Sewage metagenomics is a powerful tool for proactive pathogen surveillance and understanding microbial community dynamics. To support such efforts, we present a highly curated and accessible longitudinal dataset of 239 sewage samples collected from five European cities. The dataset, processed through metagenomic sequencing, includes rich analytical outputs such as taxonomic profiles, identified antimicrobial resistance genes, assembled contigs with annotated origins, metagenome-assembled genomes with functional gene annotations, and metadata. Given the computational intensity and time required to reproduce such analyses, we share this dataset to promote reuse and advance research. In addition to the metagenomic data, qPCR was used to identify specific pathogens, and Hi-C sequencing was performed on a subset of the samples to strengthen genomic linkage analysis. Central to this resource is a publicly available PostgreSQL database, designed to facilitate efficient exploration and reuse of the data. This comprehensive database allows users to perform targeted queries, subset data, and streamline access to this extensive resource.

The dry-curing process, performed in dedicated cabinets, is increasingly popular in restaurants and the food industry, driven by consumer demand for minimally processed seafood. This technology can promote the consumption of Neglected and Underutilized Fish Species (NUS), which provide good nutritional value and local-seasonal availability but have low commercial interest. Due to limited scientific data on dry-cured fish safety, this study monitored microbiological and physico-chemical parameters to estimate the shelf-life of three dry-cured NUS-fish-product prototypes from Liza ramada (thinlip grey mullet), Trachurus trachurus (horse mackerel), and Phycis blennoides (greater forkbeard). Fillets were cured using a curing solution (15% w/w) containing salt (37%), dextrose, sucrose, along with a mix of spices and antioxidants. After the dry-curing process, fillets were vacuum-packed and stored at 4 degrees C for 60 days and analyzed at five time points: 0 (T0), 15 (T1), 30 (T2), 45 (T3), and 60 (T4) days. The shelf-life of such products was determined at 30, 45, and 60 days for T. trachurus, L. ramada, and P. blennoides fillets, respectively. Dry-cured T. trachurus fillets exhibited the most pronounced oxidative and aromatic changes, followed by L. ramada fillets. In contrast, P. blennoides fillets remained microbiologically and physicochemically stable throughout the 60-day of storage. Enterobacteriaceae, beta-glucuronidase-positive E. coli, coagulase-positive Staphylococci, Salmonella spp., and Listeria monocytogenes were not detected. These results demonstrated the technological feasibility and sustainability of dry-cured ready-to-eat NUS products. Further research is needed to optimize dry-curing parameters and improve consumer acceptance, supporting the valorisation of NUS species and sustainable local seafood choices.

Alheira is a traditionally smoked, fermented, non-ready-to-eat meat sausage produced in the Trás-os-Montes region of Portugal, where lactic acid bacteria (LAB) are major determinants of product safety and quality. This study used whole-genome sequencing to characterize 59 LAB isolates collected from artisanal alheira produced in six municipalities. Eight species belonging to six genera were identified, and average nucleotide identity analysis resolved 24 non-redundant strain groups. Functional annotation revealed broad repertoires of carbohydrate-active enzymes, proteases, lipases, transport systems, and genes associated with tolerance to acid, oxidative, bile, heat, and salt stress. Genotype–phenotype inference was performed with a subset of 22 strain groups represented by unflagged genome assemblies. After false-discovery-rate correction, carbon-metabolism, transport, energy-metabolism, total functional-gene, acid-stress, bile-stress, and total-stress counts were positively associated with total pH decline. A mixed-effects model showed a positive association between standardized total functional-gene count and acidification (b=0.137±0.059, p=0.049; marginal R2=0.266, conditional R2=0.582), but within–between decomposition indicated that the association was detectable between species rather than within species. Regularized regression, Random Forest, AICc model comparison, and partial least-squares regression converged on carbon metabolism, transport, energy metabolism, and acid- or bile-stress functions as the principal exploratory signals; however, sparse LASSO regression for variable selection was unstable, and prediction of unobserved species was limited. None of the five targeted pathways: acetaldehyde, citrate, diacetyl, exopolysaccharide, and lactose metabolism, remained significant after multiple-testing correction. These findings provide a genomic and phenotypic basis for selecting native starter-culture candidates while emphasizing the need for independent functional and safety validation.

Fish curing is a preservation method that has evolved into a culinary practice, combining traditional techniques with modern food science. Its main aims are to reduce water activity to extend shelf life, inhibit foodborne pathogens, and enhance the flavor and texture of the products. Recently, controlled maturing techniques in dedicated cabinets, previously associated exclusively with meat, have begun to attract interest among chefs, restaurants, and food companies also in the field of preservation and flavor enhancement of fish. These methods involve exposing fish to controlled temperature, humidity, and airflow in dedicated cabinets for periods ranging from days to weeks, depending on species and desired outcomes. Despite the ongoing global spread of these methods, there is still a lack of specific guidelines for food business operators (FBOs) and regulatory references. This review offers a comprehensive assessment of the literature on fish maturation in dedicated cabinets, exploring food safety principles and identifying tools to support the economic and commercial potential of these technologies. A key distinction for FBOs and consumers is between dry-curing and dry-aging. Both encompass maturation in controlled environments, but dry-curing includes salting and the addition of spices and additives, resulting in ready-to-eat products. In contrast, for dry-aging, fish is simply degutted, scaled, and directly hung within cabinets. Although the literature remains limited, it is evident that monitoring of critical parameters (temperature, humidity, airflow) is essential to minimize spoilage, microbiological risk, and biogenic amine formation. FBOs must apply general good manufacturing practices (GMPs) and good hygiene practices (GHPs) for fish processing and some GMPs and GHPs specific to each dry-aging and dry-curing process, which must be individually validated. Further research is needed to optimize and validate processes for various species and to better understand biochemical and microbial changes. Moreover, specific guidelines for the food industry/operators to properly carry out these processes and ensure that the resulting products are safe for consumers should be drafted.

Chlamydia is the etiological agent of chlamydiosis in wild and domestic birds, mammals, and humans. In this study, Chlamydia reads were detected in the microbiome of the neck skin of 76 broiler carcasses collected in the same slaughterhouse at the end of the chilling tunnel. The carcasses originated from four different flocks of female Ross 308, reared in two broiler houses located in Northern Italy. One flock from each poultry house was sampled in 2019 and one flock in 2023. The carcass neck skin microbiome was investigated by shotgun metagenomic sequencing. Chlamydia reads displayed a mean relative abundance of 7.38%, with significant differences between carcasses obtained from the two poultry houses, sampled at both sampling times. Chlamydia ibidis was the prevalent species among time points and poultry houses. The zoonotic potential of C. ibidis and foodborne transmission have never been demonstrated. However, it is known that the genus Chlamydia has “spore”-like extracellular forms able to survive for months outside the host. Therefore, the presence of C. ibidis reads on broiler carcasses at the end of the chilling tunnel deserves further investigation. The results of this study highlight the feasibility of microbiome investigations to detect unexpected biological hazards in foods.

In pediatric acute myeloid leukemia (pAML) relapse/refractory (R/R) disease occurs frequently, but underlying mechanisms are unclear and effective second-line therapeutic options remain limited. While genomic characterization has advanced targeted treatments, their clinical implementation, particularly in the pediatric field, is hindered by toxicities or resistance due to poor correlation between preclinical and clinical studies. The use of more robust preclinical models is crucial for developing effective therapies. We established 26 pAML patient derived xenografts (PDXs) representing 14 high-risk genetic subtypes. These PDXs faithfully recapitulated the molecular complexity and heterogeneity of primary AML, and preserved the immunophenotypic, genomic, and transcriptomic profiles. Clonal and transcriptomic dynamics were tracked from patient samples to their matched PDXs, and vulnerabilities that could be exploited as therapeutic targets were identified, facilitating the evaluation of multi-faceted therapeutic strategies. We selected druggable variants and aberrantly activated pathways in KMT2A-rearranged AML and performed an in vitro drug screening of actionable targets exploiting a predictive three-dimensional co-culture model. Promising compounds were tested in vivo in AML-PDXs. Among new drugs targeting variants and pathways, we demonstrated the combination of IACS-010759, a mitochondrial complex I inhibitor, and Venetoclax, a BCL-2 inhibitor, reducing AML progression in KMT2A-r PDXs modeling both disease onset and relapse. The combination of Venetoclax+IACS-010759 with a stromal targeting drug slowed AML progression in a resistant model. Overall, our study highlights the power of AML-PDXs as a translational platform for novel targeted therapy identification. Our preclinical results testing Venetoclax+IACS-010759 in KMT2A-r AML strongly support mitochondrial targeting in this genetic AML subtype.

Human sewage is the main source of contamination of environmental waters with human enteric viruses, that can contaminate food such as shellfish. Metagenomic represents a new way of analyzing viral diversity through an a priori massive parallel sequencing approach. However, the precise identification of enteric viruses in sewage or shellfish matrices, is still challenging due to the low viral load, large diversity of viral genera and the large amounts of matrix masking viral sequences. This work compared three commercial kits using capture-based enrichment during the library preparation, for the diversity of detected enteric viruses and for the identification of viral strains in sewage and shellfish samples, focusing on four families impacting human health. Triplicate libraries were prepared for each sample and each kit. All three kits allowed the characterization of a variety of viral genera. In sewage samples, a large number of long contigs was obtained allowing a precise identification of more than 35 strains. In shellfish samples, long contigs were rarer but allowed the identification of one human astrovirus and one norovirus strains. Of the tested kits, one displayed lower variation between replicates, allowed to sequence a higher diversity of viruses from the four families of interest and yielded a higher number of nearly-whole genomes.

The Commission delegated Regulation (EU) 2024/1141 states that beef dry aging can be performed in a dedicated cabinet, at a surface temperature of-0.5 to 3 degrees C, maximum relative humidity of 85 %, air speed of 0.2-0.5 m/s and for a maximum of 35 days. Food business operators can apply different process parameters, even longer aging times, as long as they demonstrate to the competent authority that the aged meat is microbiologically safe. To support food business operators in the implementation of longer dry aging processes, in this study we investigated the behaviour of Listeria monocytogenes and Yersinia enterocolitica during beef dry aging in a patented cabinet up to 60 days. Beef loins were experimentally spiked with a mean load of 4.4 and 3.9 Log10 CFU/g of Listeria monocytogenes and Yersinia enterocolitica respectively and stored at 1 degrees C, relative humidity 78 % and ventilation 2.0 m/s for 60 days. L. monocytogenes counts did not significantly change during the dry aging process, while Y. enterocolitica significantly increased up to 3 Log10 CFU/g during the aging process and then tended to decrease at 60 days. The results of this study showed that beef dry aging at 1 f 2 degrees C, relative humidity 78 f 7 %, ventilation 2.0 m/s for 60 days does not support the growth of L. monocytogenes. On the contrary, the same process supported the growth of Y. enterocolitica up to 40 days in two of the three tested batches, showing that the impact of dry aging on the behaviour of this pathogen deserves further studies.

The assessment of histamine levels in fishery products has emerged as a paramount issue in the context of global food safety, given its profound implications for human health and the consequential impact on food quality and trade. Histamine intoxication, stemming from the ingestion of foods containing heightened histamine levels, results from the bacterial decarboxylation of histidine under conditions of improper handling, processing, or storage. This study endeavors to provide a thorough examination of histamine contamination in frozen-thawed tuna (Thunnus albacares) samples, employing an integrated approach that combines near-infrared spectroscopy (NIRS) with advanced machine learning techniques. One hundred and one samples were considered, and a systematic fortification process was applied to obtain samples with 4 histamine concentrations (0; 50; 150; 250 mg/kg); the fortification levels were confirmed by the LC-MS/MS analysis. Subsequently, NIRS spectra were collected and chemometric analyses, including modified partial least squares regression (MPLS) and support vector machine (SVM), were employed for quantitative and qualitative evaluation, respectively. Histamine quantification through MPLS utilizing the full spectrum exhibited good predictive performance in cross-validation and in hold-out validation (R2CV = 0.88; R2P = 0.74, respectively), confirming the potential of NIRS for estimating histamine levels in tuna. SVM classification models, both binary (presence/absence) and multiclass (four levels), demonstrated high accuracy (100% and 93%, respectively). The study highlights the effectiveness of NIRS combined with machine learning for rapid and accurate histamine detection in frozen-thawed tuna, offering a non-destructive, environmentally friendly alternative to traditional methods. This approach holds significant promise for food business operators and regulatory authorities, enhancing product safety, quality control, and decision-making processes related to histamine contamination in the seafood industry.

A trial was conceived to evaluate the effects of the dietary inclusion of defatted BSF larvae meal on growth performance, breast meat quality traits, cecal microbiota composition and metabolomics profile of turkeys. A total of 1512 female turkeys (B.U.T. 6) were divided into two groups (9 replicate pens) fed either a basal diet (CON group) or CON diet with 5% BSF meal from 65 days to slaughtering (105 days; INS group). The administration of BSF meal improved final body weight (10.17 vs. 10.06 kg/bird, respectively for INS and CON; P = 0.04) as well as daily weight gain and feed conversion ratio in the rearing cycle (96.22 vs. 95.23 g/bird/day and 2.127 vs. 2.141, respectively; P = 0.03). Breast meat quality traits and cecal microbiota were only slightly affected by the treatment. The cecal concentration of tyramine was significantly lower in INS turkeys, which showed higher levels of glucose and malonate (P = 0.03). The use of BSF meal tended (0.05 < P < 0.10) to increase the cecal content of isoleucine, betaine and butyrate, and to reduce the amount of 3-phenylpropionate. Overall, the dietary inclusion of 5% BSF meal from 65 to 105 days improved the growth performances of female turkeys mainly through the modulation of the gut metabolomics profile.

Next-Generation Sequencing (NGS) has transformed cancer research and clinical practice, with Whole Exome Sequencing (WES) driving advances in mutational profiling and personalized oncology. Yet, transcriptomic signatures remain essential for understanding disease mechanisms, including therapy resistance pathways. RNA sequencing (RNA-seq), however, faces unique challenges when dealing with low-input or degraded RNA, as often found in archival formalin-fixed paraffin-embedded (FFPE) tissues. Although previous studies have compared library preparation protocols, rapidly evolving technologies call for updated evaluations. Here, we present a direct comparison of two FFPE-compatible stranded RNA-seq library preparation kits: TaKaRa™ SMARTer® Stranded Total RNA-Seq Kit v2 (Kit A) and Illumina Stranded Total RNA Prep Ligation with Ribo-Zero Plus (Kit B). Both kits generated high-quality RNA-seq data, yet important differences emerged. Notably, Kit A achieved comparable gene expression quantification to Kit B while requiring 20-fold less RNA input, a crucial advantage for limited samples, albeit with increased sequencing depth. We critically discuss these results in relation to RNA availability, technical performance, cost-effectiveness, processing time, and automation potential, offering practical guidance for selecting optimal RNA-seq strategies in clinical and translational research settings.

BACKGROUND. In recent years, cancer research on genome and transcriptome sequencing tuned the scenario that was previously conceived as targetable in pediatric acute myeloid leukemia (AML), but the actual dearth of new agents approval underlined that performing robust preclinical studies for drug advance still represents a challenge. The optimization of disease models results crucial to prioritize innovative treatments. METHODS . For this study, we established and characterized AML patient-derived xenografts (PDXs) by immunophenotype, RNA-sequencing and whole-exome sequencing (WES). According to somatic mutations, we determined AML clonal dynamics from patient' AML to PDXs. In parallel, by deconvolution approach based on single-cell reference transcriptomic profiles and by Gene Set Enrichment Analysis, we dissected RNA-seq data. Based on results emerging from omics data, we selected targeted drugs, tested on a three-dimensional (3D) culture system consisting in a biomimetic scaffold co-cultured with blasts and mesenchymal stromal cells (MSCs), and in AML-PDXs. RESULTS. We generated 26 AML-PDXs representatives of 14 different AML genetic subtypes. AML cells propagated in PDXs resemble the original AML features in terms of immunophenotype, variants, and transcriptome, offering a comprehensive view of the disease complexity. By WES, we confirmed a high intra-tumoral heterogeneity, we recognized AML “founder” clones characterized by an average of 22 variants (at least one in AML driver genes) always been maintained during PDX passages and we also identified smaller clones, recording the majority of variants in NRAS, CREBBP, FAT1, TET2, FLT3, NOTCH1 and KDM5A genes. Overall, the inference of the evolutionary clonal trajectories informed about the clones-variants to be targeted for their potential to sustain AML relapse. We prioritized peculiar variants and transcriptomic signatures for pharmacological targeting. We selected Asparaginase (ASPN), IACS-010759 (IACS), Disulfiram, Gallein, 5-Azacytidine, Quizartinib, Trametinib, γ-Secretase inhibitor and ICG-001 to be tested either alone or in combination with Venetoclax (VEN) in 3D with ex vivo AML cells and AML patient derived MSCs. We highlighted ICG-001, a CBP/β-catenin inhibitor blocking the β-catenin dependent gene transcription, for its ability to reduce AML proliferation in 3D and the combinations VEN+IACS and VEN+ASPN being highly synergistic in KMT2A-rearranged AML (p<0.001). We conducted phase II-like preclinical investigations on six KMT2A-rearranged AML-PDXs treating them for 4 weeks with VEN+IACS or VEN+ASPN. Only 3 out of 6 models treated with VEN alone underwent remission, whereas it was observed in 6 out of 6 models treated with either of the 2 combinations. At day +15 after stop therapy, 6/6 models treated with VEN relapsed, in comparison with only 1 model treated with VEN+IACS and 1 with VEN+ASPN. Forty-five days after the end of combination treatments 4/6 models persisted in remission. Finally, both novel combinations promoted a significantly prolonged PDXs survival compared to mice treated with VEN alone. CONCLUSIONS. Overall, AML-PDXs capturing AML heterogeneity allow to target cancer cells in their multiple aspects and to determine a patient-specific drug profiling increasing the chance to treat leukemia. We support the use of pediatric AML-PDXs in preclinical testing for their ability to mimic the heterogeneity of drug response, aiding in the identification of tailored second-line treatments. VEN+ASPN combination represents a novel promising therapeutic strategy for advancing the treatment of KMT2A-rearranged AML.

FGFR inhibitors have been developed to inhibit FGFR activation and signal transduction; notwithstanding, currently the selection of intrahepatic cholangiocarcinoma (iCCA) patients for these drugs only relies on the detection of FGFR2 genetic alterations (GAs) in tumor tissues or circulating tumor DNAs, without concomitant assessment of FGFR2 signalling status. Accordingly, we performed multi-omic analyses of FGFR2 genes and FGFR2 signalling molecules in the tissue samples from 36 iCCA naïve patients. Gain-of-function FGFR2 GAs were detected in 7 patients, including missense mutations (n = 3; p.F276C, p.C382R and p.Y375C), translocations (n = 1) and copy number gain (n = 4; CNV ≥ 4). In contrast, among 29 patients with wild-type FGFR2, 4 cases showed activation of FGFR2 signalling, as they expressed the FGFR2 ligand FGF10 and phosphorylated FGFR2/FRS2α proteins; the remaining 25 cases resulted negative for activated FGFR2 signalling, as they lacked FGFR2 (n = 8) or phosphorylated FRS2α (n = 17) expression. Overall, we found that activation of FGFR2 signalling occurs not only in iCCA naïve patients with FGFR2 GAs, but also in a subgroup carrying wild-type FGFR2. This last finding entails that also this setting of patients could benefit from FGFR targeted therapies, widening indication of these drugs for iCCA patients beyond current approval. Future clinical studies are therefore encouraged to confirm this hypothesis.

The commercialization of processed fish products is rising in restaurants and small to medium enterprises. However, there is a lack of data related to the microbiological safety of such products. In this study total aerobic colony count and Enterobacteriaceae, as proxy of process hygiene criteria, and detection of Listeria monocytogenes and concentration of histamine, as food safety criteria, were investigated in Salmo salar (salmon), Xiphias gladius (swordfish) and Thunnus albacares (yellowfin tuna), before, during, and at the end of a dry-curing process, performed in a dedicated cabinet, at controlled temperature, relative humidity and ventilation, up to 240 h. The microbiological parameters were investigated in the tested fish products by culture methods and shotgun metagenomic, while the presence of histamine, and other biogenic amines, was quantified by High Performance Liquid Chromatography. In the raw material, and up to the end of the dry curing process, the concentration of Enterobacteriaceae was always lower than 10 CFU/g, while total aerobic colony counts ranged between 3.9 and 5.4 Log CFU/g in salmon; 5.5 and 5.9 Log CFU/g in swordfish; 4.4 and 4.8 Log CFU/g in tuna. The pH values were significantly different between fish species, in the raw materials and during processing except for T4, occurring 70 h after the start of the process for salmon and after 114 h for swordfish and tuna. Water activity was different at specific sampling points and at the end of processing. Overall, 79 % of the sequences identified in the tested fish samples were assigned to y bacteria. The most abundant phyla were Pseudomonadota, Bacillota and Mycoplasmatota. The microbial populations identified by shotgun metagenomic in the tested fish species clustered well separated one from the other. Moreover, the microbial richness was significantly higher in salmon and tuna in comparison to swordfish. Listeria monocytogenes was not detected in the raw material by using the reference cultural method and very few reads (relative abundance <0.007) were detected in swordfish and tuna by shotgun metagenomic. Histamine producing bacteria, belonging to the genera Vibrio, Morganella, Photobacterium and Klebsiella, were identified primarily in swordfish. However, histamine and other biogenic amines were not detected in any sample. To the best of our knowledge this is the first paper reporting time point determinations of microbiological quality and safety parameters in salmon, swordfish and tuna, before, during and at the end of a dry-curing process. The data collected in this paper can help to predict the risk profile of ready to eat dry-cured fish products during storage before consumption.

The market of dry aged meat is rapidly increasing. However, few papers report the impact of specific dry aging parameters on quality and microbiological safety of dry aged meat. In this study we assessed the impact of a 90 days dry aging process, performed in a commercial cabinet, on the quality parameters and microbiological safety of Holstein beef loins. Meat pH, colour indexes and fatty acids were stable during the process. Main volatile compounds’ concentration increased over time. Any oxidation of protein residues was detected and the main proteins undergoing proteolytic degradation early in the process were myoglobin, myofibrillar constituents and sarcoplasmic enzymes. The most significant histological changes started at 30 days and striation impairment was observed, not related to oxidative processes. The total bacteria count increased without signs of spoilage, Enterobacteriaceae were not enumerated and few reads of foodborne pathogens were detected by shotgun metagenomic. These results show that when the dry aging process is performed in a dedicated cabinet under strictly controlled process parameters, the microbiological safety of the meat is preserved. Our study provides the first insights of dry aged Holstein loins, whose valorisation would certainly enhance the economic, social and environmental sustainability of the Parmigiano Reggiano food system.

This systematic review aimed to compile the available body of knowledge about microbiome-related nutritional interventions contributing to improve the chicken health and having an impact on the reduction of colonization by foodborne pathogens in the gut. Original research articles published between 2012 and 2022 were systematically searched in Scopus and PubMed. A total of 1,948 articles were retrieved and 140 fulfilled the inclusion criteria. Overall, 73 papers described 99 interventions against colonization by Escherichia coli and related organisms; 10 papers described 15 interventions against Campylobacter spp.; 36 papers described 54 interventions against Salmonella; 40 papers described 54 interventions against Clostridium perfringens. A total of 197 microbiome-related interventions were identified as effective against one or more of the listed pathogens and included probiotics (n = 80), prebiotics (n = 23), phytobiotics (n = 25), synbiotics (n = 12), organic acids (n = 12), enzymes (n = 4), essential oils (n = 14) and combination of these (n = 27). The identified interventions were mostly administered in the feed (173/197) or through oral gavage (11/197), in the drinking water (7/197), in ovo (2/197), intra amniotic (2/197), in fresh or reused litter (1/197) or both in the feed and water (1/197). The interventions enhanced the beneficial microbial communities in the broiler gut as Lactic Acid Bacteria, mostly Lactobacillus spp., or modulated multiple microbial populations. The mechanisms promoting the fighting against colonization by foodborne pathogens included competitive exclusion, production of short chain fatty acids, decrease of gut pH, restoration of the microbiome after dysbiosis events, promotion of a more stable microbial ecology, expression of genes improving the integrity of intestinal mucosa, enhancing of mucin production and improvement of host immune response. All the studies extracted from the literature described in vivo trials but performed on a limited number of animals under experimental settings. Moreover, they detailed the effect of the intervention on the chicken gut without details on further impact on poultry meat safety.

Dry-aged meat is gaining popularity among food business operators and private consumers. The process is carried out in aerobic conditions by hanging beef carcasses or placing subprimal or primal cuts in a dedicated cabinet for several weeks or even months while controlling the environment through the management of process parameters such as temperature, relative humidity, and airflow. In this review, we present a critical evaluation of the literature to evaluate tools to manage the process to guarantee food safety and identify critical control points, as well as good hygiene and manufacturing practices. In controlled aging conditions, only Listeria monocytogenes and Yersinia enterocolitica can multiply, while a reduction in the number of Salmonella spp. and Escherichia coli O157:H7 is generally reported. Enterobacteriaceae usually decrease on the surface of the meat during maturation; thus, for the purpose of the hygiene evaluation of the production process, a count no higher than that of unmatured meat is expected. Besides, various studies report that the total bacterial count and the spoilage microorganisms significantly increase on the surface of the meat, up to 5-6 Log10 CFU/g in the absence of visible spoilage. Bacteria of the Pseudomonas genus tend to progressively replace other microorganisms during maturation; thus, the total mesophilic or psychrophilic bacterial load is not a good indicator of process hygiene for matured meat. Critical parameters for the control of the process are temperature, relative humidity, and ventilation, which should be monitored during the process. For this reason, equipment designed and certified for dry- aging must be used, and the manufacturer must validate the process. Food business operators must apply general good manufacturing practices (GMP) and good hygiene practices (GHP) for meat processing and some GMP and GHP specific for dry-aging. Several research needs were identified, among them the evolution of the populations of L. monocytogenes and Y. enterocolitica and the microbiology of the inner parts of the dry-aged meat.

In 2022, the number of foodborne outbreaks in Europe increased by 43.9%, highlighting the need to improve surveillance systems and design outbreak predictive tools. This review aims to assess the scientific literature describing wastewater surveillance to monitor foodborne pathogens in association with clinical data. In the selected studies, the relationship between peaks of pathogen concentration in wastewater and reported clinical cases is described. Moreover, details on analytical methods to detect and quantify pathogens as well as wastewater sampling procedures are discussed. Few papers show a statistically significant correlation between high concentrations of foodborne pathogens in wastewater and the occurrence of clinical cases. However, monitoring pathogen concentration in wastewater looks like a promising and cost-effective strategy to improve foodborne outbreak surveillance. Such a strategy can be articulated in three steps, where the first one is testing wastewater with an untargeted method, like shotgun metagenomic, to detect microorganisms belonging to different domains. The second consists of testing wastewater with a targeted method, such as quantitative polymerase chain reaction, to quantify those specific pathogens that in the metagenomic dataset display an increasing trend or exceed baseline concentration thresholds. The third involves the integrated wastewater and clinical data analysis and modeling to find meaningful epidemiological correlations and make predictions.

In this pilot study, we compared the metagenomic profiles of different types of artisanal fermented meat products collected in Italy, Greece, Portugal, and Morocco to investigate their taxonomic profile, also in relation to the presence of foodborne pathogens and antimicrobial resistance genes. In addition, technical replicates of the same biological sample were tested to estimate the reproducibility of shotgun metagenomics. The taxonomic analysis showed a high level of variability between different fermented meat products at both the phylum and genus levels. Staphylococcus aureus was identified with the highest abundance in Italian fermented meat; Escherichia coli in fermented meat from Morocco; Salmonella enterica in fermented meat from Greece; Klebsiella pneumoniae and Yersinia enterocolitica in fermented meat from Portugal. The fungi Aspergillus, Neosartoria, Emericella, Penicillum and Debaryomyces showed a negative correlation with Lactococcus, Enterococcus, Streptococcus, Leuconostoc and Lactobacillus. The resistome analysis indicated that genes conferring resistance to aminoglycoside, macrolide, and tetracycline were widely spread in all the samples. Our results showed that the reproducibility between technical replicates tested by shotgun metagenomic was very high under the same conditions of analysis (either DNA extraction, library preparation, sequencing analysis, and bioinformatic analysis), considering both the degree of overlapping and the pairwise correlation.

Fish dry-curing technologies performed within dedicated cabinets are gaining popularity in restaurants and food companies. These technologies are based on a constant control and adjustment of temperature, relative humidity and ventilation parameters and are used to transform raw fillets or whole fish into ready to eat products. To date no scientific data are available regarding the impact of these technologies on the survival of foodborne pathogens such as Listeria monocytogenes. Thus, we performed challenge tests to evaluate the behaviour of L. monocytogenes throughout the curing process conducted within a patented cabinet on salmon, yellowfin tuna and swordfish. A significant decrease of L. monocytogenes count was recorded during salting for salmon, yellowfin tuna and swordfish (0.72, 0.51 and 0.84 Log10 CFU/g; p <0.05 and <0.001) and during drying and aging for tuna and swordfish (0.77 and 0.49 Log(10) CFU/g; p < 0.01 and < 0.05). However, an increase of the pathogen was expected applying two predictive microbiology models using the parameters of the challenge tests. Further studies are therefore necessary to include into predictive models relevant parameters which may affect the behavior of L. monocytogenes, such as ventilation, relative humidity and effect of competitive microflora.