Aflatoxins (AFs) are mycotoxins produced by some species of Aspergillus. In dairy cows, ingested AFB1 is metabolized into carcinogenic AFM1 which is eliminated through milk, thus posing a risk for consumer health. Here we describe the set, validation, and application of screening (ELISA) and confirmatory (HPLC) tests carried out on milk samples collected through official control of mycotoxin levels in northern Italy over a three-year period (2012–2014). The limit of detection (LOD) was set at 5 ppt and 2 ppt for ELISA and HPLC, respectively, and the limit of quantification (LOQ) was 10 ppt for confirmatory HPLC. A total of 1668 milk samples were analyzed: ELISA identified 36 (2.2%) positive milk samples that were subsequently confirmed by HPLC. The level of AFM1 in the positive samples ranged between 18 ± 2 and 208 ± 27 ppt. Of the total samples, only eight (0.5%) were found non-compliant with the EU regulatory limit (50 ppt; range 74 ± 10 to 208 ± 27 ppt). Use of ELISA and HPLC tests in series allows for high-volume analysis of samples, thus saving time and money while guaranteeing high analytical precision and accuracy.

Staphylococcus aureus is a major cause of clinical infections in humans and its enterotoxins cause foodborne disease. In the present study, we tested a total of 51 isolates of S. aureus from small-ruminant dairy farms with artisan dairy facilities, all located in Latium, Italy. The farms have a known history of a high prevalence of methicillin-resistant S. aureus (MRSA). Most of the MRSA isolates (27 of 51) belonged to spa-type t127 (43.1%), followed by t2678 (3.9%), t044 (2%), t1166 (2%), and t1773 (2%). PFGE performed on mecA positive strains identified one cluster (≥ 80% of similarity), comprising 22 MRSA. Nine of twenty-two MRSA isolates were assigned human host origin, and 13 isolates did not belong to a specific host. During the characterization study, one strain isolated from bulk tank milk samples harbored the pvl gene; the strain was not enterotoxigenic with a non-specific host according to the biotyping scheme, highlighting the possible emerging risk of transmission of bacterial virulence factors by foods, the environment, and foodhandlers. These findings stress the importance of hygienic measures at all processing steps of the food production chain and underline that monitoring for the presence of MRSA throughout the food chain is essential for public health.

On 1 January 2018, a new regulation on ‘Novel Food’ has come into application in the EU. Insects and insect-based products are therefore included among the categories of food which constitute novel foods. Insects are nutrient-rich, produce fewer greenhouse gases and ammonia than conventional livestock, and have high feed conversion efficiency. Insects may be an alternative food source in the near future, but consideration of insects as a food requires scrutiny due to the risk of allergens. The aim of the present study was to develop a set of multiplex polymerase chain reaction (PCR) to detect nine edible insect species directly in foods. Four sets of mPCRs were designed to detect Locusta migratoria migratorioides (Reiche & Fairmaire, 1849) (Orthoptera: Acrididae), Tenebrio molitor (Linnaeus, 1758) (Coleoptera: Tenebrionidae) (mPCR-I), Acheta domesticus (Linnaeus, 1758) (Orthoptera: Gryllidae), Bombyx mori (Linnaeus, 1758) (Lepidoptera: Bombycidae (mPCR-II), Alphitobius diaperinus (Panzer, 1797) (Coleoptera: Tenebrionidae), Schistocerca gregaria (Forskål, 1775) (Orthoptera: Acrididae), Zophobas atratus (Fabricius, 1775) (Coleoptera: Tenebrionidae) (mPCR-III), Galleria mellonella (Linnaeus, 1758) (Lepidoptera: Pyralidae), and Gryllodes sigillatus (Walker, 1869) (Orthoptera: Gryllidae) (mPCR-IV). Results demonstrate that the panel of mPCRs allowed a rapid genetic identification of the insect species and has proved to be a sensible and highly discriminatory method. The assay is a potential tool in issues related to the labeling of products and food safety, in case of allergic consumers.

The primary production of fresh soft fruits was considered to be a suspected critical point for the contamination of frozen berries that were responsible for the large 2013–2014 Hepatitis A virus (HAV) outbreak in Europe. In this study, an Italian berries’ production area was studied for its agro-technical characteristics, and the fresh fruits were analyzed for the presence of enteric viruses (HAV and Norovirus (NoV) genogroup I and genogroup II (GGI and GGII)), the enumeration of hygienic quality parameters, and the prevalence of bacterial pathogens. A total of 50 producers were sampled, who specialized in the exclusive or shared cultivation of berries. Escherichia coli was detected in two blackberry samples, whereas HAV and Norovirus were not detected. The samples were negative for Salmonella spp., Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC). The farms’ attributes were not associated with positive samples, apart from the presence of E. coli and the aerobic mesophilic bacteria for blackberry that were statistically correlated. In blueberries, the high aerobic mesophilic count could likely be associated with the resistance of the outer layer to handling. However, the two pathogens (Salmonella spp. and STEC) and the targeted viruses (HAV, NoV GGI and GGII) were not detected, highlighting the low risk of foodborne pathogens and viral contamination at the primary production stage of the berry food chain in the area considered in this pilot study.

Staphylococcus aureus is a major human pathogen and an important cause of livestock infections. More than 20 staphylococcal enterotoxins with emetic activity can be produced by specific strains responsible for staphylococcal food poisoning, one of the most common food-borne diseases. Whole genome sequencing provides a comprehensive view of the genome structure and gene content that have largely been applied in outbreak investigations and genomic comparisons. In this study, six enterotoxigenic S. aureus strains were characterised using a combination of molecular, phenotypical and computational methods. The genomes were analysed for the presence of virulence factors (VFs), where we identified 110 genes and classified them into five categories: adherence (n = 31), exoenzymes (n = 28), genes involved in host immune system evasion (n = 7); iron uptake regulatory system (n = 8); secretion machinery factors and toxins’ genes (n = 36), and 39 genes coding for transcriptional regulators related to staphylococcal VFs. Each group of VFs revealed correlations among the six enterotoxigenic strains, and further analysis revealed their accessory genomic content, including mobile genetic elements. The plasmids pLUH02 and pSK67 were detected in the strain ProNaCC1 and ProNaCC7, respectively, carrying out the genes sed, ser, and selj. The genes carried out by prophages were detected in the strain ProNaCC2 (see), ProNaCC4, and ProNaCC7 (both positive for sea). The strain ProNaCC5 resulted positive for the genes seg, sei, sem, sen, seo grouped in an exotoxin gene cluster, and the strain ProNaCC6 resulted positive for seh, a transposon-associated gene. The six strains were used for the production of naturally contaminated cheeses which were tested with the European Screening Method for staphylococcal enterotoxins. The results obtained from the analysis of toxins produced in cheese, combined with the genomic features represent a portrait of the strains that can be used for the production of staphylococcal enterotoxin-positive cheese as reference material.

Staphylococcal food poisoning outbreaks are a major cause of food-borne illness in the European Union and their notification has been mandatory since 2005. Criteria for the enumeration of coagulase-positive Staphylococci (CPS) and the detection of staphylococcal enterotoxins (SEs) in cheese have been set down in Commission Regulation EC 2073/2005. Currently, few information are available about the distribution of SEs in naturally contaminated cheeses, including raw-milk and artisanal dairy products. The aim of this study was therefore to investigate at both the CPS enumeration and the succession of the enterotoxigenic Staphylococcus aureus and produced enterotoxins levels on the rind and the core of a raw-milk semi-hard cheese, produced on farm. The study has been conducted in three steps: (I) seven wheels at different time of ripening where tested for the presence of SEs. (II) from each wheel, four portions were subsequently sampled from four different areas (peripheral rind, central rind, peripheral core and central core). (III) two cheese wheels, characterized by the highest and lowest CPS numbers and SEs quantification, based on the second step of the study, were further analyzed. A significant difference has been observed in the distribution of CPS and SEs in the four areas sampled, irrespectively of the batch and the time of ripening. The results of this study provided a set of previously unknown information on the influence of natural conditions on the distribution of CPS and SEs thereof in the cheese matrix, filling a gap in the understanding of SEs biosynthesis process.