Susceptibility and transcriptomic response to plasma-activated water of Listeria monocytogenes planktonic and sessile cells.

Plasma-Activated Water (PAW) was generated from tap water using a surface dielectric barrier discharge at different discharge power (26 and 36 W) and activation time (5 and 30 min). The inactivation of a three-strain Listeria monocytogenes cocktail in planktonic and biofilm state was evaluated. PAW generated at 36 W-30 min showed the lowest pH and the highest hydrogen peroxide, nitrates, nitrites contents and effectiveness against cells on planktonic state, resulting in 4.6 log reductions after a 15-min treatment. Although the antimicrobial activity in biofilms formed on stainless steel and on polystyrene was lower, increasing the exposure time to 30 min allowed an inactivation >4.5 log cycles. The mechanisms of action of PAW were investigated using chemical solutions that mimic its physico-chemical characteristics and also RNA-seq analysis. The main transcriptomic changes affected carbon metabolism, virulence and general stress response genes, with several overexpressed genes belonging to the cobalamin-dependent gene cluster.

Novel methods of microbiome analysis in the food industry.

The study of the food microbiome has gained considerable interest in recent years, mainly due to the wide range of applications that can be derived from the analysis of metagenomes. Among these applications, it is worth mentioning the possibility of using metagenomic analyses to determine food authenticity, to assess the microbiological safety of foods thanks to the detection and tracking of pathogens, antibiotic resistance genes and other undesirable traits, as well to identify the microorganisms responsible for food processing defects. Metataxonomics and metagenomics are currently the gold standard methodologies to explore the full potential of metagenomes in the food industry. However, there are still a number of challenges that must be solved in order to implement these methods routinely in food chain monitoring, and for the regulatory agencies to take them into account in their opinions. These challenges include the difficulties of analysing foods and food-related environments with a low microbial load, the lack of validated bioinformatics pipelines adapted to food microbiomes and the difficulty of assessing the viability of the detected microorganisms. This review summarizes the methods of microbiome analysis that have been used, so far, in foods and food-related environments, with a specific focus on those involving Next-Generation Sequencing technologies.

Stress resistance associated with multi-host transmission and enhanced biofilm formation at 42 °C among hyper-aerotolerant generalist Campylobacter jejuni.

One of the emerging conundrums of Campylobacter food-borne illness is the bacterial ability to survive stressful environmental conditions. We evaluated the heterogeneity among 90 C. jejuni and 21 C. coli isolates from different sources in Egypt with respect to biofilm formation capabilities (under microaerobic and aerobic atmosphere) and resistance to a range of stressors encountered along the food chain (aerobic stress, refrigeration, freeze-thaw, heat, peracetic acid, and osmotic stress). High prevalence (63%) of hyper-aerotolerant (HAT) isolates was observed, exhibiting also a significantly high tolerance to heat, osmotic stress, refrigeration, and freeze-thaw stress, coupled with high biofilm formation ability which was clearly enhanced under aerobic conditions, suggesting a potential link between stress adaptation and biofilm formation. Most HAT multi-stress resistant and strong biofilm producing C. jejuni isolates belonged to host generalist clonal complexes (ST-21, ST-45, ST-48 and ST-206). These findings highlight the potential role of oxidative stress response systems in providing cross-protection (resistance to other multiple stress conditions) and enhancing biofilm formation in Campylobacter and suggest that selective pressures encountered in hostile environments have shaped the epidemiology of C. jejuni in Egypt by selecting the transmission of highly adapted isolates, thus promoting the colonization of multiple host species by important disease-causing lineages.

Development and validation of a regression model for Listeria monocytogenes growth in roast beefs.

Food business operators are responsible for food safety and assessment of shelf lives for their ready-to-eat products. For assisting them, a customized software based on predictive models, ListWare, is being developed. The aim of this study was to develop and validate a predictive model for the growth of Listeria monocytogenes in sliced roast beef. A challenge study was performed comprising 51 different combinations of variables. The growth curves followed the Baranyi and Roberts model with no clear lag phase and specific growth rates in the range <0.005-0.110 hr-1. A linear regression model was developed based on 528 observations and had an adjusted R-square of 0.80. The significant predictors were storage temperature, sodium lactate, interactions between sodium acetate and temperature, and MAP packaging and temperature. The model was validated in four laboratories in three countries. For conditions where the model predicted up to + log 2 cfu/g Listeria concentration, the observed concentrations were true or below the predicted concentration in 90% of the cases. For the remaining 10%, the roast beef was coated with spices and therefore different from the others. The model will be implemented in ListWare web-application for calculation of "Listeria shelf life".

Selection for Antimicrobial Resistance in Foodborne Pathogens through Exposure to UV Light and Nonthermal Atmospheric Plasma Decontamination Techniques.

This study was aimed at assessing whether the repeated exposure of 12 strains of Salmonella spp., Escherichia coli, and Listeria monocytogenes to alternative nonthermal decontamination techniques with UV light (UV-C) and nonthermal atmospheric plasma (NTAP) may cause the emergence of variants showing increased resistance to clinically relevant antibiotics (ampicillin, cefotaxime, ciprofloxacin, gentamicin, streptomycin, tetracycline, erythromycin, vancomycin, and colistin). UV-C and NTAP treatments were applied on the surface of inoculated brain heart infusion (BHI) agar plates. Survivors were recovered and after 24 h of growth in BHI broth were again subjected to the decontamination treatment; this was repeated for 10 consecutive cycles. A total of 174 strain/decontamination technique/antibiotic combinations were tested, and 12 variant strains with increased resistance to one of the antibiotics studied were identified, with the increases in the MICs in Mueller-Hinton broth ranging from 2- to 256-fold. The variant strains of Salmonella spp. isolated were further characterized through phenotypic screenings and whole-genome sequencing (WGS) analyses. Most changes in susceptibility were observed for antibiotics that act at the level of protein synthesis (aminoglycosides, tetracyclines, and glycylcyclines) or DNA replication (fluoroquinolones), as well as for polymyxins. No changes in resistance to ß-lactams were detected. WGS analyses showed the occurrence of sequence alterations in some antibiotic cellular targets (e.g., gyrA for ciprofloxacin-resistant variants, rpsL for a streptomycin-resistant variant), accompanied by variations in stress response regulators and membrane transporters likely involved in the nonselective efflux of antibiotics, which altogether resulted in a low- to medium-level increase in microbial resistance to several antibiotics.IMPORTANCE The emergence and spread of antibiotic resistance along the food chain can be influenced by the different antimicrobial strategies used from farm to fork. This study evidences that two novel, not yet widely used, nonthermal microbial decontamination techniques, UV light and nonthermal atmospheric plasma, can select variants with increased resistance to various clinically relevant antibiotics, such as ciprofloxacin, streptomycin, tetracycline, and erythromycin. Whole-genome analysis of the resistant variants obtained for Salmonella spp. allowed identification of the genetic changes responsible for the observed phenotypes and suggested that some antimicrobial classes are more susceptible to the cross-resistance phenomena observed. This information is relevant, since these novel decontamination techniques are being proposed as possible alternative green techniques for the decontamination of environments and equipment in food and clinical settings.

A Systematic Review of Bacterial Foodborne Outbreaks Related to Red Meat and Meat Products.

Our investigation focused on foodborne outbreaks related to meat and meat products, published in peer-reviewed journals in the period 1980-2015. Most of the outbreaks, investigated in this study, were caused by Escherichia coli and Salmonella, causing 33 and 21 outbreaks, respectively, mostly in Europe and the United States. In the E. coli outbreaks, the total number of reported cases was 1966, of which 1543 were laboratory confirmed. The number of cases requiring hospitalization was 476, of whom 233 cases had a hemolytic-uremic syndrome (HUS), and the reported deaths were 32. All of the E. coli outbreaks, except four, were caused by serovar O157:H7. The other four outbreaks were caused by the following serovars: O111:H8, O26:H11, O111, and O103:H25. Fresh processed meat products were the category most frequently implicated. In the Salmonella outbreaks, the total number of all reported cases was 2279, of whom 1891 were laboratory confirmed. The number of reported cases requiring hospitalization was 94, and seven were reported dead. Regarding Salmonella, eight serovars caused those outbreaks. The most common serovar causing Salmonella-related outbreaks was Salmonella Typhimurium. The food category most frequently implicated in those outbreaks was raw-cured fermented sausages. Other organisms linked to meat-associated outbreaks, but less frequently reported, were Staphylococcus aureus, Bacillus cereus, Clostridium perfringens, Clostridium botulinum, and Listeria monocytogenes. Issues of the burden of outbreaks, the challenges of comparing global outbreaks, food attribution, and how the meat industry works to meet consumer demands while maintaining food safety are discussed.

Antimicrobial resistance and virulence characterization of Staphylococcus aureus and coagulase-negative staphylococci from imported beef meat.

BACKGROUND: The objectives of this study were to characterize the diversity and magnitude of antimicrobial resistance among Staphylococcus species recovered from imported beef meat sold in the Egyptian market and the potential mechanisms underlying the antimicrobial resistance phenotypes including harboring of resistance genes (mecA, cfr, gyrA, gyrB, and grlA) and biofilm formation. RESULTS: The resistance gene mecA was detected in 50% of methicillin-resistant non-Staphylococcus aureus isolates (4/8). Interestingly, our results showed that: (i) resistance genes mecA, gyrA, gyrB, grlA, and cfr were absent in Staphylococcus hominis and Staphylococcus hemolyticus isolates, although S. hominis was phenotypically resistant to methicillin (MR-non-S. aureus) while S. hemolyticus was resistant to vancomycin only; (ii) S. aureus isolates did not carry the mecA gene (100%) and were phenotypically characterized as methicillin- susceptible S. aureus (MSS); and (iii) the resistance gene mecA was present in one isolate (1/3) of Staphylococcus lugdunensis that was phenotypically characterized as methicillin-susceptible non-S. aureus (MSNSA). CONCLUSIONS: Our findings highlight the potential risk for consumers, in the absence of actionable risk management information systems, of imported foods and advice a strict implementation of international standards by different venues such as CODEX to avoid the increase in prevalence of coagulase positive and coagulase negative Staphylococcus isolates and their antibiotic resistance genes in imported beef meat at the Egyptian market.

Effects of high hydrostatic pressure on Escherichia coli ultrastructure, membrane integrity and molecular composition as assessed by FTIR spectroscopy and microscopic imaging techniques.

High hydrostatic pressure (HHP) is a novel food processing technology that is considered as an attractive alternative to conventional heat treatments for the preservation of foods, due to its lethal effects on pathogenic and spoilage microorganisms, while causing minor effects on food quality and sensorial attributes. This study is aimed at investigating how HHP treatments at varying intensities in the range 50-900 MPa affect the viability, membrane integrity, ultrastructure and molecular composition of Escherichia coli. Results of membrane integrity tests (measurement of cellular leakage and monitoring of propidium iodide uptake through fluorescence microscopy) and ultrastructural observations by transmission electron microscopy demonstrated that HHP gave rise to cellular enlargement, membrane damage or detachment, DNA and protein denaturation and loss of intracellular contents. Fourier-transform infrared (FTIR) spectroscopy analyses evidenced minor changes in molecular composition in response to high pressures, which were mostly observed on the spectral region w4 (1200-900 cm-1), mainly informative of carbohydrates and polysaccharides of the cell wall. These findings suggest that exposure of E. coli cells to HHP causes alterations in their physical integrity while producing minor modifications in biochemical cellular composition. The current study increases the knowledge on the mechanisms of E. coli inactivation by HHP and provides valuable information for the design of more effective food preservation regimes based on the integration of mild HHP in combination with other food preservation strategies into a multi-target hurdle technology approach.

The detailed analysis of the microbiome and resistome of artisanal blue-veined cheeses provides evidence on sources and patterns of succession linked with quality and safety traits.

BACKGROUND: Artisanal cheeses usually contain a highly diverse microbial community which can significantly impact their quality and safety. Here, we describe a detailed longitudinal study assessing the impact of ripening in three natural caves on the microbiome and resistome succession across three different producers of Cabrales blue-veined cheese. RESULTS: Both the producer and cave in which cheeses were ripened significantly influenced the cheese microbiome. Lactococcus and the former Lactobacillus genus, among other taxa, showed high abundance in cheeses at initial stages of ripening, either coming from the raw material, starter culture used, and/or the environment of processing plants. Along cheese ripening in caves, these taxa were displaced by other bacteria, such as Tetragenococcus, Corynebacterium, Brevibacterium, Yaniella, and Staphylococcus, predominantly originating from cave environments (mainly food contact surfaces), as demonstrated by source-tracking analysis, strain analysis at read level, and the characterization of 613 metagenome-assembled genomes. The high abundance of Tetragenococcus koreensis and Tetragenococcus halophilus detected in cheese has not been found previously in cheese metagenomes. Furthermore, Tetragenococcus showed a high level of horizontal gene transfer with other members of the cheese microbiome, mainly with Lactococcus and Staphylococcus, involving genes related to carbohydrate metabolism functions. The resistome analysis revealed that raw milk and the associated processing environments are a rich reservoir of antimicrobial resistance determinants, mainly associated with resistance to aminoglycosides, tetracyclines, and ß-lactam antibiotics and harbored by aerobic gram-negative bacteria of high relevance from a safety point of view, such as Escherichia coli, Salmonella enterica, Acinetobacter, and Klebsiella pneumoniae, and that the displacement of most raw milk-associated taxa by cave-associated taxa during ripening gave rise to a significant decrease in the load of ARGs and, therefore, to a safer end product. CONCLUSION: Overall, the cave environments represented an important source of non-starter microorganisms which may play a relevant role in the quality and safety of the end products. Among them, we have identified novel taxa and taxa not previously regarded as being dominant components of the cheese microbiome (Tetragenococcus spp.), providing very valuable information for the authentication of this protected designation of origin artisanal cheese. Video Abstract.

Persistence of microbiological hazards in food and feed production and processing environments.

Listeria monocytogenes (in the meat, fish and seafood, dairy and fruit and vegetable sectors), Salmonella enterica (in the feed, meat, egg and low moisture food sectors) and Cronobacter sakazakii (in the low moisture food sector) were identified as the bacterial food safety hazards most relevant to public health that are associated with persistence in the food and feed processing environment (FFPE). There is a wide range of subtypes of these hazards involved in persistence in the FFPE. While some specific subtypes are more commonly reported as persistent, it is currently not possible to identify universal markers (i.e. genetic determinants) for this trait. Common risk factors for persistence in the FFPE are inadequate zoning and hygiene barriers; lack of hygienic design of equipment and machines; and inadequate cleaning and disinfection. A well-designed environmental sampling and testing programme is the most effective strategy to identify contamination sources and detect potentially persistent hazards. The establishment of hygienic barriers and measures within the food safety management system, during implementation of hazard analysis and critical control points, is key to prevent and/or control bacterial persistence in the FFPE. Once persistence is suspected in a plant, a 'seek-and-destroy' approach is frequently recommended, including intensified monitoring, the introduction of control measures and the continuation of the intensified monitoring. Successful actions triggered by persistence of L. monocytogenes are described, as well as interventions with direct bactericidal activity. These interventions could be efficient if properly validated, correctly applied and verified under industrial conditions. Perspectives are provided for performing a risk assessment for relevant combinations of hazard and food sector to assess the relative public health risk that can be associated with persistence, based on bottom-up and top-down approaches. Knowledge gaps related to bacterial food safety hazards associated with persistence in the FFPE and priorities for future research are provided.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 19: Suitability of taxonomic units notified to EFSA until September 2023.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was Enterococcus faecium, 5 were Escherichia coli, 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: Ensifer adhaerens and Heyndrickxia faecalis did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and Burkholderia ubonensis also due to its ability to generate biologically active compounds with antimicrobial activity; Klebsiella pneumoniae, Serratia marcescens and Pseudomonas putida due to safety concerns. K. pneumoniae is excluded from future QPS evaluations. Chlamydomonas reinhardtii is recommended for QPS status with the qualification 'for production purposes only'; Clostridium tyrobutyricum is recommended for QPS status with the qualification 'absence of genetic determinants for toxigenic activity'; Candida oleophila has been added as a synonym of Yarrowia lipolytica. The Panel clarifies the extension of the QPS status for genetically modified strains.

Re-evaluation of certain aspects of the EFSA Scientific Opinion of April 2010 on risk assessment of parasites in fishery products, based on new scientific data. Part 1: ToRs1-3.

Surveillance data published since 2010, although limited, showed that there is no evidence of zoonotic parasite infection in market quality Atlantic salmon, marine rainbow trout, gilthead seabream, turbot, meagre, Atlantic halibut, common carp and European catfish. No studies were found for greater amberjack, brown trout, African catfish, European eel and pikeperch. Anisakis pegreffii, A. simplex (s. s.) and Cryptocotyle lingua were found in European seabass, Atlantic bluefin tuna and/or cod, and Pseudamphistomum truncatum and Paracoenogonimus ovatus in tench, produced in open offshore cages or flow-through ponds or tanks. It is almost certain that fish produced in closed recirculating aquaculture systems (RAS) or flow-through facilities with filtered water intake and exclusively fed heat-treated feed are free of zoonotic parasites. Since the last EFSA opinion, the UV-press and artificial digestion methods have been developed into ISO standards to detect parasites in fish, while new UV-scanning, optical, molecular and OMICs technologies and methodologies have been developed for the detection, visualisation, isolation and/or identification of zoonotic parasites in fish. Freezing and heating continue to be the most efficient methods to kill parasites in fishery products. High-pressure processing may be suitable for some specific products. Pulsed electric field is a promising technology although further development is needed. Ultrasound treatments were not effective. Traditional dry salting of anchovies successfully inactivated Anisakis. Studies on other traditional processes - air-drying and double salting (brine salting plus dry salting) - suggest that anisakids are successfully inactivated, but more data covering these and other parasites in more fish species and products is required to determine if these processes are always effective. Marinade combinations with anchovies have not effectively inactivated anisakids. Natural products, essential oils and plant extracts, may kill parasites but safety and organoleptic data are lacking. Advanced processing techniques for intelligent gutting and trimming are being developed to remove parasites from fish.

Exploitation of microbial activities at low pH to enhance planetary health.

Awareness is growing that human health cannot be considered in isolation but is inextricably woven with the health of the environment in which we live. It is, however, under-recognized that the sustainability of human activities strongly relies on preserving the equilibrium of the microbial communities living in/on/around us. Microbial metabolic activities are instrumental for production, functionalization, processing, and preservation of food. For circular economy, microbial metabolism would be exploited to produce building blocks for the chemical industry, to achieve effective crop protection, agri-food waste revalorization, or biofuel production, as well as in bioremediation and bioaugmentation of contaminated areas. Low pH is undoubtedly a key physical-chemical parameter that needs to be considered for exploiting the powerful microbial metabolic arsenal. Deviation from optimal pH conditions has profound effects on shaping the microbial communities responsible for carrying out essential processes. Furthermore, novel strategies to combat contaminations and infections by pathogens rely on microbial-derived acidic molecules that suppress/inhibit their growth. Herein, we present the state-of-the-art of the knowledge on the impact of acidic pH in many applied areas and how this knowledge can guide us to use the immense arsenal of microbial metabolic activities for their more impactful exploitation in a Planetary Health perspective.

Microbiome mapping in beef processing reveals safety-relevant variations in microbial diversity and genomic features.

The microbiome of surfaces along the beef processing chain represents a critical nexus where microbial ecosystems play a pivotal role in meat quality and safety of end products. This study offers a comprehensive analysis of the microbiome along beef processing using whole metagenomics with a particular focus on antimicrobial resistance and virulence-associated genes distribution. Our findings highlighted that microbial communities change dynamically in the different steps along beef processing chain, influenced by the specific conditions of each micro-environment. Brochothrix thermosphacta, Carnobacterium maltaromaticum, Pseudomonas fragi, Psychrobacter cryohalolentis and Psychrobacter immobilis were identified as the key species that characterize beef processing environments. Carcass samples and slaughterhouse surfaces exhibited a high abundance of antibiotic resistance genes (ARGs), mainly belonging to aminoglycosides, ß-lactams, amphenicols, sulfonamides and tetracyclines antibiotic classes, also localized on mobile elements, suggesting the possibility to be transmitted to human pathogens. We also evaluated how the initial microbial contamination of raw beef changes in response to storage conditions, showing different species prevailing according to the type of packaging employed. We identified several genes leading to the production of spoilage-associated compounds, and highlighted the different genomic potential selected by the storage conditions. Our results suggested that surfaces in beef processing environments represent a hotspot for beef contamination and evidenced that mapping the resident microbiome in these environments may help in reducing meat microbial contamination, increasing shelf-life, and finally contributing to food waste restraint.

Improved sampling and DNA extraction procedures for microbiome analysis in food-processing environments.

Deep investigation of the microbiome of food-production and food-processing environments through whole-metagenome sequencing (WMS) can provide detailed information on the taxonomic composition and functional potential of the microbial communities that inhabit them, with huge potential benefits for environmental monitoring programs. However, certain technical challenges jeopardize the application of WMS technologies with this aim, with the most relevant one being the recovery of a sufficient amount of DNA from the frequently low-biomass samples collected from the equipment, tools and surfaces of food-processing plants. Here, we present the first complete workflow, with optimized DNA-purification methodology, to obtain high-quality WMS sequencing results from samples taken from food-production and food-processing environments and reconstruct metagenome assembled genomes (MAGs). The protocol can yield DNA loads >10 ng in >98% of samples and >500 ng in 57.1% of samples and allows the collection of, on average, 12.2 MAGs per sample (with up to 62 MAGs in a single sample) in ~1 week, including both laboratory and computational work. This markedly improves on results previously obtained in studies performing WMS of processing environments and using other protocols not specifically developed to sequence these types of sample, in which <2 MAGs per sample were obtained. The full protocol has been developed and applied in the framework of the European Union project MASTER (Microbiome applications for sustainable food systems through technologies and enterprise) in 114 food-processing facilities from different production sectors.

Selection for loss of RpoS in Cronobacter sakazakii by growth in the presence of acetate as a carbon source.

We demonstrate that growth of Cronobacter sakazakii in the presence of acetate as a carbon source promotes loss of RpoS, with a consequent reduction in stress tolerance. This suggests that C. sakazakii is capable of regulating cell fitness through mutation of the rpoS gene.

Structure-activity relationship of synthetic variants of the milk-derived antimicrobial peptide αs2-casein f(183-207).

Template-based studies on antimicrobial peptide (AMP) derivatives obtained through manipulation of the amino acid sequence are helpful to identify properties or residues that are important for biological activity. The present study sheds light on the importance of specific amino acids of the milk-derived αs2-casein f(183-207) peptide to its antibacterial activity against the food-borne pathogens Listeria monocytogenes and Cronobacter sakazakii. Trimming of the peptide revealed that residues at the C-terminal end of the peptide are important for activity. Removal of the last 5 amino acids at the C-terminal end and replacement of the Arg at position 23 of the peptide sequence by an Ala residue significantly decreased activity. These findings suggest that Arg23 is very important for optimal activity of the peptide. Substitution of the also positively charged Lys residues at positions 15 and 17 of the αs2-casein f(183-207) peptide also caused a significant reduction of the effectiveness against C. sakazakii, which points toward the importance of the positive charge of the peptide for its biological activity. Indeed, simultaneous replacement of various positively charged amino acids was linked to a loss of bactericidal activity. On the other hand, replacement of Pro residues at positions 14 and 20 resulted in a significantly increased antibacterial potency, and hydrophobic end tagging of αs2-casein f(193-203) and αs2-casein f(197-207) peptides with multiple Trp or Phe residues significantly increased their potency against L. monocytogenes. Finally, the effect of pH (4.5 to 7.4), temperature (4°C to 37°C), and addition of sodium and calcium salts (1% to 3%) on the activity of the 15-amino-acid αs2-casein f(193-207) peptide was also determined, and its biological activity was shown to be completely abolished in high-saline environments.

Survival of acid adapted and non-acid adapted Salmonella Typhimurium in pasteurized orange juice and yogurt under different storage temperatures.

The survival capacity of Salmonella enterica serovar Typhimurium acid adapted and non-acid adapted cells was monitored in pasteurized yogurt (pH 4.1) and orange juice (pH 3.6) during storage at different temperatures (4, 10, 25 and 37 ). Acid adapted and non-acid adapted cells were obtained by means of their growth for 36 h in Brain Heart Infusion broth acidified at pH 4.8 with citric acid and buffered (pH 7.0) Brain Heart Infusion broth, respectively. S. typhimurium showed a great ability to survive in both foodstuffs and, especially, in yogurt, where both acid adapted and non-acid adapted populations suffered only a reduction of about 1.3-1.9 log10 cycles after 43 days of storage in the range of temperatures 4-25 . At 37 a higher bacterial inactivation was observed (4.0-4.4 log10 cycles). In orange juice, a different behaviour was observed for acid-adapted and non-acid adapted cells. Whereas non-acid adapted cells survived better than acid adapted cells at 4 and 10 , acid adapted cells showed enhanced survival abilities at higher temperatures (25 and 37 ). Thus, the times required to achieve a 5 log10 cycles reduction for non-acid adapted and acid adapted cells were 10.2 and 6.0 (4 ), 6.3 and 4.2 (10 ), 0.6 and 1.0 (25 ) and 0.10 and 0.15 (37 ) days, respectively. Evidence found in this study demonstrates that refrigeration temperatures protect S. typhimurium from inactivation in acid foods and indicates that S. typhimurium acid tolerance response (ATR) is determined by storage temperature and food composition.

Assessment of a plasmid conjugation procedure to monitor horizontal transfer of an extended-spectrum ß-lactamase resistance gene under food chain scenarios.

Plasmids are relevant reservoirs of antimicrobial resistance genes (ARGs) which confer adaptive advantages to their host and can be horizontally transferred. The aims of this study were to develop a conjugation procedure to monitor the horizontal transfer of a 193 kb plasmid containing the extended-spectrum ß-lactamase production gene bla CTX-M-14 between two Escherichia coli strains under a range of food chain-related scenarios, including temperature (20-37 °C), pH (5.0-9.0) or the presence of some biocidal agents (benzalkonium chloride, sodium hypochlorite or peracetic acid). The average conjugation rate in LB broth after 18 h at 37 °C was 2.09e-04 and similar rates were observed in a food matrix (cow's milk). The conjugation was reduced at temperatures below 37 °C, at alkaline pH (especially at pH 9.0) or in the presence of benzalkonium chloride. Peracetic acid and sodium hypochlorite slightly increased conjugation rates, which reached 5.59e-04 and 6.77e-03, respectively. The conjugation procedure described can be used to identify risk scenarios leading to an enhanced ARGs transmission via plasmid conjugation, as well as to identify novel intervention strategies impairing plasmid conjugation and tackling antimicrobial resistance.

High pressure processing at the early stages of ripening enhances the safety and quality of dry fermented sausages elaborated with or without starter culture.

To study the quality of chorizo de León dry fermented sausages (DFS), high pressure processing (HPP) applied at the early stages of ripening and the use of a functional starter culture were evaluated as additional safety measures. Furthermore, the ability to control the populations of artificially inoculated Listeria monocytogenes and Salmonella Typhimurium was investigated and the evolution of microbial communities was assessed by amplicon 16S rRNA metataxonomics. The use of HPP and the starter culture, independently or combined, induced a reduction of Listeria monocytogenes of 1.5, 4.3 and > 4.8 log CFU/g respectively, as compared to control. Salmonella Typhimurium counts were under the detection limit (<1 log) in all treated end-product samples. Both additional measures reduced the activity of undesirable microbiota, such as Serratia and Brochothrix, during the production of DFS. Moreover, the starter culture highly influencedthe taxonomic profile of samples.No adverse sensory effects were observed, and panelists showed preference for HPP treated DFS. In conclusion, this new approach of applying HPP at the early stages of ripening of DFS in combination with the use of a defined starter culture improved the safety and quality of the meat product.