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The commercialization of processed fish products is rising in restaurants and small to medium enterprises. However, there is a lack of data related to the microbiological safety of such products. In this study total aerobic colony count and Enterobacteriaceae, as proxy of process hygiene criteria, and detection of Listeria monocytogenes and concentration of histamine, as food safety criteria, were investigated in Salmo salar (salmon), Xiphias gladius (swordfish) and Thunnus albacares (yellowfin tuna), before, during, and at the end of a dry-curing process, performed in a dedicated cabinet, at controlled temperature, relative humidity and ventilation, up to 240 h. The microbiological parameters were investigated in the tested fish products by culture methods and shotgun metagenomic, while the presence of histamine, and other biogenic amines, was quantified by High Performance Liquid Chromatography. In the raw material, and up to the end of the dry curing process, the concentration of Enterobacteriaceae was always lower than 10 CFU/g, while total aerobic colony counts ranged between 3.9 and 5.4 Log CFU/g in salmon; 5.5 and 5.9 Log CFU/g in swordfish; 4.4 and 4.8 Log CFU/g in tuna. The pH values were significantly different between fish species, in the raw materials and during processing except for T4, occurring 70 h after the start of the process for salmon and after 114 h for swordfish and tuna. Water activity was different at specific sampling points and at the end of processing. Overall, 79 % of the sequences identified in the tested fish samples were assigned to y bacteria. The most abundant phyla were Pseudomonadota, Bacillota and Mycoplasmatota. The microbial populations identified by shotgun metagenomic in the tested fish species clustered well separated one from the other. Moreover, the microbial richness was significantly higher in salmon and tuna in comparison to swordfish. Listeria monocytogenes was not detected in the raw material by using the reference cultural method and very few reads (relative abundance <0.007) were detected in swordfish and tuna by shotgun metagenomic. Histamine producing bacteria, belonging to the genera Vibrio, Morganella, Photobacterium and Klebsiella, were identified primarily in swordfish. However, histamine and other biogenic amines were not detected in any sample. To the best of our knowledge this is the first paper reporting time point determinations of microbiological quality and safety parameters in salmon, swordfish and tuna, before, during and at the end of a dry-curing process. The data collected in this paper can help to predict the risk profile of ready to eat dry-cured fish products during storage before consumption., Competing Interests: Declaration of competing interest None of the authors declare to have financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Current sampling plans assume a random distribution of microorganisms in food. However, food-borne pathogens are estimated to be heterogeneously distributed in powdered foods. This spatial distribution together with very low level of contaminations raises concern of the efficiency of current sampling plans for the detection of food-borne pathogens like Cronobacter and Salmonella in powdered foods such as powdered infant formula or powdered eggs. An alternative approach based on a Poisson distribution of the contaminated part of the lot (Habraken approach) was used in order to evaluate the probability of falsely accepting a contaminated lot of powdered food when different sampling strategies were simulated considering variables such as lot size, sample size, microbial concentration in the contaminated part of the lot and proportion of contaminated lot. The simulated results suggest that a sample size of 100g or more corresponds to the lower number of samples to be tested in comparison with sample sizes of 10 or 1g. Moreover, the number of samples to be tested greatly decrease if the microbial concentration is 1CFU/g instead of 0.1CFU/g or if the proportion of contamination is 0.05 instead of 0.01. Mean contaminations higher than 1CFU/g or proportions higher than 0.05 did not impact on the number of samples. The Habraken approach represents a useful tool for risk management in order to design a fit-for-purpose sampling plan for the detection of low levels of food-borne pathogens in heterogeneously contaminated powdered food. However, it must be outlined that although effective in detecting pathogens, these sampling plans are difficult to be applied since the huge number of samples that needs to be tested. Sampling does not seem an effective measure to control pathogens in powdered food., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In our study we aimed at deriving performance objectives (POs) for Salmonella in fresh pork meat intended to be eaten cooked, using loin chop as a model. Moreover, sampling plans to verify the compliance of meat lots to such POs are presented. Ten lots of product, collected in the same slaughterhouse along a one-year period, were tested for Salmonella detection under four different storage stages through the product shelf life. The POs were considered as different target values from Salmonella prevalence and were calculated at the 50th percentile of prevalence distributions under each stage. Results obtained indicated that values increased between a minimum of 26.10% of positives after final storage at 14°C to a maximum of 46.70% of positives after storage at retail. The number of samples to be tested in order to detect at least one positive and verify the compliance to the estimated POs ranged between five, for samples after storage at retail, and ten, for samples stored at 14°C before the expiration date. For risk management purposes, percentiles different from the 50th can be selected in order to derive the POs as well as the number of samples to be tested in order to verify their fulfillment. Thus, the approach presented in this paper offers different options to risk managers for improving the decision-making process., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In 2002, the Regulation (EC) 178 of the European Parliament and of the Council states that, in order to achieve the general objective of a high level of protection of human health and life, food law shall be based on risk analysis. However, the Commission Regulation No 2073/2005 on microbiological criteria for foodstuffs requires that food business operators ensure that foodstuffs comply with the relevant microbiological criteria. Such criteria define the acceptability of a product, a batch of foodstuffs or a process, based on the absence, presence or number of micro-organisms, and/or on the quantity of their toxins/metabolites, per unit(s) of mass, volume, area or batch. The same Regulation describes a food safety criterion as a mean to define the acceptability of a product or a batch of foodstuff applicable to products placed on the market; moreover, it states a process hygiene criterion as a mean indicating the acceptable functioning of the production process. Both food safety criteria and process hygiene criteria are not based on risk analysis. On the contrary, the metrics formulated by the Codex Alimentarius Commission in 2004, named Food Safety Objective (FSO) and Performance Objective (PO), are risk-based and fit the indications of Regulation 178/2002. The main aims of this review are to illustrate the key differences between microbiological criteria and the risk-based metrics defined by the Codex Alimentarius Commission and to explore the opportunity and also the possibility to implement future European Regulations including PO and FSO as supporting parameters to microbiological criteria. This review clarifies also the implications of defining an appropriate level of human protection, how to establish FSO and PO and how to implement them in practice linked to each other through quantitative risk assessment models. The contents of this review should clarify the context for application of the results collected during the EU funded project named BASELINE (www.baselineeurope.eu) as described in the papers of this special issue. Such results show how to derive POs for specific food/biological hazard combinations selected among fish, egg, dairy, meat and plant products., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The aim of this paper was to provide suitable microbiological criteria (MC) for Campylobacter in broiler carcasses and a sampling plan to verify compliance with such criteria. Data were gathered in the presence and concentration of Campylobacter in broiler carcasses collected in three different Italian slaughterhouses, labelled as A, B and C. The sampling plan to be validated in each slaughterhouse included the analysis of three different carcasses collected immediately after chilling from 30 different lots, for a total of 90 samples per slaughterhouse. The number of positive samples containing above 100 CFU/g and above 1,000 CFU/g throughout the 30 tested lots was determined to estimate between-lot variability. Based on this information, the performance of four MC was evaluated for lot compliance: i) n=3; c=0; m=100 CFU/g; ii) n=3; c=0; m=1,000 CFU/g; iii) n=3; c=1; m=1,000 CFU/g and iv) n=3; c=2; m=1,000 CFU/g. Positive Campylobacter samples were found in 60% of the lots tested in slaughterhouses A and C and in 73.3% of lots from slaughterhouse B. The differences among the three slaughterhouses in the mean Campylobacter levels found in positive samples were not significant and were used to evaluate the performance of the MC. The level of lot compliance to different MC was calculated and for the most stringent one (n=3; c=0; m=100 CFU/g) was 40% at slaughterhouses A and C but only 26.7% at slaughterhouse B. The results of this study show an alternative approach to establish MC for Campylobacter in broilers. According to (1) Campylobacter prevalence and concentration in Italy, (2) applied experimental plan and (3) selected slaughterhouses, the number of compliant lots to the suggested MC ranged between 26.7 and 100%. The selection of the fit for purpose MC is a risk manager decision, based on a reasonable balance between public health and cost for poultry industries., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Raw sheep milk cured cheese produced in the Castilla y Leon region (Spain) constitutes a traditional semi-hard aromatic cheese typically aged for three to six months. This product is catalogued as ready-to-eat since it is not submitted to any further treatment before consumption. Thus, foodborne pathogens such as Listeria monocytogenes can represent a health concern for susceptible consumers. This study was aimed at evaluating the survival of L. monocytogenes on raw sheep milk cured cheese under different storage temperatures. Log-linear+shoulder and Weibull type models were fitted to data observed in order to estimate kinetic parameters. The Arrhenius relationship was further used to predict the impact of temperature on L. monocytogenes behavior during storage at 4, 12 and 22°C. Additionally, growth of lactic acid bacteria (LAB) as a representative group of the indigenous microbiota was evaluated. Results obtained indicated that the time to eradication (time when absence of L. monocytogenes in the analyzed samples was observed) was 114, 104, and 77 days for cheese samples stored at 4, 12 and 22°C, respectively. The LAB population showed an increase at 12 and 22°C during storage. However, an increase of 1 log CFU/g was observed during the first 2 weeks irrespectively of the storage temperature. The log-linear+shoulder model indicated a good fit to observed data. Likewise, the Arrhenius relationship explained sufficiently the dependency of temperature on L. monocytogenes behavior. This study demonstrated that cheese storage at ambient temperatures could lead to the preservation of its quality properties as well as its safety against L. monocytogenes., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Eggs and egg products have been described as the most critical food vehicles of salmonellosis. The prevalence and level of contamination of Salmonella on table eggs are low, which severely affects the sensitivity of sampling plans applied voluntarily in some European countries, where one to five pools of 10 eggs are tested by the culture based reference method ISO 6579:2004. In the current study we have compared the testing-sensitivity of the reference culture method ISO 6579:2004 and an alternative real-time PCR method on Salmonella contaminated egg-pool of different sizes (4-9 uninfected eggs mixed with one contaminated egg) and contamination levels (10°-10(1), 10(1)-10(2), 10(2)-10(3)CFU/eggshell). Two hundred and seventy samples corresponding to 15 replicates per pool size and inoculum level were tested. At the lowest contamination level real-time PCR detected Salmonella in 40% of contaminated pools vs 12% using ISO 6579. The results were used to estimate the lowest number of sample units needed to be tested in order to have a 95% certainty not falsely to accept a contaminated lot by Monte Carlo simulation. According to this simulation, at least 16 pools of 10 eggs each are needed to be tested by ISO 6579 in order to obtain this confidence level, while the minimum number of pools to be tested was reduced to 8 pools of 9 eggs each, when real-time PCR was applied as analytical method. This result underlines the importance of including analytical methods with higher sensitivity in order to improve the efficiency of sampling and reduce the number of samples to be tested., (Copyright © 2013 Elsevier B.V. All rights reserved.)

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The goal of this study was to define a practical approach to derive risk management measures, such as performance objectives (POs), for Listeria monocytogenes in pork cuts intended to be eaten cooked. Moreover, sampling plans to verify the compliance of meat lots to such POs are presented. The POs were estimated as prevalence and/or concentration values that should not be exceeded at time of consumption. To derive possible POs for L. monocytogenes, ten lots of pork cuts, collected within the same slaughterhouse along a one-year period, were tested for the presence and concentration of the pathogen under four different scenarios through the product shelf life. Our results indicated that the median values of the prevalence distributions ranged between 0.41 and 0.68. The number of samples to be tested in order to verify lot compliance ranged between six, for samples tested immediately after packaging, and three, for samples tested at the end of the shelf life. The concentration values ranged between 2.02 log10 CFU/g, for samples tested immediately after packaging, up to 3.14 log10 CFU/g for samples tested after final storage at 14°C. The concentration of L. monocytogenes in the samples contaminated by less than 10 CFU/g was estimated between 7 CFU/10g to 7 CFU/g, after storage at retail and abuse temperature, respectively. Basing on the knowledge of log normal distributions, the maximum contamination level of the lots in order to achieve the suggested POs, was calculated. It was obtained that mean concentration estimated as PO should be between -0.43 and 0.48 log10 CFU/g. Other risk management options are further evaluated and discussed. These results would help food operators and authorities to establish safety targets and corrective actions regarding inhibition of L. monocytogenes in fresh pork meat., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In the present study, prevalence and concentration of Salmonella spp. and Listeria monocytogenes in fresh pork cuts were determined through the analysis of twelve lots for one year. Five samples were analyzed at retail and after storage at 4 and 12°C up to the end of shelf-life (7 days). Results obtained for Salmonella spp. indicated that a total of 15 samples (8.33%) were positive, which represents 4 (25%) sampling events positive (i.e. at least one sample was positive in at least one of the sampling scenarios). Salmonella was randomly distributed and direct correlation with storage time and temperature was not obtained. For L. monocytogenes, 26 samples (14.44%) were positive, which represents 5 (41.67%) positive sampling lots. For this pathogen, a group of samples were only positive at the end of the shelf-life but not immediately after purchasing indicating clearly that the contamination was not only heterogeneously distributed but also close to the levels of detection, and in all the cases below the limit of contamination. As neither Salmonella spp. nor L. monocytogenes was enumerated by direct plating (<10 cfu/g) a probabilistic approach basing on Binomial and Poisson distributions was subsequently performed to estimate microbial concentration from presence/absence data. Estimated concentration values were below 40 cfu/kg for both pathogens in more than 80% of the tested lots. The data collected in this study add new knowledge on this very important and difficult to control segment of the farm-to-fork chain., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Listeria monocytogenes has been detected in fresh as well as dry and semidry fermented sausages, rendering preparation and consumption of these products as a potential risk to human health. The aims of this study were (1) to evaluate the L. monocytogenes prevalence in 288 fresh and 237 fermented sausages produced in northern Italy; (2) to quantify the average pathogen Most Probable Number (MPN) per g of sausage; (3) to evaluate the sausage strain genetic diversity by automated PvuII ribotyping; and (4) to predict the pathogenicity lineage of these isolates determining their DuPont Identification Library Codes (DUP-IDs) by EcoRI ribotyping. The overall prevalence of L. monocytogenes in the sampled sausages was 28.2%. The percentage of L. monocytogenes positive fresh sausages was significantly higher than that of fermented sausages (i.e. 38.9 vs 15.2%), which had a pathogen load always lower than 10 MPN/g. In contrast, 16.1% of fresh sausages were contaminated by 10 to 100 MPN/g and 20.5% had more than 100 MPN/g. PvuII successfully discriminated sausage isolates with a Simpson's numerical index of discrimination of 0.637. A total of 12 and 9 different PvuII ribogroups were identified among 47 fresh and 24 fermented randomly selected sausage strains, respectively. Six of those ribogroups were shared between strains contaminating both kinds of sausages. According to the evaluation of the strain DUP-IDs, the majority of the isolates investigated in this study were part of the type II L. monocytogenes pathogenicity lineage, but type I lineage strains were identified among fermented sausage isolates. In conclusion, L. monocytogenes prevalence in Italian sausages was estimated to be around 28.2%. However, 84.2% of the samples were contaminated by less than 100 MPN of L. monocytogenes per g and the majority of L. monocytogenes contaminating strains would be classified in the type II pathogenicity lineage, including serotypes 1/2a, 1/2c and 3a.

In this study 1656 Gorgonzola cheeses, collected from October 2003 to April 2004 in the same industrial plant located in Lombardia (Italy), were analysed in order to evaluate their level of contamination with Listeria monocytogenes after packaging, as well as at the end of the shelf life. A subset of Gorgonzola isolates was submitted to automated EcoRI ribotyping to evaluate their DUP-IDs (DuPont identification library code) and their level of genetic diversity. The isolate ribotyping profiles were included in an on-line database named PathogenTracker database. The strain DUP-IDs and the similarities between Gorgonzola isolates and PathogenTracker human sporadic strains allowed to evaluate the potential virulence of Gorgonzola-associated strains. The L. monocytogenes detection rates observed in the cheese samples monitored after packaging and at the end of the shelf life were 2.1% and 4.8%, respectively. Seventy percent of the strains genotyped were classified in the same ribotype, labelled as 204 S5, indicating that L. monocytogenes population associated to Gorgonzola cheese shows a low level of genetic diversity. Ninety percent of the strains were classified in DUP-IDs belonging to the II pathogenicity lineage identified for L. monocytogenes. That lineage includes serotype 1/2a, 1/2c and 3c strains, associated to the 35% of the human sporadic isolates described in the literature as causing listeriosis. Moreover, 16.7% of Gorgonzola isolates showed a similarity >or=99% with PathogenTracker human sporadic strains. The results of this study showed that the incidence of L. monocytogenes in Gorgonzola cheeses commercialised by the plant tested was lower than that observed in other Italian blue-veined cheeses by other authors. However, it increased during cheese storage and it become double at the end of the cheese shelf life, ranging from 30 to 60 days after packaging.

The Campylobacter detection is performed by conventional culture methods and the identification of Campylobacter jejuni and Campylobacter coli is principally based on the hippurate hydrolysis test. The two major drawbacks of this biochemical test for species identification include the inconsistency of the results and the presence of atypical strains, which can lead to the misidentification of an isolate. As an alternative, multiplex polymerase chain reaction (mPCR) protocols for the simultaneous detection and identification of different Campylobacter species have been developed. This study examined the performances of an experimental BAX System assay for the C. jejuni and C. coli identification in comparison to a multiplex PCR protocol recently published. The samples tested were represented by 106 environmental swabs collected on Teflon strips and tables, stainless steel saws, hooks and trays, ceramic floors and walls, as well as equipment surfaces, located in a swine (N=50) and a poultry (N=56) slaughterhouse. The highest Campylobacter detection rate was obtained after 48 h of enrichment by using both the PCR procedures. After 24 h, the BAX System provides a more rapid and accurate Campylobacter detection and identification assay than the multiplex PCR. Except for two samples, all the broths where Campylobacter cells were detected after 24 or 48 h of enrichment, with at least one of the PCR protocols, gave Campylobacter colonies using the culture method.