used for a meat product, can be fruitfully used in cheese characterized by active competitive microbiota and progressive drying during ripening.

Listeria monocytogenes in poultry: Detection and strain characterization along an integrated production chain in Italy.

In this study, thirteen batches of broiler chicken from an integrated Italian poultry company were investigated for the detection of Listeria monocytogenes. The prevalence was evaluated in faeces samples at farm level and after transport, caecal contents and carcass neck skin from 2 slaughterhouses (M1 and M2), for a total of 2080 samples, throughout a 27-month period. No positive results were recorded in faeces, while the overall prevalence of contamination in carcass neck skin was 26.7%. Then, 123 isolates out of 139 positive skin samples, with the prevalent serotypes 4b (76%) and 1/2b (94%) from slaughterhouses M1 and M2 respectively, were PFGE characterized, showing the presence of 18 different pulsotypes and 8 genetic clusters. The same pulsotypes were found in carcasses from different farms, but slaughtered in the same abattoir, highlighting the environmental origin of contamination. The persistence of the pathogen over long time seemed to be very likely, considering that undistinguishable pulsotypes were found in carcasses slaughtered in the same slaughterhouse after periods up to 18 months long. The implementation of cleaning and sanitation at slaughterhouse level could represent the main factor for the control of such pathogen in the poultry meat processing line.

Campylobacter jejuni and Campylobacter coli: prevalence, contamination levels, genetic diversity and antibiotic resistance in Italy.

A research was carried out in Italy with the aim of assessing Campylobacter contamination in broilers from breeding to slaughter, of defining the genetic diversity of isolates and their antibiotic resistance. Sampling was carried out in a slaughterhouse, and in farms representative of the most common broiler production in Italy. At farm, the 78.8% (95% C.I.: 74.5%­82.5%) of cloacal samples tested positive for Campylobacter spp. C. jejuni showed higher prevalence in winter than in spring and summer (p < 0.00001, χ2 = 32.9), while C. coli showed an opposite trend (p < 0.00001, χ2= 41.1). At slaughterhouse, the 32.3% (95% C.I.: 30.2%­35.2%) and the 23.9% (95% C.I.: 21.7%­26.3%) of skin samples tested positive for C. jejuni for C. coli, respectively. C. coli showed higher prevalence than C. jejuni at washing (p < 0.05, χ2 = 11.11) and at chilling (p < 0.05, χ2 = 9.26). PFGE revealed high heterogeneity among isolates. Some clones were identified within the same farm in more than one season, suggesting environmental conditions able to support their persistence; other clones resulted to be spatially distant, suggestive of cross­contamination. Both Campylobacter species showed high resistance to nalidixic acid and ciprofloxacin, while resistance to erythromycin was more frequent in C. coli than C. jejuni (p < 0.05; χ2 test).

Production of monoclonal antibodies binding to the VP7 protein of African horse sickness virus.

Monoclonal antibodies (MAbs) against AHSV were produced by immunising BALB/c mice with AHSV serotype 9 and six clones able to recognize specifically the VP7-AHSV with a strong reactivity were selected. The specificity of the MAbs was assessed in i-ELISA against a commercial VP7-AHSV and in immunoblot against a home-made VP7-AHSV, expressed by a Baculovirus expression system; potential cross-reactions with related orbiviruses (Bluetongue virus and Epizootic Haemorrhagic Disease virus) were investigated as well. One of the six MAbs selected, MAb 7F11E14, was tested in direct immunofluorescence and reacted with all nine AHSV serotypes, but didn't cross-react with BTV and EHDV. MAb 7F11E14 was also used to develop a competitive ELISA and was able to detect AHSV antibodies in the sera of AHS infected animals.

Challenge test studies on Listeria monocytogenes in ready-to-eat iceberg lettuce.

Shelf-life studies in ready-to-eat (RTE) modified atmosphere packaged (MAP) precut iceberg lettuce (minimally processed) were carried out in order to evaluate the natural microflora of the product and survival or multiplication of Listeria monocytogenes (L. monocytogenes), taking into consideration the impact of the production steps resulting in a reduction of the shelf life of the fresh-cut produce, due to the accelerated enzymatic activity, moisture loss, and microbial proliferation. The research first aimed to evaluate the characteristics of the natural microflora of the product, and then, L. monocytogenes dynamics were studied via challenge tests. L. monocytogenes concentration was studied at 8 and 12°C storage temperature for 10 days, 6 days longer than their shelf life. The number of L. monocytogenes in samples stored both at 8°C and 12°C increased gradually, more evidently in samples stored at 12°C. L. monocytogenes dynamics were studied to define maximum growth rate (µmax) at 8°C (0.0104 log10CFU/g/h) and 12°C (0.0183 log10CFU/g/h). Data obtained from the study were used to develop and validate a specific predictive model able to predict the behavior of L. monocytogenes in RTE MAP iceberg lettuce. According to the model, an increase in storage temperature of 6°C (e.g., from 8 to 14°C) would lead to an increase in L. monocytogenes concentration of more than 6 log10CFU/g at the 10th day of the challenge test (12th days of shelf life). Storage at 4°C allowed to increase L. monocytogenes enumeration from 3.30 log10CFU/g at D0 to 3.60 log10CFU/g at D10. The model could be applied to microorganisms other than L. monocytogenes, modifying the coefficients of the polynomial equation on which it is based.

Development of an indirect ELISA for the serological diagnosis of dourine.

Dourine is a parasitic venereal disease of equines caused by T. equiperdum. Humoral antibodies are found in infected animals, but diagnosis of dourine must include history, clinical, and pathological findings in addition to serology. Complement Fixation Test (CFT) is the Office International des Epizooties (OIE) recommended test for international trade; however, some uninfected equines may give inconsistent or nonspecific reactions in CFT due to the anticomplementary effects of their sera. In this study an Indirect Enzyme Linked Immunosorbent Assay (iELISA) was developed. This test could be used to confirm positive serological cases of dourine or to solve inconclusive results obtained by CFT, in addition to Indirect Fluorescent Antibody Test (IFAT) and a Chemiluminescent Immunoblotting Assay (cIB). Six-hundred-and-six CFT negative sera and 140 sera positive to CFT and IFAT were tested by iELISA using OVI T. equiperdum as antigen. Results were expressed as percentage of positivity and the optimum cut-off value determined sensitivity and specificity of 100%. All positive sera, tested by cIB, were confirmed as positive. Additionally, twenty seven sera, low-positive at CFT and negative by IFAT, were tested with iELISA and cIB. All samples resulted negative by cIB and one of them was positive in ELISA. Our results suggest that iELISA and cIB may be used as alternative or supplementary confirmatory tests whenever other recommended serological methods are inconclusive or doubtful.

Validation of the Socially Acceptable Behaviour (SAB) test in a Central­Italy pet dog population.

Aggressiveness is reported to be the most important public health issue related to both owned and free­roaming dogs. Common approaches to assess canine aggressiveness are temperament tests. The main aim of this study was to evaluate whether one such test, the Socially Acceptable Behaviour (SAB) test, created to evaluate aggressive and fearful behaviour in dogs in the Netherlands, could be used reliably to assess dog aggression and fear in a population of owned dogs in Central Italy. Reactions to the test were recorded and compared to the owners' perception of their dog's aggressiveness using a validated questionnaire (C­BARQ). Dogs showing aggressive reactions during the test obtained significantly higher (more aggressive) scores on the C­BARQ subscales 'stranger­directed aggression' (SDA p<0.001), 'owner­directed aggression' (ODA p = 0.03), and 'familiar dog aggression' (FDA p = 0.006), than dogs who did not react aggressively. Logistic regression analysis revealed that 7 of the SAB­subtests were predictive of the SDA score. The findings indicated that aggression directed toward unfamiliar people can be reliably assessed using the SAB test for a population of Italian pet dogs.

Listeria monocytogenes in Smoked Salmon and Other Smoked Fish at Retail in Italy: Frequency of Contamination and Strain Characterization in Products from Different Manufacturers.

Seven hundred seventy-eight samples of packaged smoked fish (774 smoked salmon and 4 smoked swordfish) on sale in Italy, from 50 different manufacturers located in 12 European Union countries, were purchased from the Italian market between May and December 2011. The surface temperatures of the samples on sale ranged from 0 to 13°C (3.4 ± 1.5°C, mean ± SD). Six hundred eighty (87.4%) of 778 samples were stored at ≤4°C. One hundred fifty-seven samples (20.2%, 95% confidence interval 17.5 to 23.1%) were contaminated by Listeria monocytogenes , with 26 samples (3.3%, 95% confidence interval 2.3 to 4.8%) at levels >100 CFU/g. The maximum level of contamination was 1.3 ×106 CFU/g. The differences in the level of contamination of smoked fish between countries (χ2 = 91.54, P < 0.05) and manufacturers (χ2 = 193.22, P < 0.05) were significant. The frequency of detection for products from different manufacturing premises ranged from 0 to 76.9%. Serotyping by serological agglutination revealed that the main serotypes detected were 1/2a (65.3%) and 1/2b (22.4%). Pulsed-field gel electrophoresis typing with restriction enzymes AscI and ApaI yielded 36 pulsotypes from 144 isolates, clustering into 17 groups. Eight main pulsotypes accounted for 70.8% of the isolates. Three of the main pulsotypes were exclusively from products of a single manufacturer. In general, products from the same manufacturer showed genetic homogeneity, with one strongly prevalent pulsotype. Different manufacturers usually showed very different levels of contamination of the final product, confirming the importance of the management of process hygiene for controlling L. monocytogenes contamination.

Predicting the kinetics of Listeria monocytogenes and Yersinia enterocolitica under dynamic growth/death-inducing conditions, in Italian style fresh sausage.

Traditional Italian pork products can be consumed after variable drying periods, where the temporal decrease of water activity spans from optimal to inactivating values. This makes it necessary to A) consider the bias factor when applying culture-medium-based predictive models to sausage; B) apply the dynamic version (described by differential equations) of those models; C) combine growth and death models in a continuous way, including the highly uncertain growth/no growth range separating the two regions. This paper tests the applicability of published predictive models on the responses of Listeria monocytogenes and Yersinia enterocolitica to dynamic conditions in traditional Italian pork sausage, where the environment changes from growth-supporting to inhibitory conditions, so the growth and death models need to be combined. The effect of indigenous lactic acid bacteria was also taken into account in the predictions. Challenge tests were carried out using such sausages, inoculated separately with L. monocytogenes and Y. enterocolitica, stored for 480h at 8, 12, 18 and 20°C. The pH was fairly constant, while the water activity changed dynamically. The effects of the environment on the specific growth and death rate of the studied organisms were predicted using previously published predictive models and parameters. Microbial kinetics in many products with a long shelf-life and dynamic internal environment, could result in both growth and inactivation, making it difficult to estimate the bacterial concentration at the time of consumption by means of commonly available predictive software tools. Our prediction of the effect of the storage environment, where the water activity gradually decreases during a drying period, is designed to overcome these difficulties. The methodology can be used generally to predict and visualise bacterial kinetics under temporal variation of environments, which is vital when assessing the safety of many similar products.