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

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

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

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

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

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

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

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

Biogenic Amines in Meat and Meat Products: A Review of the Science and Future Perspectives.

Biogenic amines (BAs) can be found in a wide range of meat and meat products, where they are important as an index for product stability and quality, but also for their impact on public health. This review analyzes the scientific evidence gathered so far on the presence and role of biogenic amines in meat and meat products, also considering the effect of technological conditions on BAs accumulation or decrease. The data provided can be useful for developing solutions to control BAs formation during the shelf-life, for example by novel starters for dry cured products, as well as by packaging technologies and materials for fresh meats. Further research, whose trends are reviewed in this paper, will fill the knowledge gaps, and allow us to protect such perishable products along the distribution chain and in the home environment.

Update on Biogenic Amines in Fermented and Non-Fermented Beverages.

The formation of biogenic amines in food and beverages is mainly due to the presence of proteins and/or free amino acids that represent the substrates for microbial or natural enzymes with decarboxylation or amination activity. Fermentation occurring in many alcoholic beverages, such as wine, beer, cider, liqueurs, as well as coffee and tea, is one of the main processes affecting their production. Some biogenic amines can also be naturally present in some fruit juices or fruit-based drinks. The dietary intake of such compounds should consider all their potential sources by both foods and drinks, taking in account the health impact on some consumers that represent categories at risk for a deficient metabolic activity or assuming inhibiting drugs. The most important tool to avoid their adverse effects is based on prevention through the selection of lactic acid bacteria with low decarboxylating activity or good manufacturing practices hurdling the favoring conditions on biogenic amines' production.

Detection of ethinyl estradiol and resorcylic acid lactones in poultry meat according to the national residue plan.

The aim of this study was the detection of ethinyl estradiol and resorcylic acid lactones (zeranol and taleranol) in poultry meat samples as unauthorized substances included in the Italian national residue plan. The samples were purified by a solid phase extraction using a C18 column combined with alumina, and the analytes were detected by two specific enzyme-linked immunosorbent assay (ELISA) kits. As they were collected in the frame of official control activities, the method was also validated according to the Commission Decision 2002/657/EC requirements for screening methods with qualitative purpose. Specificity and detection capability were the performance criteria considered for the validation study, and the latter parameter showed a value of 0.5 µg/kg for both the investigated compounds. Such result was well comparable with the data reported by using chromatographic techniques as confirmation methods, and therefore, the ELISA kits tested in this study could be used for the screening of large numbers of samples.

An Overview of Histamine and Other Biogenic Amines in Fish and Fish Products.

The occurrence of biogenic amines in fish is directly associated with microorganisms with decarboxylase activity. These compounds are generally detoxified by oxidases in the intestinal tract of humans, but some conditions, such as alcohol consumption, enzyme deficiency, or monoamino-oxidase antidepressant use, can make their intake by food dangerous. Due to its toxicity, histamine is a unique biogenic amine with regulatory limits for fishery products. This review focuses on biogenic amines in fish, with a detailed picture of the number of alert notifications or intoxication events reported in the last years. The favoring conditions for their formation, as well as the main preventive and control measures to ensure public health, are also reviewed.

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

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

Rapid Methods for Assessing Food Safety and Quality.

Food safety represents a central issue for the global food chain and a daily concern for all people. Contaminated food by physical, biological or chemical hazards can harm consumers, increasing demand for health services, government expenditure on public health and other social costs. The quality assurance programs are based on the continuous monitoring of raw matter, production process, storage and distribution of the end products, including the purpose for which they are intended. Such programs represent an important objective for food producers, not only for the potential risk to human health, but also for the economic losses to which they can be subjected. The development and use of rapid analytical methods able to identify the main failures in food production can benefit food companies by saving time and costs for the good and fast control of products through the entire food chain.

Corrigendum: Changes in Polyphenolic Concentrations of Table Olives (cv. Itrana) Produced Under Different Irrigation Regimes During Spontaneous or Inoculated Fermentation.

[This corrects the article DOI: 10.3389/fmicb.2018.01287.].

Accreditation Procedure for Trichinella spp. Detection in Slaughterhouses: The Experience of an Internal Laboratory in Italy.

Trichinellosis is a severe foodborne zoonotic disease due to the consumption of undercooked meat containing Trichinella spp. larvae. According to Commission Regulation (EU) No 1375/2015, domestic pigs, farmed wild boar, and horses must be tested for the presence of the parasite in the muscles as part of post-mortem examination. In this study, the accreditation procedure and the maintenance of the certificate for internal laboratory attached to a slaughterhouse are described. The main advantages of such accreditation are represented by the possibility to obtain fast results in order to process carcasses quickly, whereas the difficulties for the technician are linked to performing proficiency testing and following training courses. This program can be considered particularly useful for surveillance and food safety purposes.

Changes in Polyphenolic Concentrations of Table Olives (cv. Itrana) Produced Under Different Irrigation Regimes During Spontaneous or Inoculated Fermentation.

Irrigation is widely used for the production of table olives because it increases fruit size and yield. However, irrigation also determines less accumulation of total phenols, an increase in water content, a decrease of firmness, lower concentrations of soluble sugars in the mesocarp, thus positively or negatively affecting the fermentation process for the production of table olives. In this study we tested the hypothesis that green fruits of cultivar Itrana obtained by different irrigation regimes had different phenolic concentration that responded differentially to spontaneous or inoculated fermentation. Fruits were harvested from two orchards in the Latina province of Latium, Italy, which had been irrigated with different volumes of water during the growing season to compare the evolution of spontaneous and inoculated fermentation processes. We measured fruit characteristics at harvest, changes in the concentrations of secoiridoids and lignans, and main microbial groups abundance during fermentation. At harvest and during fermentation the concentration of phenolic compounds was higher in fruits sampled from trees that had received less water in the field. Differences were observed between spontaneous and inoculated fermentations, with a prevalence of lactic acid bacteria (LAB) in inoculated samples. In particular, oleuropein concentration completely disappeared only from samples inoculated with the two selected strains used as starters. The inoculum with selected LAB positively influenced the fermentation process of green olives, whereas the irrigation regime previously experienced by trees did not alter fermentation.

Influence of Iodine Feeding on Microbiological and Physico-Chemical Characteristics and Biogenic Amines Content in a Raw Ewes' Milk Cheese.

Iodine is an essential trace element involved in the regulation of thyroid metabolism and antioxidant status in humans and animals. The aim of this study was to evaluate the effect of ewes’ dietary iodine supplementation on biogenic amines content as well as microbiological and physico-chemical characteristics in a raw milk cheese at different ripening times (milk, curd, and 2, 7, 15, 30, 60, and 90 days). Two cheese-making trials were carried out using milk from ewes fed with unifeed (Cheese A) or with the same concentrate enriched with iodine (Cheese B). The results indicated that the counts of principal microbial groups and physico-chemical characteristics were quite similar in both cheeses at day 90. Cheese B was characterized by a higher content of biogenic amines and propionic acid. Propionic bacteria were found in both cheeses mainly in Trial B in agreement with the higher content of propionic acid detected.

Impact of Saccharomyces cerevisiae strains on traditional sparkling wines production.

Sparkling wine fermentation is a challenge for yeasts due to the hostile conditions. A phenotype sought in starters is flocculation, because it reduces riddling time. For this reason, six flocculent Saccharomyces cerevisiae wine strains with different flocculation degree and autolytic activity and two commercial strains were tested for traditional sparkling wine production in a winery. Yeast viability, free aminoacids and high molecular weight nitrogen release and physico-chemical composition of sparkling wines were evaluated. Moreover, strains were tested for their aromatic potential. Obtained data revealed that flocculent yeasts presented oenological performances (in terms of fermentation rate, maximum pressure reached, free aminoacids - AAN and high molecular weight nitrogen - HMWN release) similar to the commercial strains. All considered strains were able to complete fermentation and viable cells of all strains were detected in all sparkling wines produced even after 6 months. F6789 and F10471 strains showed slow fermentation kinetics reaching the maximum of pressure at 180 days. Regarding nitrogen compounds release, FI strain was characterized by the highest amount of AAN and HMWN released, followed by F6789. Strains showed a considerable diversification in terms of number and amount of aroma molecules produced and sparkling wines obtained with autochthonous flocculent strains presented a higher amount of alcohols and esters already after 3 months. Further studies are necessary to select starter strains to improve traditional sparkling wines production.

Determination of Lipophilic Marine Biotoxins in Mussels Harvested from the Adriatic Sea by LC-MS/MS.

Lipophilic marine biotoxins include okadaic acid, pectenotoxin, yessotoxin and azaspiracid groups. The consumption of contaminated molluscs can lead to acute food poisoning syndromes depending on the exposure level. Regulatory limits have been set by Regulation (European Community, 2004a) No 853/2004 and LC-MS/MS is used as the official analytical method according to Regulation (European Community, 2011) No 15/2011. In this study specimens of mussels (Mytilus galloprovincialis) were collected along the coasts of the central Adriatic Sea during the years 2015-2017 and analyzed by the European harmonized Standard Operating Procedure. The method was validated for linearity, specificity, repeatability and reproducibility and it revealed able to be used for the detection of the lipophilic marine biotoxins. Levels of okadaic acid, pectenotoxin, yessotoxin and its analogs were detected at different concentrations in 148 (37%) out of a total of 400 samples, always below the maximum limits, except for 11 (4.3%) of them that were non-compliant because they exceeded the regulatory limit. Moreover, some samples were exposed to a multi-toxin mixture with regards to okadaic acid, yessotoxin and 1-Homo yessotoxin. Following these results, the aquaculture farms from which the non-compliant samples derived were closed until the analytical data of two consecutive samplings returned favorable. Besides the potential risk of consumption of mussels contaminated by lipophilic marine biotoxins, these marine organisms can be considered as bio-indicators of the contamination status of the marine ecosystem.

Development of a rapid method for the detection of Yersinia enterocolitica serotype O:8 from food.

In this study, a new and alternative method based on monoclonal antibodies (MAbs) for the rapid detection of Yersinia enterocolitica O:8 was developed. This microorganism is an emerging foodborne pathogen causing gastrointestinal disease in humans. The transmission can occur through contaminated food such as raw or undercooked meat, milk and dairy products, water and fresh vegetables. Nine MAbs (46F7, 54B11, 54C11, 62D10, 64C7, 64C10, 72E8, 72E10, 72G6) were characterized and selected versus Y. enterocolitica O:8, and only 2 of them showed also a weak cross-reaction with Campylobacter jejuni. The MAb 54B11 was used for the development of Y. enterocolitica capture-ELISA in food matrices, i.e. meat and dairy products (n = 132). The method was validated by ISO 16140:2003 and compared with the official method for the detection of presumptive pathogenic Y. enterocolitica (ISO 10273:2003). Relative accuracy, sensitivity and specificity corresponded to 100%. The selectivity was evaluated on other food samples (n = 126) showing a lower confidence limit of 90.3% and an upper confidence limit of 100%. The results from this study demonstrated that the developed method was rapid and cheap, specific and sensitive for the screening of the pathogen in food.

Simultaneous determination of aflatoxins, T-2 and HT-2 toxins, and fumonisins in cereal-derived products by QuEChERS extraction coupled with LC-MS/MS.

A fast, easy, and cheap method for the simultaneous determination and quantification of aflatoxins (B1, B2, G1, G2), T-2 and HT-2 toxins, and fumonisins (B1, B2) in cereal-derived products was developed. This method involved a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction coupled with liquid chromatography-tandem mass spectrometry. The method validation was performed by analyzing samples spiked at four levels, and the recoveries ranged from 83.6 to 102.9%, whereas the maximum values of repeatability and within-laboratory reproducibility were 14.3 and 15.7% following the performance criteria set by the European legislation. The method was then applied for the analysis of 21 cereal-derived products purchased on the Italian market, which were correctly packaged and labeled as intended for human consumption. The co-occurrence of more than one mycotoxin in the analyzed samples could represent a risk for consumers, and the described method could be a valid alternative for their simultaneous detection in the framework of official control. Graphical Abstract ᅟ.

Histamine Food Poisoning.

The consumption of food containing high amounts of histamine and other biogenic amines can cause food poisoning with different symptoms linked to the individual sensitivity and the detoxification activity. Histamine is the only biogenic amine with regulatory limits set by the European Commission in fish and fishery products, because it can lead to a fatal outcome. However, also fermented foods can be involved in outbreaks and sporadic cases of intoxication. The factors affecting the presence of histamine in food are variable and product specific including the availability of the precursor amino acid, the presence of microorganisms producing decarboxylases, and the conditions allowing their growth and enzyme production. Generally, the good quality of raw material and hygienic practices during food processing as well as the use of histidine decarboxylase-negative starter cultures can minimize the occurrence of histamine. Further studies are necessary to estimate the human exposure and the relationship between the total amount of the biogenic amines ingested with food and health effects.