Comprehensive Review on the Biocontrol of Listeria monocytogenes in Food Products.

Listeria monocytogenes is a foodborne pathogen that causes listeriosis, a group of human illnesses that appear more frequently in countries with better-developed food supply systems. This review discusses the efficacy of actual biocontrol methods combined with the main types of food involved in illnesses. Comments on bacteriophages, lactic acid bacteria, bacteriocins, essential oils, and endolysins and derivatives, as main biological antilisterial agents, are made bearing in mind that, using them, food processors can intervene to protect consumers. Both commercially available antilisterial products and solutions presented in scientific papers for mitigating the risk of contamination are emphasized. Potential combinations between different types of antilisterial agents are highlighted for their synergic effects (bacteriocins and essential oils, phages and bacteriocins, lactic acid bacteria with natural or synthetic preservatives, etc.). The possibility to use various antilisterial biological agents in active packaging is also presented to reveal the diversity of means that food processors may adopt to assure the safety of their products. Integrating biocontrol solutions into food processing practices can proactively prevent outbreaks and reduce the occurrences of L. monocytogenes-related illnesses.

Hand hygiene practices during meal preparation-a ranking among ten European countries.

BACKGROUND: The aim of this paper was to map consumers' food hygiene practices from 10 European countries and evaluate which demographic groups are more likely to be exposed to foodborne pathogens and establish a ranking of adherence to food hygiene practices in 10 European countries. METHODS: The research design consisted of a cross-national quantitative consumer survey regarding food safety and hygiene practices during meal preparation (SafeConsume project) and was conducted in ten European countries (France, Denmark, Germany, Greece, Hungary, Norway, Portugal, Romania, Spain and UK). The survey questions were based on recommended hand hygiene practices and on observed practices from a field study performed in 90 European households from six of the countries covered by the survey (France, Hungary, Norway, Portugal, Romania, and UK). SPSS Statistics 26 (IBM Software Group, Chicago, IL) was used for the descriptive and regression analyses of the data. Regression analyses were used to check the relation between demographic characteristics, country of origin and self-reported hand hygiene practices. RESULTS: According to the regression models, families with elderly members aged over 65 showed a higher tendency to follow proper hand washing practices compared to families without elderly members. Meanwhile, families with children under the age of 6 reported being up to twice as likely to wash their hands at critical moments compared to families without children. Overall, taking into consideration the likelihood of washing hands after touching raw chicken and the percentages scores for proper hand cleaning methods and key moments for hand washing, the rank of the countries regarding proper hand hygiene practices was the following: Denmark, Greece, Norway, Romania, Hungary, Germany, UK, Portugal, France, and Spain. CONCLUSIONS: Information and education should point both at the key moments as suggested by the Royal Society for Public Health (RSPH) and the International Scientific Forum on Home Hygiene (IFH) and safe practices. Public health burden generated by improper hand washing may be significantly reduced if education is targeted on consumers' behaviour and practices.

Deterministic Approach and Monte Carlo Simulation to Predict Listeria monocytogenes Time to Grow on Refrigerated Ham: A Study Supporting Risk-based Decisions for Consumers' Health.

This study assessed the growth of Listeria monocytogenes in ready-to-eat (RTE) ham during storage under conditions simulating domestic practices with the intention to offer support in the elaboration of food safety policies that should better protect consumers against food poisoning at home. RTE ham, artificially contaminated at either medium (102-103 CFU/g) or high (104-105 CFU/g) concentration, was stored at both isothermal (4℃ in a refrigerator able to maintain a relatively constant temperature and 5℃ and 7℃ in a refrigerator with fluctuating temperature) and dynamic (5℃ and 7℃ with intermittent exposure to ambient temperature, e.g. 25℃) conditions. Under isothermal conditions, the increasing storage temperature determined a significantly increased (p < 0.05) capacity of L. monocytogenes to grow. The kinetic growth parameters were derived by fitting the Baranyi and Roberts model to the experimental data and, based on the maximum specific growth rates, it was estimated the temperature dependence of L. monocytogenes growth in RTE ham. At medium contamination level, sanitary risk time calculation revealed that, unlike storage at 5℃ and 7℃, storage at 4℃ of the RTE ham extends the time period during which the product is safe for consumption by ∼40 and 52%, respectively. However, the real temperature fluctuations included in the Monte Carlo simulations at low L. monocytogenes counts (1, 5 and 10 CFU/g) have shortened the safety margins. Stochastic models also proved to be useful tools for describing the pathogen's behavior when refrigeration of the RTE ham alternates with periods of ham being kept at room temperature, considered dynamic conditions of growth.

Kitchen layouts and consumers' food hygiene practices: Ergonomics versus safety.

Our paper emphasizes the importance of the kitchen layout in facilitating consumers' food hygiene practices. A significant correlation was found between the sink placement (inside or outside the kitchen) and hygienic practices during food handling based on a survey performed on consumers from ten European countries, indicating that those who had the sink in the kitchen were more likely to perform proper hygiene practices than those who have not. The self-reported practices were supported by observed practices in 64 households from five European countries. The observational study combined with the examination of kitchen layouts revealed that the kitchen work triangle with its apexes represented by the kitchen sink, cooking stove and refrigerator, which is recommended for ergonomic reasons by architects and designers, did not necessarily support food hygiene practices in kitchens. Cross-contamination events were associated with the sink - countertop distances longer than 1 m. Based on this, a new kitchen triangle with its apexes represented by the kitchen sink, working place (usually countertop) and cooking stove, with the distance between the sink and the working place less than 1 m is proposed to be used as norm in kitchen designs for combining ergonomics with safety. This triangle is proposedly named the food safety triangle and is aimed to mitigate the risks of foodborne illnesses by creating an arrangement that facilitates hygiene practices. This study is the first to highlight the importance of implementing the concept of food safety in the kitchen design based on significant correlations between kitchen equipment placement and consumers' food safety practices.

Data on European kitchen layouts belonging to vulnerable consumers (elderly people and young families with children or pregnant women) and risk-takers (young single men).

The data presented here capture the structure of kitchen layouts belonging to consumers vulnerable to foodborne diseases and food risk-takers. Data were collected in the frame of the SafeConsume project by multidisciplinary research teams that visited consumers during preparing a meal and had the possibility to examine their cooking routines. Distances between sink and stove, sink and refrigerator, stove and refrigerator, sink and working place (countertop or table), stove and working place were analyzed to correlate food safety practices applied during cooking with kitchen arrangements. The results arising from analyzing the ergonomics of kitchens versus potential cross-contamination events are presented in Mihalache et al., [1]. These data contribute to a better understanding of real kitchen layouts and can be used as a starting point for future research regarding food safety-oriented arrangements instead of ergonomics-focused designs, for food safety risk assessments, as study cases for explaining specific measures that can be established to improve food handling and hygiene practices in homes and for sociological research pointing consumers' behavior during cooking.

Efficacy of Removing Bacteria and Organic Dirt from Hands-A Study Based on Bioluminescence Measurements for Evaluation of Hand Hygiene When Cooking.

The objective of this study was to evaluate the efficacy of dirt removal (bacteria and organic matter) of several hand-cleaning procedures. The results from the hand hygiene experiment indicated that washing hands with warm water and soap for 20 s is the most effective method investigated when hands are either dirty or greasy. Even if not proper washing, rinsing under running water for 5 s is a cleaning procedure that may significantly reduce the probability of cross-contamination, as it removes 90% of the hands' dirt. Although less effective than water and soap, the usage of antibacterial wipes was significantly more effective than wet wipes, indicating that they are a better choice when water and soap are not available. The results of this study enable us to inform consumers about the effectiveness of hand-cleaning procedures applied in their homes when cooking. Moreover, it can make consumers understand why, during the COVID-19 pandemic, authorities recommended washing hands as a preventive measure of infection and using an anti-bacterial hand gel or wiping hands with an antimicrobial wipe if water and soap are not available.

Cross-contamination of lettuce with Campylobacter spp. via cooking salt during handling raw poultry.

Campylobacter spp. are the most common bacterial pathogens associated with human gastroenteritis in industrialized countries. Contaminated chicken is the food vehicle associated with the majority of reported cases of campylobacteriosis, either by the consumption of undercooked meat or via cross- contamination of ready-to-eat (RTE) foods during the handling of contaminated raw chicken parts and carcasses. Our results indicate that cooking salt (used for seasoning) is a potential vehicle for Campylobacter spp. cross-contamination from raw chicken to lettuce, through unwashed hands after handling contaminated chicken. Cross-contamination events were observed even when the chicken skin was contaminated with low levels of Campylobacter spp. (ca. 1.48 Log CFU/g). The pathogen was recovered from seasoned lettuce samples when raw chicken was contaminated with levels ≥ 2.34 Log CFU/g. We also demonstrated that, once introduced into cooking salt, Campylobacter spp. are able to survive in a culturable state up to 4 hours. After six hours, although not detected following an enrichment period in culture medium, intact cells were observed by transmission electron microscopy. These findings reveal a "novel" indirect cross-contamination route of Campylobacter in domestic settings, and a putative contamination source to RTE foods that are seasoned with salt, that might occur if basic food hygiene practices are not adopted by consumers when preparing and cooking poultry dishes.

Salmonella in eggs: From shopping to consumption-A review providing an evidence-based analysis of risk factors.

Nontyphoidal salmonellae are among the most prevalent foodborne pathogens causing gastrointestinal infections worldwide. A high number of cases and outbreaks of salmonellosis are associated with the consumption of eggs and egg products, and several of these occur at the household level. The aim of the current study is to critically evaluate the current status of knowledge on Salmonella in eggs from a consumer's perspective, analyzing the hazard occurrence and the good practices that should be applied to reduce salmonellosis risk. Following a HACCP (Hazard Analysis and Critical Control Point) based approach, some steps along the food journey were identified as Critical Consumer Handling (CCH)-steps in which consumers, through their behavior or choice, can significantly reduce the level of Salmonella in eggs and egg products. From shopping/collecting to consumption, each of these steps is discussed in this review to provide an evidence-based overview of risk factors of human salmonellosis related to egg consumption. The main message to consumers is to choose Salmonella-free eggs (those that some official entity or producer guarantees that does not contain Salmonella), when available, especially for dishes that are not fully heat treated. Second, as guaranteed Salmonella-free eggs are only available in a few countries, refrigerated storage from the point of collection and proper cooking will significantly reduce the risk of salmonellosis. This will require a revision of the actual recommendations/regulations, as not all ensure that eggs are maintained at temperatures that prevent growth of Salmonella from collection until the time of purchasing.

The complete genome sequence of Listeria monocytogenes strain S2542 and expression of selected genes under high-pressure processing.

OBJECTIVES: The study aims to generate the whole genome sequence of L. monocytogenes strain S2542 and to compare it to the genomes of strains RO15 and ScottA. In addition, we aimed to compare gene expression profiles of L. monocytogenes strains S2542, ScottA and RO15 after high-pressure processing (HPP) using ddPCR. RESULTS: The whole genome sequence of L. monocytogenes S2542 indicates that this strain belongs to serotype 4b, in contrast to the previously reported serotype 1/2a. Strain S2542 appears to be more susceptible to the treatment at 400 MPa compared to RO15 and ScottA strains. In contrast to RO15 and ScottA strains, viable cell counts of strain S2542 were below the limit of detection after HPP (400 MPa/8 min) when stored at 8 °C for 24 and 48 h. The transcriptional response of all three strains to HPP was not significantly different.

Consumer practices and prevalence of Campylobacter, Salmonella and norovirus in kitchens from six European countries.

About 40% of foodborne infections are acquired in the home. The aim of the present study was to track contamination of pathogens during domestic food preparation and link the contamination to preparation practices. Research participants from 87 households in six European countries were observed and interviewed during shopping and preparation of a chicken and vegetable meal. The presence of Salmonella spp., Campylobacter spp. and norovirus on raw chicken, kitchen surfaces, cloths and sponges was determined. The prevalence of Campylobacter on raw chicken varied from 8.3% in Norway (NO) to 80% in France (FR) and Portugal (PT), with a mean prevalence of 57%. Campylobacter was found on half of the products that had been frozen and appeared to be less prevalent on chicken from supermarkets than other sources. Salmonella was found in 8.6% of raw chicken samples, exclusively from Hungary (HU). A relationship between observed practices and spread of pathogens to kitchen surfaces was found only for the use of cutting boards for chicken and/or vegetables. After food preparation, Campylobacter and Salmonella were isolated from 23% (samples derived from HU, RO, UK) and 8.7% (HU), respectively of cutting boards. Research participants in France and Portugal were more likely to buy products that fitted their recipe, with less need for using cutting boards. Using the same board and knife for vegetables after using it for chicken and without washing with detergent was common in Portugal and Romania, but not in the other countries. Contamination with Campylobacter to other kitchen surfaces or washing utensils were found in five households (UK, RO, PT). Rinsing chicken in sinks was common in three countries (PT, HU, RO), and washing vegetables in the same sink was also usual. Prevalence of Norovirus was low, with detection in one out of 451 samples. The participants' awareness of the risk posed by pathogens from raw chicken differed among the six countries, with higher awareness in Norway and the UK than the other countries studied. In conclusion, practices intended to avoid cross-contamination from chicken to kitchen surfaces and washing utensils are not established among consumers in all European countries. Nevertheless, cross-contamination events that disseminate infectious doses of pathogens seems to be rare, probably due to the relatively low levels of pathogens in food combined with food preferences. Food safety interventions must consider the national food culture, preferences, practices and the prevalence and levels of pathogens in food. Emphasis should be on providing and promoting chicken products with lower risk (prevalence of pathogens, ready-to-cook) and safe use of cutting boards.

High-pressure processing-induced transcriptome response during recovery of Listeria monocytogenes.

BACKGROUND: High-pressure processing (HPP) is a commonly used technique in the food industry to inactivate pathogens, including L. monocytogenes. It has been shown that L. monocytogenes is able to recover from HPP injuries and can start to grow again during long-term cold storage. To date, the gene expression profiling of L. monocytogenes during HPP damage recovery at cooling temperature has not been studied. In order identify key genes that play a role in recovery of the damage caused by HPP treatment, we performed RNA-sequencing (RNA-seq) for two L. monocytogenes strains (barotolerant RO15 and barosensitive ScottA) at nine selected time points (up to 48 h) after treatment with two pressure levels (200 and 400 MPa). RESULTS: The results showed that a general stress response was activated by SigB after HPP treatment. In addition, the phosphotransferase system (PTS; mostly fructose-, mannose-, galactitol-, cellobiose-, and ascorbate-specific PTS systems), protein folding, and cobalamin biosynthesis were the most upregulated genes during HPP damage recovery. We observed that cell-division-related genes (divIC, dicIVA, ftsE, and ftsX) were downregulated. By contrast, peptidoglycan-synthesis genes (murG, murC, and pbp2A) were upregulated. This indicates that cell-wall repair occurs as a part of HPP damage recovery. We also observed that prophage genes, including anti-CRISPR genes, were induced by HPP. Interestingly, a large amount of RNA-seq data (up to 85%) was mapped to Rli47, which is a non-coding RNA that is upregulated after HPP. Thus, we predicted that Rli47 plays a role in HPP damage recovery in L. monocytogenes. Moreover, gene-deletion experiments showed that amongst peptidoglycan biosynthesis genes, pbp2A mutants are more sensitive to HPP. CONCLUSIONS: We identified several genes and mechanisms that may play a role in recovery from HPP damage of L. monocytogenes. Our study contributes to new information on pathogen inactivation by HPP.

Genomic characterization of the most barotolerant Listeria monocytogenes RO15 strain compared to reference strains used to evaluate food high pressure processing.

BACKGROUND: High pressure processing (HPP; i.e. 100-600 MPa pressure depending on product) is a non-thermal preservation technique adopted by the food industry to decrease significantly foodborne pathogens, including Listeria monocytogenes, from food. However, susceptibility towards pressure differs among diverse strains of L. monocytogenes and it is unclear if this is due to their intrinsic characteristics related to genomic content. Here, we tested the barotolerance of 10 different L. monocytogenes strains, from food and food processing environments and widely used reference strains including clinical isolate, to pressure treatments with 400 and 600 MPa. Genome sequencing and genome comparison of the tested L. monocytogenes strains were performed to investigate the relation between genomic profile and pressure tolerance. RESULTS: None of the tested strains were tolerant to 600 MPa. A reduction of more than 5 log10 was observed for all strains after 1 min 600 MPa pressure treatment. L. monocytogenes strain RO15 showed no significant reduction in viable cell counts after 400 MPa for 1 min and was therefore defined as barotolerant. Genome analysis of so far unsequenced L. monocytogenes strain RO15, 2HF33, MB5, AB199, AB120, C7, and RO4 allowed us to compare the gene content of all strains tested. This revealed that the three most pressure tolerant strains had more than one CRISPR system with self-targeting spacers. Furthermore, several anti-CRISPR genes were detected in these strains. Pan-genome analysis showed that 10 prophage genes were significantly associated with the three most barotolerant strains. CONCLUSIONS: L. monocytogenes strain RO15 was the most pressure tolerant among the selected strains. Genome comparison suggests that there might be a relationship between prophages and pressure tolerance in L. monocytogenes.

Characterization of the biofilm phenotype of a Listeria monocytogenes mutant deficient in agr peptide sensing.

Listeria monocytogenes is a food-borne human pathogen and a serious concern in food production and preservation. Previous studies have shown that biofilm formation of L. monocytogenes and presence of extracellular DNA (eDNA) in the biofilm matrix varies with environmental conditions and may involve agr peptide sensing. Experiments in normal and diluted (hypoosmotic) complex media at different temperatures revealed reduced biofilm formation of L. monocytogenes EGD-e ΔagrD, a mutant deficient in agr peptide sensing, specifically in diluted Brain Heart Infusion at 25°C. This defect was not related to reduced sensitivity to DNase treatment suggesting sufficient levels of eDNA. Re-analysis of a previously published transcriptional profiling indicated that a total of 132 stress-related genes, that is 78.6% of the SigB-dependent stress regulon, are differentially expressed in the ΔagrD mutant. Additionally, a number of genes involved in flagellar motility and a large number of other surface proteins including internalins, peptidoglycan binding and cell wall modifying proteins showed agr-dependent gene expression. However, survival of the ΔagrD mutant in hypoosmotic conditions or following exposure to high hydrostatic pressure was comparable to the wild type. Also, flagellar motility and surface hydrophobicity were not affected. However, the ΔagrD mutant displayed a significantly reduced viability upon challenge with lysozyme. These results suggest that the biofilm phenotype of the ΔagrD mutant is not a consequence of reduced resistance to hypoosmotic or high pressure stress, motility or surface hydrophobicity. Instead, agr peptide sensing seems to be required for proper regulation of biosynthesis, structure and function of the cell envelope, adhesion to the substratum, and/or interaction of bacteria within a biofilm.

Resistance of Listeria monocytogenes to Stress Conditions Encountered in Food and Food Processing Environments.

Listeria monocytogenes is a human food-borne facultative intracellular pathogen that is resistant to a wide range of stress conditions. As a consequence, L. monocytogenes is extremely difficult to control along the entire food chain from production to storage and consumption. Frequent and recent outbreaks of L. monocytogenes infections illustrate that current measures of decontamination and preservation are suboptimal to control L. monocytogenes in food. In order to develop efficient measures to prevent contamination during processing and control growth during storage of food it is crucial to understand the mechanisms utilized by L. monocytogenes to tolerate the stress conditions in food matrices and food processing environments. Food-related stress conditions encountered by L. monocytogenes along the food chain are acidity, oxidative and osmotic stress, low or high temperatures, presence of bacteriocins and other preserving additives, and stresses as a consequence of applying alternative decontamination and preservation technologies such high hydrostatic pressure, pulsed and continuous UV light, pulsed electric fields (PEF). This review is aimed at providing a summary of the current knowledge on the response of L. monocytogenes toward these stresses and the mechanisms of stress resistance employed by this important food-borne bacterium. Circumstances when L. monocytogenes cells become more sensitive or more resistant are mentioned and existence of a cross-resistance when multiple stresses are present is pointed out.

Technological and sensorial role of yeast ß-glucan in meat batter reformulations.

This study shows that apart from acting as nutritional value improver, yeast ß-glucan can be successfully used to reformulate meat products. When added to meat batters, yeast derived ingredients containing ß-glucans (GOLDCELL® IY B and GOLDCELL® BETA GLUCAN) improved the emulsifying capacity (up to 5 increments), the water holding capacity (up to 8 increments) as well as the emulsion stability. A decrease in total fluid release up to 4.30% and 3.99%, respectively with GOLDCELL® IY B and GOLDCELL® BETA GLUCAN respectively, at 1.5% addition level was observed. A significant decrease in hardness and fracturability values was also observed, while maintaining the structural cohesiveness of the samples, in part due to the increase in humidity content. A maximum level of 3% ingredient mixture can be added to meat batter formulations without significant impact on sensory characteristics. Adding yeast ß-glucan to meat batters can allow food to decrease the NaCl and polyphosphate content in meat products.

Tracking Foodborne Pathogenic Bacteria in Raw and Ready-to-Eat Food Illegally Sold at the Eastern EU Border.

Food illegally brought into the European Union, mainly in the personal luggage of travelers, represents a potential threat to consumers' health. The aim of this study was to investigate the presence of five pathogens in food brought into the European Union by Moldavian citizens as personal goods and illegally sold in Romania in the vicinity of the border. The occurrence of Staphylococcus aureus and Listeria monocytogenes was 7.5% and 8%, while Campylobacter spp., Escherichia coli O157:H7, and Salmonella spp. were absent in all samples. L. monocytogenes sequence type 2, 9, 121, and 155, highly prevalent among foodstuffs worldwide, was also present among isolates from ready-to-eat food illegally sold in Romania, even at the same date of sampling, indicating cross-contamination during food handling. S. aureus spa types t449, t304, and t524 were most often isolated from raw-milk cheeses contaminated with 10(3)-10(5) colony-forming units per gram, evidencing a contamination at herd level or unhygienic conditions during processing. S. aureus t011 and t3625, both included in the livestock-associated CC398, were isolated from pork lard and poultry meat. This study shows that cross-border trade from nonmember states represents a neglected route of transmission of foodborne pathogens into the European Union that could lead to sporadic or family-associated cases of disease.

Dynamics of Listeria monocytogenes colonisation in a newly-opened meat processing facility.

This study determined the colonisation scenario of Listeria monocytogenes in a newly-opened ready-to-eat meat processing facility using a combination of classical microbiology and molecular biology techniques. Samples (n=183), including food contact surfaces, non-food contact surfaces, raw materials and food samples, collected on four sampling occasions, were analysed for L. monocytogenes by the ISO 11290:1996 standard method and by real-time PCR applied to the second enrichment broth from the ISO method. No L. monocytogenes were detected on the first sampling occasion, but by the second sampling occasion a persistent clone had colonised the facility. Analysis of the second enrichment of the ISO method by real-time PCR was more sensitive for the detection of L. monocytogenes than the ISO method alone. In order to reduce the risk of cross contamination and the public health risk, awareness and proactive measures are required to control L. monocytogenes from the first days of production in a newly opened meat processing facility.

Putative Cross-Contamination Routes of Listeria monocytogenes in a Meat Processing Facility in Romania.

Putative routes of Listeria monocytogenes contamination, based on the workflow of the employees, were studied in a meat processing facility by investigating 226 samples collected from food contact surfaces, non-food contact surfaces, raw materials, and ready-to-eat meat products on four occasions over a 1-year period. In total, 19.7% of non-food contact surfaces, 22.9% of food contact surfaces, 45% of raw materials, and 20% of ready-to-eat meat products were positive for L. monocytogenes (analyzed by the International Organization for Standardization standard method ISO 11290). Pulsed-field gel electrophoresis (PFGE) profiles were determined for a representative subset of these isolates, and 11 distinct pulsotypes were identified, two of which were frequently isolated (T4 and T8) and considered persistent. Strains from the various pulsotypes were screened for the presence of bcrABC and qacH, the genes responsible for tolerance responses to quaternary ammonium compounds. Two strains harbored bcrABC, and these strains had a higher benzalkonium chloride tolerance; however, they were not considered persistent strains. The frequently isolated PFGE pulsotype T8 strains were highly adhesive to abiotic surfaces at 10 and 20°C; however, the pulsotype T6 strain, which was isolated only at the last sampling time, had the highest adhesion ability, and the pulsotype T4 strain (the second most persistent pulsotype) had only modest adhesion. Four putative cross-contamination routes were confirmed by mapping the persistent and other isolates. This information could allow a food safety manager to adjust the work flow to improve the hygienic conditions in a meat processing facility. This study revealed the prevalence and persistence of L. monocytogenes strains in a meat processing facility and established the importance of developing strategies to avoid cross-contamination, recalls, and outbreaks of listeriosis.

High sensitive multiresidue analysis of pharmaceuticals and antifungals in surface water using U-HPLC-Q-Exactive Orbitrap HRMS. Application to the Danube river basin on the Romanian territory.

The occurrence of 67 pharmaceutical and antifungal residues in the Danube river on the Romanian territory was studied by using solid-phase extraction (SPE) and LC-Q Exactive Orbitrap high resolution MS in both full scan (FS) MS and targeted MS/MS modes. A single-laboratory validation procedure was carried out for the determination of 67 compounds in FSMS mode evaluating selectivity, sensitivity, linearity, precision and accuracy. The method showed satisfactory analytical performance. The evaluation of the recovery concluded that 75% of the compounds show recoveries between 85 and 115% and 10% of the compounds show recoveries between 85% and 65%. The level of detection was lower than 5 ng l(-1) for 66% of the compounds, between 5 and 10 ng l(-1) for 22% and between 10 and 25 ng l(-1) for 14% of the compounds. The coefficients of determination R(2) were higher than 0.99 for 79% of the compounds, over a linearity range of 2.5-50 ng l(-1). Targeted MS/MS analysis, performed in addition to the full scan acquisition was used for confirmatory purpose. Twenty samples of Danube water and three of the main tributaries were collected in May, July, August and October 2014. Analysis of the selected water samples revealed the occurrence of 23 compounds such as diclofenac, carbamazepine, sulfamethoxazole, tylosin, indomethacin, ketoprofen, piroxicam, together with antifungals like thiabendazole, and carbendazim. Carbamazepine was detected in 17 samples, the maximum concentration being 40 ng l(-1). The highest concentration reached was 166 ng l(-1) for diclofenac.

Foods from black market at EU border as a neglected route of potential methicillin-resistant Staphylococcus aureus transmission.

The illegal entrance of foods to EU through black markets at the EU borders can constitute a neglected route of dissemination of foodborne pathogens, and in particular of methicillin-resistant Staphylococcus aureus (MRSA). In this study, we have assessed the presence of MRSA in foods sold in a black market at an EU border (the southeast part of Romania, on the border with Republic of Moldavia). We performed a search for MRSA among 200 food samples collected from 2012 to 2013. All S. aureus were studied by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing. MRSA isolates were further characterized by multilocus sequence typing (MLST) and SCCmec typing, and tested for the presence of Panton-Valentine leukocidin (PVL) virulence factors. Overall, 32 S. aureus isolates were recovered from 16 food samples (8%). One isolate detected in a pork lard sample was MRSA (0.5%). PFGE with the restriction enzyme SmaI revealed 12 genotypes among the 32 S. aureus isolates. The MRSA isolate belonged to sequence type 398, harbored SCCmec type V, tested negative for the presence of the PVL genes and was resistant to ciprofloxacin, tetracycline and cefazolin, besides all ß-lactams. Among 31 methicillin-sensitive S. aureus (MSSA), 29% were resistant to penicillin, 9.7% to tetracycline and 3.2% to ciprofloxacin. In conclusion, in this study we report the presence of livestock-associated MRSA in foods sold in a black market at an EU border: ST398-MRSA-V. These results confirm the potential role of food in the dissemination of MRSA lineages among population, and the potential role of illegally introduced food to EU in the prevalence and evolution of MRSA clones in the community.