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Listeria monocytogenes raises major challenges for the food industry. Due to its capacity to form biofilms, this pathogen can persist in processing environments and contaminate the final products. Neutral electrolyzed water (NEW) may offer a promising and eco-friendly method for controlling L. monocytogenes biofilms, though current in vitro studies on its antibiofilm activity are limited and often focused on reference strains. In this study, we assessed the effect of NEW on biofilms formed by meat-related and reference L. monocytogenes strains on polystyrene and stainless steel. Forty wild-type strains isolated from meat products and processing environments were firstly screened for their biofilm-forming abilities and classified as weak (30 %; 12/40), moderate (55 %; 22/40), and strong (15 %; 6/40) biofilm producers. Twenty-two wild-type and two reference strains were selected for the eradication assays, performed by treating the biofilms with NEW for 9 minutes of total contact time. In silico functional enrichment analysis and the visualization of biofilms by scanning electron microscopy (SEM) were also performed. The NEW treatment resulted in a greater average reduction of viable cells in biofilms formed on polystyrene (4.3 ± 1.0 log 10 CFU/cm 2 ) compared to stainless steel (2.9 ± 2.0 log 10 CFU/cm 2 ), and a remarkable intraspecies variability was observed. SEM images revealed higher structural damage on biofilms formed on polystyrene. Functional enrichment analysis suggested that clustered regularly interspaced short palindromic repeats (CRISPR)-associated elements could be involved in resistance to the treatments. NEW could be a promising additional tool to mitigate L. monocytogenes biofilms in meat processing environments, although its effect varied with surface material and strain-specific characteristics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Food processing lines represents a suitable environment for bacterial biofilm formation. One of the most common biofilm-forming genera in dairy processing plants is Bacillus , which includes species that may have a negative impact on safety and/or quality of dairy products. In the current study, we evaluated the biofilm forming ability and molecular characteristics of dairy Bacillus spp. isolates ( B. cereus and B. subtilis ). Reference strains ( B. cereus ATCC 14579 and B. subtilis NCTC 3610) were also included in the experiment. All isolates were screened by micro-titer plate (96 wells) to assess their ability to form biofilm. Then, they were tested on two common food contact surfaces (polystyrene and stainless steel) by using 6-well plates and AISI 316 stainless steel coupons. Biofilm formation, expressed as biofilm production index (BPI), was higher on polystyrene than stainless steel (except for B. cereus ATCC 14579). These observations were further confirmed by scanning electron microscopy, which allowed the microscopy observation of biofilm structure. Moreover, a possible correlation among total viable cell counts (CFU) and BPI was examined, as well as a connection among biofilm formation and bacterial cell hydrophobicity. Finally, whole genome sequencing was performed highlighting a genetic similarity among the strains belonging to the same species. The presence of selected genes involved in biofilm formation was also examined showing that strains with a greater presence of these genes were able to produce more biofilm in the tested materials. Additionally, for B. cereus strains enterotoxin genes were detected., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Catania, Di Ciccio, Ferrocino, Civera, Cannizzo and Dalmasso.)

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., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Rubiola, Chiesa, Dalmasso, Di Ciccio and Civera.)

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/cm 2 ) of Listeria monocytogenes increased to 3 log CFU/cm 2 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., (Copyright © 2020 Di Ciccio, Rubiola, Grassi, Civera, Abbate and Chiesa.)

The objective of this study was to compare the biofilm-forming capabilities of different genotypes of Staphylococcus aureus dairy isolates from Switzerland and northern Italy, including Staph. aureus genotype B (GTB) and methicillin-resistant Staph.aureus (MRSA). We hypothesized that biofilm formation might be more pronounced in the contagious GTB isolates compared with other genotypes affecting individual animals. Twenty-four dairy isolates, including 9 MRSA, were further characterized by genotyping by using ribosomal spacer PCR, spa typing, biofilm formation under static and dynamic conditions, and scanning electron microscopy. The GTB isolates (n = 6) were more able to form biofilms than other genotypes at 37°C and at 20°C after 48 and 72 h of incubation in the static assay using polystyrene microtiter plates. This result was supported by scanning electron micrographs showing a GTB isolate producing strong biofilm with extracellular matrix in contrast to a genotype C isolate. Furthermore, none of the MRSA isolates formed strong biofilms in the static assay. However, some MRSA produced low or moderate amounts of biofilm depending on the applied conditions. Under dynamic conditions, a much more diverse situation was observed. The ability of GTB isolates to be strong biofilm formers was not observed in all cases, emphasizing the importance of growth conditions for the expression of biofilm-related genes. No specific genotype, spa type, or MRSA isolate could be categorized significantly into one level of biofilm formation. Nineteen percent of isolates behaved similarly under static and dynamic conditions. The results of this study expand our knowledge of different dairy-related Staph. aureus subtypes and indicate the benefit of genotyping when biofilms are studied., (The Authors. Published by the Federation of Animal Science Societies and Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).)

The capability to produce biofilm is an important persistence and dissemination mechanism of some foodborne bacteria. This paper investigates the relationship between some molecular characteristics (SCCmec, ST, spa-type, agr-type, cna, sarA, icaA, icaD, clfA, fnbA, fnbB, hla, hlb) of 22 food-related methicillin-resistant Staphylococcus aureus (MRSA) strains and their ability to form biofilm on stainless steel and polystyrene. Five (22.7%, 5/22) strains were able to synthesize biofilm on polystyrene, and one of these (4.5%, 1/22) strains was also able to synthesize biofilm on stainless steel. The largest amount of biofilm was formed on polystyrene by 2 MRSA strains isolated from cows' milk, thus raising concern about the dairy industry. The majority of MRSA biofilm producers carried SCCmec type IVa, suggesting that the presence of SCCmecIVa and/or agr type III could be related to the ability to form biofilm. In conclusion, in order to achieve an acceptable level of food safety, Good Hygiene Practices should be strictly implemented along the food chain to reduce the risk of colonization and dissemination of MRSA biofilm-producing strains in the food industry., Practical Application: In this study, some assayed isolates of food-related MRSA demonstrated the capacity to form biofilm. Biofilm formation differed according to surface characteristics and MRSA strains. A relationship was observed between some molecular characteristics and the ability to form biofilms. Few studies have investigated the ability of MRSA to form biofilms, and the majority of these studies have investigated clinical aspects. This work was performed to investigate whether or not there is a difference between MRSA food isolates and MRSA clinical isolates in their ability to form biofilm. These initial findings could provide information that will contribute to a better understanding of these aspects., (© 2017 Institute of Food Technologists®.)

To evaluate the occurrence of Toxoplasma gondii and to genetically characterize its isolates in carcasses of industrial fattening pigs, blood, diaphragm, and heart samples were collected from 375 carcasses of pigs slaughtered to be processed for Parma ham production. Pigs had been bred on approved farms (n = 75) located in the so-called Food Valley in Italy. Sera were examined for immunoglobulin G antibodies to T. gondii by modified agglutination test (MAT). Both heart and diaphragm samples from seropositive carcasses were processed for the presence of T. gondii DNA (B1 locus) by real-time PCR and high resolution melting (HRM) assay. Anti-Toxoplasma antibodies were detected in 2.1% of pig carcasses, with titers from 1:10 to 1:320. T. gondii DNA was detected in all (eight) seropositive carcasses and in 11 (5 heart and 6 diaphragm samples) of 16 samples; that is, it was detected in heart tissue in two subjects, in diaphragm tissue in three subjects, and in both muscle tissues in three subjects. Toxoplasma genotypes were determined in seven of eight carcasses: type III was identified in four carcasses, type II in two, and both III and II in one carcass. The serological findings and the molecular detection of T. gondii strains suggest that cured meat products obtained from industrially bred pigs may be potential sources of toxoplasmosis for humans. Our results provide novel, important information regarding the seroprevalence and molecular prevalence of T. gondii in intensively reared pigs within this specific region of Italy, particularly because Parma ham from this region is known and consumed worldwide. On-farm preventive measures combined with slaughterhouse monitoring of carcasses of pigs bred for cured meat production should never be overlooked to prevent the introduction of T. gondii into the food chain and to ensure safety for consumers of these products.

There is increasing concern about the public health impact of methicillin-resistant Staphylococcus aureus. Food and animal are vectors of transmission, but the contribution of a contaminated environment is not well characterized. With regard to this, staphylococcal biofilms serve as a virulence factor, allowing MRSA strains to adhere to surfaces and other materials used in the food industry. Methicillin resistance and biofilm-forming capacity may contribute to the success of S. aureus as a human pathogen in both health care and community settings and the food production chain. This review summarizes current knowledge about the significance of food- and animal-derived MRSA strains and provides data on attachment and biofilm formation of MRSA. In addition, the impact of quorum sensing on MRSA gene expression and biofilm formation is examined., (Copyright © 2016. Published by Elsevier Masson SAS.)

The aim of this survey was to obtain data on microbiological contamination of pig carcasses and environments in three large-scale Italian slaughterhouses (identified as A-B-C) located in Northern Italy. Each slaughterhouse was visited six times. Five carcasses and three different sites of the slaughterhouse (before and during slaughter) were sampled on each sampling day. A single pooled caecal sample was taken on each sampling day. A total of 90 carcasses, 108 environmental samples and 18 caecal samples were collected. Samples from pig carcasses and slaughterhouse environment were analyzed for total viable count (TVC), Enterobacteriaceae count (EBC) and Salmonella. The caecal contents were examined for Salmonella. Carcasses from slaughterhouse A presented the greatest TVC and EBC mean log value, whereas environmental samples collected during slaughter activities from slaughterhouse C showed the greatest TVC and EBC mean log value. As far as the environmental samples collected before slaughter activities are concerned, an average up to 6 log 10 colony forming unit (CFU)/cm 2 TVC in two slaughter plants (A and C) and 5 log 10 CFU/cm 2 TVC in one slaughter plant (B) was detected. Salmonella was recovered in two slaughterhouses (A and B). Four different Salmonella serotypes were detected in the positive samples (11). Within serotype S. Rissen and S. Typhimurium monophasic-variant isolates, two pulsed-field gel electrophoresis patterns were identified. The findings in this survey suggest that carcass contamination is influenced by the slaughterhouse plant and this could be a result of differences in line speed. The results of environmental sampling have not shown an association with the slaughterhouse plant.

Listeria monocytogenes is a foodborne pathogen able to persist in food industry and is responsible for a severe illness called listeriosis. The ability of L. monocytogenes to persist in environments is due to its capacity to form biofilms that are a sessile community of microorganisms embedded in a self-produced matrix of extracellular polymeric substances (EPS's). In this review, we summarized recent efforts performed in order to better characterize the polymeric substances that compose the extracellular matrix (ECM) of L. monocytogenes biofilms. EPS extraction and analysis led to the identification of polysaccharides, proteins, extracellular DNA, and other molecules within the listerial ECM. All this knowledge will be useful for increasing food protection, suggesting effective strategies for the minimization of persistence of L. monocytogenes in food industry environments., Competing Interests: The authors declare no conflict of interest.

The aim of the present study was to investigate the sources of Listeria monocytogenes contamination in a cold smoked salmon processing environment over a period of six years (2003-2008). A total of 170 samples of raw material, semi-processed, final product and processing surfaces at different production stages were tested for the presence of L. monocytogenes. The L. monocytogenes isolates were characterized by multiplex PCR for the analysis of virulence factors and for serogrouping. The routes of contamination over the six year period were traced by PFGE. L. monocytogenes was isolated from 24% of the raw salmon samples, 14% of the semi-processed products and 12% of the final products. Among the environmental samples, 16% were positive for L. monocytogenes. Serotyping yielded three serovars: 1/2a, 1/2b, 4b, with the majority belonging to serovars 1/2a (46%) and 1/2b (39%). PFGE yielded 14 profiles: two of them were repeatedly isolated in 2005-2006 and in 2007-2008 mainly from the processing environment and final products but also from raw materials. The results of this longitudinal study highlighted that contamination of smoked salmon occurs mainly during processing rather than originating from raw materials, even if raw fish can be a contamination source of the working environment. Molecular subtyping is critical for the identification of the contamination routes of L. monocytogenes and its niches into the production plant when control strategies must be implemented with the aim to reduce its prevalence during manufacturing., (Copyright © 2012 Elsevier B.V. All rights reserved.)

There is increasing concern about the impact on public health of methicillin-resistant Staphylococcus aureus (MRSA) associated with animal food products. MRSA remains a serious problem because of the high incidence and multidrug resistance of the strains, even for strains isolated from foods, food environments and food handlers. The objectives of this study are: (i) to evaluate the susceptibility of S. aureus strains isolated from food, food handlers and food-processing environments to 14 antibiotics currently used in veterinary and human therapy; (ii) to assess the presence of the mecA gene. A total of 1007 samples were collected from food, food handlers, and environments and were analyzed for the presence of S. aureus. S. aureus was present in 165 of the 1007 samples. A total of 157 isolates were methicillin-susceptible S. aureus (MSSA) and 8 isolates were MRSA. In particular, out of 8 MRSA strains detected, 4 strains harboured the mecA gene. All MRSA strains were resistant to at least one of the tested antibiotics and 6 strains demonstrated multi-resistance. Considering the high level of resistances in S. aureus and the isolation of MRSA strains, the surveillance of antimicrobial resistance and the spreading of this pathogen is of crucial importance in the food production chain. These data are useful in improving background data on antimicrobial resistance of S. aureus isolated from food, processing environments and food handlers, supporting the prudent use of antibiotics and the development of international control programs.

Listeria monocytogenes is a foodborne pathogen which is able to cause serious disease both in humans and in animals. Several studies have demonstrated variations in the levels of virulence among L. monocytogenes strains. Invasion and growth ability of L. monocytogenes into cultured cells have been used to evaluate its pathogenicity. In particular, invasiveness and growth ability have been typically investigated using HeLa cell line. This study aimed to provide further insights on the virulence potential as well as on the molecular and phenotypic characteristics of L. monocytogenes isolated both from food sources and food environments. Thirty-eight isolates were tested for cell invasion and intracellular growth. Among the latter, 15 strains exhibited a high invasion index (I.I.); 18 strains showed intermediate II and 5 isolates revealed a low II. Regarding intracellular growth, all tested isolates had a replication time between 2 and 6h. Furthermore, nine virulence-associated genes (hlyA, actA, inlA, inlB, iap, plcA, plcB, mpl, prfA) were investigated by the multiplex PCR assay. All tested virulence genes were detected in all strains. Interestingly, a polymorphism was observed in the actA gene. However, the polymorphism could not be related to a different level of invasion or intracellular growth. In conclusion, data presented in this study have revealed considerable differences in the ability of L. monocytogenes strains to invade host cells and suggest the presence of additional factors that may contribute to adhesion and invasion. Virulence of L. monocytogenes is still not fully understood in some respects. Further studies focused on the mechanisms of L. monocytogenes pathogenicity together with the development of more reliable and efficient methods for virulence determination in this species are still required., (Copyright 2009 Elsevier B.V. All rights reserved.)