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.

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.

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.

Heavy metal (As, Cd, Hg, Pb, Cu, Zn, Se) concentrations in muscle and bone of four commercial fish caught in the central Adriatic Sea, Italy.

Heavy metal (As, Cd, Cu, Pb, Zn, Hg and Se) concentrations in the muscle and bone of four fish species (Mullus barbatus, Merluccius merluccius, Micromesistius poutassou, and Scomber scombrus) from the central Adriatic Sea were measured and the relationships between fish size (length and weight) and metal concentrations in the tissues were investigated. Samples were analyzed by inductively coupled plasma-atomic emission spectrophotometry with automatic dual viewing. In the muscle, results of linear regression analysis showed that, except for mercury, significant relationships between metal concentrations and fish size were negative. Only mercury levels were positively correlated with Atlantic mackerel size (p < 0.05). No significant variations of heavy metal concentrations were observed in muscles of the examined species, but a significant difference (p < 0.01) was found for As, Cd, Pb, and Se concentrations in bone. All the investigated metals showed higher values in the muscle than in bone, except for lead and zinc. Regarding cadmium, lead, and mercury maximum levels, set for the edible portion by European legislation, several samples exceeded these values, confirming the heavy metal presence in species caught near the Jabuka Pit.

Nutritional quality and safety related to trace element content in fish from Tyrrhenian Sea.

This study analyzed for the detection of the following trace elements: As, Cd, Cu, Hg, Pb, Se, Zn, in muscle of four widely traded and consumed marine fish species (Mullus surmuletus L., Merluccius merluccius L., Micromesistius poutassou (Risso, 1827), Scomber scombrus L.) selected for their wide trade and consumption. Mean concentrations found in fish muscle, irrespective of species, ranged from 3.61 to 105.49 (As); 0.01-0.08 (Cd); 0.74-4.71 (Cu); 0.09-1.10 (Hg); 0.02-0.58 (Pb); 0.30-2.88 (Se); 11.56-73.37 (Zn) mg/kg wet weight. As, Hg, Cu and Se concentrations showed a significant difference (p < 0.01) among the analyzed species in winter and in summer. Cd exhibited a significant difference (p < 0.01) only in summer. The maximum levels set for Hg, Cd and Pb by European Regulation No 1881/2006 were exceeded by 5 (8.9%), 1 (1.8%) and 4 (7.1%) samples, respectively. In particular the species exceeding the legal limits were red mullet for Cd and Pb, Atlantic mackerel and blue whiting for Hg and Pb.

Official controls for the determination of lipophilic marine biotoxins in mussels farmed along the Adriatic coast of Central Italy.

In the present study, 334 samples of mussels (Mytilus galloprovincialis) harvested along the coasts of the Central Adriatic Sea during the years 2020-2021 were analyzed for the presence of lipophilic marine biotoxins according to the European Harmonized Standard Operating Procedure. The results showed that 74 (22%) and 84 (25%) samples were positive to okadaic acid and yessotoxin groups, respectively. Among them, only 11 (3.3%) samples resulted as non-compliant, as they exceeded the maximum limits (160 µg okadaic acid equivalent/kg) established by the Regulation (EC) 853/2004. The method applied in this study was able to detect and quantify lipophilic marine biotoxins concentrations, in order to monitor their presence in molluscs and avoid the risk of consumer exposure.

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.

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.

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.

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.

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.

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.

Levels of total mercury in marine organisms from Adriatic Sea, Italy.

The presence of total mercury in fish, crustacean and cephalopod from Adriatic Sea, was investigated. The highest concentrations were observed in decreasing order in: Norway lobster (0.97 +/- 0.24 mg/kg; mean +/- SE), European hake (0.59 +/- 0.14 mg/kg), red mullet (0.48 +/- 0.09 mg/kg), blue whiting (0.38 +/- 0.09 mg/kg), Atlantic mackerel (0.36 +/- 0.08 mg/kg) and European flying squid (0.25 +/- 0.03 mg/kg). A significant difference (p < 0.01) was found between the levels of total mercury in Norway lobster and those detected in all other species. The 25% of all samples exceeded the maximum limit fixed by Commission Regulation (EC) No 1881/2006. The results show that fish and fishery products can exceed the maximum levels and stress the need of more information for consumers in particular for people that eat large amount of fish.

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.

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.

Investigation of Nonionophoric Coccidiostat Residues in Feed as a Consequence of Carryover.

Residues of nonionophoric coccidiostats at carryover concentrations in feedstuffs collected from feed mills or animal farms in central Italy were detected as part of the official controls carried out from 2011 through 2016. The 118 samples were collected on the production line or during feed distribution and storage to determine the sampling sites at major risk of cross-contamination. For determination of nonionophoric coccidiostats, a fast, easy, and cheap method was developed and validated. Feed samples were extracted with acetonitrile-methanol and directly injected for liquid chromatography with tandem mass spectrometry. A total of 24 samples (20.3%) were positive, but only 5 (4.2%) of these samples exceeded the maximum limit set by European legislation. Most of the positive samples were collected from a batch of feed produced immediately following processing of another batch to which the coccidiostat robenidine had been added.

Investigation of phenylbutazone and its metabolite oxyphenbutazone in horse meat products during years 2013-2017.

This study showed the results of a monitoring program aimed at detecting the fraudulent use of horse meat in samples collected in the framework of official controls during the years 2013-2017 after the so-called horse meat scandal that caused a decreasing consumer confidence in the food industry, particularly in meat products. A total of 125 samples diversely distributed in the 5 years of monitoring were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was validated according to Commission Decision 657/2002/EC with regard to the following parameters: linearity, specificity, decision limit (CCα), detection capability (CCß), recovery, and precision (repeatability and within-laboratory reproducibility). Even if the results of this study revealed no positive sample, the need for constant assurance of food safety and consumers' health protection requires the implementation of preventive as well as corrective actions in the management of risks linked to the food chain in a more global context.

Polycyclic aromatic hydrocarbons in marine organisms from the Gulf of Naples, Tyrrhenian Sea.

Polycyclic aromatic hydrocarbons (PAHs) were determined by an HPLC method with fluorescence detection in bivalves (Mitylus galloprovincialis), cephalopods (Todarodes sagittatus), crustaceans (Aristeus antennatus), and fish (Mullus surmeletus, Scomber scombrus, Micromesistius poutassou, and Merluccius merluccius) caught in the Gulf of Naples (Tyrrhenian Sea, Italy). Anthracene, fluoranthene, pyrene, benz(a)anthracene, chrysene, benzo(b)fluoranthene, and benzo(k)fluoranthene were detected, at different concentrations, in all of the examined marine organisms, whereas benzo(a)pyrene, dibenz(a,h)anthracene, benzo(g,h,i)perylene, and indeno(1,2,3-cd)pyrene were found only in Mediterranean mussels. Of mussels collected in winter 71.43% exceeded the maximum residual levels (MRL) fixed for the benzo(a)pyrene in European Regulation 208/2005/EC, whereas all samples collected in summer reported values lower than this limit. In comparison to the other marine organisms, the mussels showed the highest PAH concentrations (p < 0.01). Fish showed total PAH levels lower than those of cephalopods and, in particular, European hake showed the lowest values (6.06 ng/g of fresh weight).