Microbial contamination pathways in a poultry abattoir provided clues on the distribution and persistence of Arcobacter spp.

The consumption of contaminated poultry meat is a significant threat for public health, as it implicates in foodborne pathogen infections, such as those caused by Arcobacter. The mitigation of clinical cases requires the understanding of contamination pathways in each food process and the characterization of resident microbiota in the productive environments, so that targeted sanitizing procedures can be effectively implemented. Nowadays these investigations can benefit from the complementary and thoughtful use of culture- and omics-based analyses, although their application in situ is still limited. Therefore, the 16S-rRNA gene-based sequencing of total DNA and the targeted isolation of Arcobacter spp. through enrichment were performed to reconstruct the environmental contamination pathways within a poultry abattoir, as well as the dynamics and distribution of this emerging pathogen. To that scope, broiler's neck skin and caeca have been sampled during processing, while environmental swabs were collected from surfaces after cleaning and sanitizing. Metataxonomic survey highlighted a negligible impact of fecal contamination and a major role of broiler's skin in determining the composition of the resident abattoir microbiota. The introduction of Arcobacter spp. in the environment was mainly conveyed by this source rather than the intestinal content. Arcobacter butzleri represented one of the most abundant species and was extensively detected in the abattoir by both metataxonomic and enrichment methods, showing higher prevalence than other more thermophilic Campylobacterota. In particular, Arcobacter spp. was recovered viable in the plucking sector with high frequency, despite the adequacy of the sanitizing procedure.IMPORTANCEOur findings have emphasized the persistence of Arcobacter spp. in a modern poultry abattoir and its establishment as part of the resident microbiota in specific environmental niches. Although the responses provided here are not conclusive for the identification of the primary source of contamination, this biogeographic assessment underscores the importance of monitoring Arcobacter spp. from the early stages of the production chain with the integrative support of metataxonomic analysis. Through such combined detection approaches, the presence of this pathogen could be soon regarded as hallmark indicator of food safety and quality in poultry slaughtering.

Detection of Sarcocystis hominis, Sarcocystis bovifelis, Sarcocystis cruzi, Sarcocystis hirsuta and Sarcocystis sigmoideus sp. nov. in carcasses affected by bovine eosinophilic myositis.

Bovine eosinophilic myositis is an inflammatory myopathy characterized by multiple focal or diffuse grey to green patches leading to condemnation of affected carcasses. Although its etiology is still uncertain, there is evidence that Sarcocystis species may play a role in the development of eosinophilic myositis. The goal of the present study was to identify Sarcocystis spp. in intralesional and extralesional tissues of condemned cattle carcasses, in order to evaluate the possible role of different bovine Sarcocystis spp. in the etiology of bovine eosinophilic myositis. Muscle samples (n = 100) of 26 affected carcasses were collected in Northern Italy. One to five samples with lesions and two aliquots of tissue without lesions were collected from each carcass; lesions were grossly categorized in green focal lesions and green diffuse patches. Genomic DNA was extracted and analyzed by multiplex-PCR targeting different Sarcocystis spp. Unidentified species were characterized morphologically (light microscopy, histology), ultrastructurally (scanning and transmission electron microscopy) and on the molecular level (complete 18S rRNA gene and partial cox1 gene sequencing). A bovine eosinophilic myositis prevalence of 0.017% was visually assessed by routine carcass inspection between 2014 and 2019 in Italy (184/1,108,150 slaughtered cattle). Out of 26 carcasses, 25 revealed the presence of at least one Sarcocystis species (96.2%). The presence of Sarcocystis spp. DNA was significantly more frequent in intralesional than in extralesional samples. Considering the different species, Sarcocystis bovifelis and Sarcocystis hominis were significantly more frequent in intralesional (41.7% and 50%, respectively) than in extralesional samples (1.9% and 15.4%, respectively), while there was no significant difference between the presence of Sarcocystis cruzi and Sarcocystis hirsuta in intralesional (27.1% and 2.1%, respectively) and extralesional (30.8% and 1.9%, respectively) samples. The presence of an unnamed Sarcocystis sp. showing thick-walled (3.7-5.4 µm) cysts with densely packed, flattened, undulating and narrow protrusions, which showed an S-shape in side view, was recorded in the diaphragm of two carcasses. Genomic DNA from individual sarcocysts isolated from the diaphragm was successfully amplified and further sequenced. Sequence comparison revealed <94.6% and 83.4% identity at 18S rRNA and cox1 genes, respectively, with other named Sarcocystis spp., while the phylogenetic analysis clearly separated the unnamed Sarcocystis sp. from the other Sarcocystis spp. using cattle as intermediate hosts. The present study contributes to the understanding of the importance of different Sarcocystis spp. in the pathogenesis of bovine eosinophilic myositis. The results emphasize the association of Sarcocystis hominis and Sarcocystis bovifelis with bovine eosinophilic myositis and highlight the presence of a new Sarcocystis sp. using cattle as intermediate hosts. The name Sarcocystis sigmoideus sp. nov. is proposed for the newly described Sarcocystis species.

Case-report: Massive infection by Cysticercus longicollis in a captive Lemur catta from Italy.

An adult male ring-tailed lemur (Lemur catta) from a biopark of northern Italy was submitted to necropsy. A multi-organ parasitic infection was macroscopically evident. Abundant sero-hemorrhagic fluid with larval parasites was present in all cavities. The microscopic evaluation of parasites and the molecular characterization revealed the presence of Cysticercus longicollis (the larval stage of Taenia crassiceps). Histology of liver, lungs, intestine and urinary bladder revealed several larval parasites surrounded by a severe lymphocytic infiltrate, fibrous tissue and hemorrhages. This is the first report of a ring-tailed lemur with an infection of C. longicollis in Italy. The source of infection is still not known however, the discovery of this parasite in a captive lemur poses more attention on the control of parasitic diseases implementing monitoring tests and biosecurity measures.

Molecular differentiation of Sarcocystis miescheriana and Sarcocystis suihominis using a new multiplex PCR targeting the mtDNA cox1 gene in wild boars in southern Italy.

The increase of wild boar populations density and their meat consumption across Europe could expose humans to a plethora of foodborne diseases as sarcocystosis, caused by the zoonotic protozoan Sarcocystis suihominis. Humans become infected by eating raw or undercooked pig (Sus scrofa domesticus) containing S. suihominis sarcocysts. Despite this, to date very few data are available on the risk of infection by this parasite to wild boar (Sus scrofa) meat consumers. Thus, the present study aimed to assess the occurrence of Sarcocystis spp. in wild boars from southern Italy, applying both histology and a new multiplex PCR assay targeting the cox1 gene. Between 2019 and 2020, 997 muscle tissues (i.e., n = 269 oesophagus, n = 277 diaphragms, n = 298 hearts, n = 153 tongues) from 311 wild boars were collected and screened by a combined histological and molecular approach. Overall, 251 (80.7%) animals tested were positive for Sarcocystis spp., and S. miescheriana whose definitive hosts are canids, was the only molecularly identified species. A statistically significant difference (p < 0.05) in the prevalence of Sarcocystis infection was found according to the wild boar age and muscle tissue. Findings outlined the low zoonotic potential of infection to humans via wild boar meat consumption in Italy and the importance of the application of new molecular methods in distinguishing different Sarcocystis species.

Macroscopic sarcocystosis in a pig carcass from an Italian abattoir.

Different food-safety institutions, including the European Food Safety Authority, encourage monitoring and characterising Sarcocystis spp. in animals and foodstuffs; among meat-producing animals, domestic pigs (Sus scrofa domesticus) can host two different Sarcocystis spp., that is Sarcocystis miescheriana and the zoonotic Sarcocystis suihominis. Herein, we report for the first time the presence of macrocysts of Sarcocystis miescheriana in a domestic pig resulting in carcass condemnation. In North-West Italy, in June 2022 the carcass of a clinically healthy sow was condemned due to the detection of multifocal macroscopic whitish fusiform lesions. Affected muscle samples were submitted to histological and molecular analyses targeting the mtDNA cox1 and 18S rRNA genes. At gross examination and histology, well demarcated, oval or elongated macrocysts up to 8 mm in length characterized by a calcified central core surrounded by fibrosis were detected. The molecular amplification and sequencing of the cox1 mtDNA and 18S rRNA genes revealed the presence of Sarcocystis miescheriana DNA in all sampled macrocysts. Our study provides the first molecularly confirmed case of Sarcocystis miescheriana infection in a domestic pig in Italy. The present report highlights the need to increase data related to the occurrence and the prevalence of Sarcocystis spp. in meat-producing animals, and in wild and domestic pigs in particular, taking into account the zoonotic potential of Sarcocystis suihominis and the possible financial losses related to carcass discard due to macroscopic Sarcocystis spp. cysts.

Validation Parameters of the Magnetic Stirrer Method for Pooled Sample Digestion for Trichinella spp. in Horse Meat Based on Proficiency Tests Results.

Meat of horses may be infested with nematodes of the genus Trichinella spp. and can cause serious disease in humans. Rules for the carcasses sampling of species susceptible to Trichinella spp. infection and examination are laid down in Commission Regulation 1375/2015, where the magnetic stirrer method for pooled-sample digestion is recommended (Commission Regulation 1478/2020). All personnel involved in the examination should be properly trained and participate in quality control programs. Proficiency tests (PTs) play a key role in the quality verification process. This paper presents the results of PTs organized for 68 Polish laboratories in 2014-2019. Results were assessed qualitatively at three levels of sample contamination (0, 3, 5 larvae) and quantitatively at one level (5 larvae). The laboratories have achieved the average correct qualitative results 100%, 96.2% and 96.8% for the samples contaminated with 0, 3 and 5 larvae, respectively. In the quantitative evaluation, an average 94.1% of the reported results were correct. The data from PTs enabled us to define, for the first time, validation parameters of the digestion method for the horse meat matrix in a large-scale experiment including: specificity (100%), sensitivity (95.6%), accuracy (97.1%), the limit of detection (LOD) (1.14 ≈ 1) and the limit of quantification (LOQ) (3.42 ≈ 3).

Understanding the Effect of Ozone on Listeria monocytogenes and Resident Microbiota of Gorgonzola Cheese Surface: A Culturomic Approach.

The occurrence of Listeria monocytogenes on Gorgonzola cheese surface was reported by many authors, with risks arising from the translocation of the pathogen inside the product during cutting procedures. Among the novel antimicrobial strategies, ozone may represent a useful tool against L. monocytogenes contamination on Gorgonzola cheese rind. In this study, the effect of gaseous ozone (2 and 4 ppm for 10 min) on L. monocytogenes and resident microbiota of Gorgonzola cheese rind stored at 4 °C for 63 days was evaluated. A culturomic approach, based on the use of six media and identification of colonies by MALDI-TOF MS, was used to analyse variations of resident populations. The decrease of L. monocytogenes was less pronounced in ozonised rinds with final loads of ~1 log CFU/g higher than controls. This behaviour coincided with a lower maximum population density of lactobacilli in treated samples at day 28. No significant differences were detected for the other microbial determinations and resident microbiota composition among treated and control samples. The dominant genera were Candida, Carnobacterium, Staphylococcus, Penicillium, Saccharomyces, Aerococcus, Yarrowia, and Enterococcus. Based on our results, ozone was ineffective against L. monocytogenes contamination on Gorgonzola rinds. The higher final L. monocytogenes loads in treated samples could be associated with a suppressive effect of ozone on lactobacilli, since these are antagonists of L. monocytogenes. Our outcomes suggest the potential use of culturomics to study the ecosystems of complex matrices, such as the surface of mould and blue-veined cheeses.

Shotgun metagenomic sequencing of bulk tank milk filters reveals the role of Moraxellaceae and Enterobacteriaceae as carriers of antimicrobial resistance genes.

In the present context of growing antimicrobial resistance (AMR) concern, understanding the distribution of AMR determinants in food matrices such as milk is crucial to protect consumers and maintain high food safety standards. Herein, the resistome of different dairy farms was investigated through a shotgun metagenomic sequencing approach, taking advantage of in-line milk filters as promising tools. The application of both the reads-based and the assembly-based approaches has allowed the identification of numerous AMR determinants, enabling a comprehensive resolution of the resistome. Notably most of the species harboring AMR genes were predicted to be Gram-negative genera, namely Enterobacter, Acinetobacter, Escherichia, and Pseudomonas, pointing out the role of these bacteria as reservoirs of AMR determinants. In this context, the use of de novo assembly has allowed a more holistic AMR detection strategy, while the reads-based approach has enabled the detection of AMR genes from low abundance bacteria, usually undetectable by assembly-based methods. The application of both reads-based and assembly-based approaches, despite being computationally demanding, has facilitated the comprehensive characterization of a food chain resistome, while also allowing the construction of complete metagenome assembled genomes and the investigation of mobile genetic elements. Our findings suggest that milk filters can successfully be used to investigate the resistome of bulk tank milk through the application of the shotgun metagenomic sequencing. In accordance with our results, raw milk can be considered a source of AMR bacteria and genes; this points out the importance of properly informing food business operators about the risk associated with poor hygiene practices in the dairy production environment and consumers of the potential microbial food safety risks derived from raw milk products consumption. Translating these findings as risk assessment outputs heralds the next generation of food safety controls.

Effect of gaseous ozone treatment on biofilm of dairy-isolated Pseudomonas spp. strains.

Microbial biofilms existing in food industries have been implicated as important contamination sources of spoilage and pathogenic microorganisms in the finished products. Among the innovative strategies proposed to contrast biofilms in food environments, ozone is recognised as an environmentally friendly technology but there are few studies about its effect against bacterial biofilms. The objective of this study was to evaluate the effect of gaseous ozone (50 ppm for 6 h) in inhibition and eradication of biofilm formed by twenty-one dairyisolated Pseudomonas spp. strains. Before ozone treatments, all isolates were screened for biofilm formation according to a previously described method. Strains were then divided in four groups: weak, weak/moderate, moderate/strong, and strong biofilm producers based on the biofilm biomass value of each isolate determined using the optical density (OD - 595 nm). Inhibition treatment was effective on the strain (C1) belonging to the weak producers' group, on all strains classified as weak/moderate producers, on two strains (C8 and C12) belonging to the group of moderate/strong producers and on one strain (C13) classified as strong producer. Conversely, eradication treatments were ineffective on all strains tested, except for the strain C4 which reduced its biofilm-forming abilities after exposure to ozone gas. In conclusion, gaseous ozone may be used to enhance existing sanitation protocols in food processing environments, but its application alone not seems sufficient to contrast Pseudomonas spp. established biofilms.

Comparison Between Full-Length 16S rRNA Metabarcoding and Whole Metagenome Sequencing Suggests the Use of Either Is Suitable for Large-Scale Microbiome Studies.

Since the number of studies of the microbial communities related to food and food-associated matrices almost completely reliant on next-generation sequencing techniques is rising, evaluations of these high-throughput methods are critical. Currently, the two most used sequencing methods to profile the microbiota of complex samples, including food and food-related matrices, are the 16S ribosomal RNA (rRNA) metabarcoding and the whole metagenome sequencing (WMS), both of which are powerful tools for the monitoring of foodborne pathogens and the investigation of the microbiome. Herein, the microbial profiles of 20 bulk tank milk filters from different dairy farms were investigated using both the full-length 16S (FL-16S) rRNA metabarcoding, a third-generation sequencing method whose application in food and food-related matrices is yet in its infancy, and the WMS, to evaluate the correlation and the reliability of these two methods to explore the microbiome of food-related matrices. Metabarcoding and metagenomic data were generated on a MinION platform (Oxford Nanopore Technologies) and on a Illumina NovaSeq 6000 platform, respectively. Our findings support the greater resolution of WMS in terms of both increased detection of bacterial taxa and enhanced detection of diversity; in contrast, FL-16S rRNA metabarcoding has proven to be a promising, less expensive, and more practical tool to profile most abundant taxa. The significant correlation of the two technologies both in terms of taxa diversity and richness, together with the similar profiles defined for both highly abundant taxa and core microbiomes, including Acinetobacter, Bacillus, and Escherichia genera, highlights the possible application of both methods for different purposes. This study allowed the first comparison of FL-16S rRNA sequencing and WMS to investigate the microbial composition of a food-related matrix, pointing out the advantageous use of FL-16S rRNA to identify dominant microorganisms and the superior power of WMS for the taxonomic detection of low abundant microorganisms and to perform functional analysis of the microbial communities.

Biofilm formation and genomic features of Listeria monocytogenes strains isolated from meat and dairy industries located in Piedmont (Italy).

Listeria monocytogenes is considered a major challenge for the food industry as it can persist for long periods in food processing plants by forming biofilms. The aims of this study were: i) to assess the biofilm producing ability of 57 Listeria monocytogenes isolates previously subjected to whole-genome sequencing (WGS); ii) to compare the levels of biofilm formation with the presence or absence of biofilm associated genes. To determine the presence or absence of a known set of biofilm associated genes, a comparative genomic analysis was performed on each strain. Among Listeria monocytogenes isolates, 58 %, 38.5 % and 3.5 % exhibited weak, moderate or strong biofilm production, respectively. No difference in biofilm production was observed between food and environmental isolates. The percentage of Listeria monocytogenes strains isolated from meat products (57 %) classified as moderate or strong biofilm producers was higher than the percentage obtained for strains isolated from dairy products (28 %). The presence of the Stress Survival Islet 1, the arsD stress gene and the truncated inlA protein was significantly associated with increased levels of biofilm. Combining biofilm phenotype with molecular and genotyping data may provide the opportunity to better understand the relationship between genes linked to biofilm formation in Listeria monocytogenes.

Correction: Rubiola et al. Risk Factors for Bovine Cysticercosis in North-West Italy: A Multi-Year Case-Control Study. Animals 2021, 11, 3049.

Error in Institutional Review Board Statement [...].

The Use of Ozone as an Eco-Friendly Strategy against Microbial Biofilm in Dairy Manufacturing Plants: A Review.

Managing spoilage and pathogenic bacteria contaminations represents a major challenge for the food industry, especially for the dairy sector. Biofilms formed by these microorganisms in food processing environment continue to pose concerns to food manufacturers as they may impact both the safety and quality of processed foods. Bacteria inside biofilm can survive in harsh environmental conditions and represent a source of repeated food contamination in dairy manufacturing plants. Among the novel approaches proposed to control biofilm in food processing plants, the ozone treatment, in aqueous or gaseous form, may represent one of the most promising techniques due to its antimicrobial action and low environmental impact. The antimicrobial effectiveness of ozone has been well documented on a wide variety of microorganisms in planktonic forms, whereas little data on the efficacy of ozone treatment against microbial biofilms are available. In addition, ozone is recognized as an eco-friendly technology since it does not leave harmful residuals in food products or on contact surfaces. Thus, this review intends to present an overview of the current state of knowledge on the possible use of ozone as an antimicrobial agent against the most common spoilage and pathogenic microorganisms, usually organized in biofilm, in dairy manufacturing plants.

Risk Factors for Bovine Cysticercosis in North-West Italy: A Multi-Year Case-Control Study.

Taenia saginata is the causative agent of bovine cysticercosis, a zoonotic parasitic disease with a worldwide distribution. Bovine cysticercosis is considered to be an important food safety issue responsible for human taeniasis and a major economic concern since infected carcasses undergo condemnation, freezing and downgrading. The aim of the current investigation was to assess the presence of farm-level risk factors for bovine cysticercosis in an endemic area in North-West Italy. A questionnaire was designed to collect information relating to several farm structural features, as well as management practices, environmental characteristics and attitudes of farmers. The questionnaire was administered in two separate time intervals by direct interview to previously selected case and control farms. Overall, 32 case farms and 131 control farms were included between 2005 and 2011 and 50 case farms and 192 control farms were included between 2014 and 2020. The present survey showed a significant association between the detection of bovine cysticercosis cases at slaughter and farm proximity to picnic spots, closeness of wastewater treatment plant effluents, loose-housing systems and presence of employees along with the family members, highlighting the need for targeted awareness raising policies.

Effect of Gaseous Ozone on Listeria monocytogenes Planktonic Cells and Biofilm: An In Vitro Study.

Among food-borne pathogens, Listeria monocytogenes continues to pose concerns to food business operators due to its capacity to form biofilm in processing environments. Ozone may be an eco-friendly technology to control microbial contaminations, but data concerning its effect on Listeria monocytogenes biofilm are still limited. In this study, the effect of gaseous ozone at 50 ppm on planktonic cells and biofilm of reference and food-related Listeria monocytogenes strains was evaluated. Ozone caused a reduction in microbial loads of 3.7 ± 0.4 and 3.9 ± 0.4 Log10 CFU/mL after 10 and 30 min, respectively. A complete inactivation of planktonic cells after 6 h of treatment was observed. Biofilm inhibition and eradication treatments (50 ppm, 6 h) resulted in a significant decrease of the biofilm biomass for 59% of the strains tested, whilst a slight dampening of live cell loads in the biofilm state was observed. In conclusion, gaseous ozone is not sufficient to completely counteract Listeria monocytogenes biofilm, but it may be useful as an additional tool to contrast Listeria monocytogenes free-living cells and to improve the existing sanitization procedures in food processing environments.

Molecular detection of cattle Sarcocystis spp. in North-West Italy highlights their association with bovine eosinophilic myositis.

BACKGROUND: Cattle are intermediate hosts of six Sarcocystis species, among which Sarcocystis hominis and Sarcocystis heydorni can infect humans through the consumption of raw or undercooked meat. In addition to the zoonotic potential, there is increasing interest in these protozoa because of the evidence supporting the role of Sarcocystis spp. in the occurrence of bovine eosinophilic myositis (BEM), a specific inflammatory myopathy which leads to carcass condemnation and considerable economic losses. Actually, all the prevalence studies carried out on cattle in Italy have been based on either morphological or 18S rDNA-based molecular techniques, most likely leading to misidentification of closely related species. Therefore, there is a strong need for new data on the prevalence of the different Sarcocystis spp. in cattle in Italy and their association with bovine eosinophilic myositis. METHODS: To reach our aim, individual striated muscle samples from BEM condemned carcasses (N = 54) and diaphragm muscle samples from randomly sampled carcasses (N = 59) were obtained from Northwest Italy slaughterhouses. Genomic DNA was extracted and analyzed by multiplex-PCR targeting 18S rDNA and cox1 genes. PCR products amplified using the genus-specific primer set in absence of the specific fragment for S. hirsuta, S. cruzi, S. hominis or S. bovifelis were sequenced to achieve species identification. RESULTS: Sarcocystis DNA was detected in 67.8% of the samples from slaughter cattle and in 90.7% of the samples from BEM condemned carcasses. S. cruzi was identified as the most prevalent species in slaughter cattle (61%), followed by S. bovifelis (10.2%), S. hominis (8.5%) and S. hirsuta (1.7%). Notably, among the different Sarcocystis spp. detected, the presence of S. bovifelis and S. hominis was significantly higher in samples isolated from BEM condemned carcasses (46.3% and 40.7% respectively), while there was no statistically significant difference between the presence of S. cruzi or S. hirsuta in BEM condemned carcasses (42.6% and 1.8%, respectively) and randomly sampled carcasses. Furthermore, DNA sequence analysis revealed the presence of a putative new species in two carcasses. CONCLUSIONS: Our study contributes to updating the data on the prevalence of the different Sarcocystis spp. in cattle in Italy, highlighting the presence of three Sarcocystis spp., S. cruzi, S. hominis and S. bovifelis, in BEM lesions and allowing us to speculate on the possible role of S. hominis and S. bovifelis as the major sarcosporidian species involved in bovine eosinophilic myositis.

16S rRNA Sequencing Analysis of the Gut Microbiota in Broiler Chickens Prophylactically Administered with Antimicrobial Agents.

In poultry production, gut microbiota (GM) plays a pivotal role and influences different host functions related to the efficiency of production performances. Antimicrobial (AM) use is one of the main factors affecting GM composition and functions. Although several studies have focused their attention on the role of AMs as growth promoters in the modulation of GM in broilers, the consequences of higher AM concentrations administered during prophylactic treatments need to be better elucidated. For this purpose, 16S rRNA gene sequencing was performed to evaluate the impact of different prophylactic AM protocols on the composition and diversity of the broiler GM. Diversity analysis has shown that AM treatment significantly affects alpha diversity in ileum and beta diversity in both ileum and caecum. In ileal samples, the Enterobacteriaceae family has been shown to be particularly affected by AM treatments. AMs have been demonstrated to affect GM composition in broiler. These findings indicate that withdrawal periods were not enough for the restoral of the original GM. Further studies are needed for a better elucidation of the negative effects caused by an altered GM in broilers.

Detection of Antimicrobial Resistance Genes in the Milk Production Environment: Impact of Host DNA and Sequencing Depth.

Over the past decades, antimicrobial resistance (AMR) has been recognized as one of the most serious threats to public health. Although originally considered a problem to human health, the emerging crisis of AMR requires a "One Health" approach, considering human, animal, and environmental reservoirs. In this regard, the extensive use of antibiotics in the livestock production systems to treat mastitis and other bacterial diseases can lead to the presence of AMR genes in bacteria that contaminate or naturally occur in milk and dairy products, thereby introducing them into the food chain. The recent development of high-throughput next-generation sequencing (NGS) technologies is improving the fast characterization of microbial communities and their functional capabilities. In this context, whole metagenome sequencing (WMS), also called shotgun metagenomic sequencing, allows the generation of a vast amount of data which can be interrogated to generate the desired evidence, including the resistome. However, the amount of host DNA poses a major challenge to metagenome analysis. Given the current absence of literature concerning the application of WMS on milk to detect the presence of AMR genes, in the present study, we evaluated the effect of different sequencing depths, host DNA depletion methods and matrices to characterize the resistome of a milk production environment. WMS was conducted on three aliquots of bulk tank milk and three aliquots of the in-line milk filter collected from a single dairy farm; a fourth aliquot of milk and milk filter was bioinformatically subsampled. Two commercially available host DNA depletion methods were applied, and metagenomic DNA was sequenced to two different sequencing depth. Milk filters proved to be the most suitable matrices to evaluate the presence of AMR genes; besides, the pre-extraction host DNA depletion method was the most efficient approach to remove host reads. To our knowledge, this is the first study to evaluate the limitations posed by the host DNA in investigating the milk resistome with a WMS approach, confirming the circulation of AMR genes in the milk production environment.

Fate of Listeria monocytogenes in the Presence of Resident Cheese Microbiota on Common Packaging Materials.

Literature data regarding the survival of microorganisms on materials used for food package purposes are scarce. The aim of the current study is to assess the survival of Listeria monocytogenes on different packaging materials for dairy products during extended storage at different temperatures. Three packaging materials (5 × 5 cm) were contaminated with a cocktail of five strains of Listeria monocytogenes suspended in a cheese homogenate, including the cheese's native microbial population. Contaminated samples were incubated at 37°, 12°, and 4°C and periodically analyzed up to 56 days. The evolution of the total viable count and pathogen population was evaluated. At 37°C, the results showed that Listeria monocytogenes was no longer detected on polyethylene-coated nylon (B) by day 4 and on polyethylene-coated parchment (A) and greaseproof paper (C) by day 7. Interestingly, the initial cell population (ranging between 2.5 and 2.7 log CFU/cm2) of Listeria monocytogenes increased to 3 log CFU/cm2 within 4 days of storage at 12°C on A and C. During storage, the number remained fairly constant at 12°C and 4°C on two materials (A-C) and decreased slowly on the third one (B). This study shows that survival of Listeria monocytogenes on packaging materials for dairy products will be higher when stored at 4 or 12°C compared to 37°C. The survival of Listeria monocytogenes on the packaging materials raises concerns of cross-contamination during food handling and preparation at catering and retail premises and within the home, highlighting the importance of treating the packaging materials as a potential source of cross-contamination. These initial findings may aid in quantifying risks associated with contamination of food packaging materials.

A Set of Multiplex Polymerase Chain Reactions for Genomic Detection of Nine Edible Insect Species in Foods.

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.