A challenge test on Pseudomonas spp. as spoiling microorganism in fish fillets.

Fish fillets are highly susceptible to spoilage, with Pseudomonas spp. bacteria being among the main culprits. To maintain products' quality and safety, it is important to control the load of these microorganisms and understand their growth potential in fish fillets. However, setting up challenge tests might be hard due to the difficulty of differentiating intentionally inoculated bacteria from those already present on the fillets. To overcome this obstacle, a pilot study using Pseudomonas aeruginosa, a clinically significant bacterial species that is rare in food, was conducted. Vacuum-packed Northern cod, salmon, and plaice fish fillets were experimentally inoculated and subjected to trials at both refrigeration (4 °C) and thermal abuse temperatures (from +4 °C to +6 °C and then to +8 °C). The results showed that the growth potential of Pseudomonas aeruginosa in all the fish fillets was less than 0.5 Log10 CFU/g. This confirms that vacuum packaging could reduce the multiplication of Pseudomonas spp. in the fish fillets and underlines as it is crucial to have fish fillets containing initial loads of Pseudomonas between 104-105 CFU/g or lower at the beginning of the shelf life in order to control the deterioration rate of the product. This study provides a basis for developing further challenge tests for Pseudomonas spp. in the fish industry and highlights the importance of controlling initial loads of Pseudomonas to prevent product deterioration during the shelf life.

Microbiological safety of dry-cured fish from the raw material to the end of processing.

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.

Systematic review on microbiome-related nutritional interventions interfering with the colonization of foodborne pathogens in broiler gut to prevent contamination of poultry meat.

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.

Reduction of the microbial load in meat maturation rooms with and without alkaline electrolyzed water fumigation.

Dry aging is a process during which meat is stored within maturation chambers at low temperatures and low relative humidity, resulting in improved tenderness and flavor development. The cuts are exposed to the atmosphere by hanging them or setting them on racks in the maturation chamber without any protective packaging. Animals and humans are usually the major sources of bacterial food contamination in the meat industry, but other routes might be involved. Therefore, procedures to reduce or eliminate pathogens from surfaces are crucial for an effective hazard analysis critical control point program in the food industry and other environments. This study aimed to assess the survival of Listeria monocytogenes, Escherichia coli, Salmonella spp., and Staphylococcus aureus on the inner surface of dry aging chambers. Moreover, we tested the efficacy of alkaline electrolyzed water (REW) for its application within a procedure aimed at reducing foodborne pathogens during meat storage. Environmental conditions inside the dry aging cabinet determine a reduction of circa 3 log CFU/cm2 of the considered microorganisms on the inner surface in 24 hours. Additionally, the nebulization of REW with the smoking system increased the count reduction in 24 hours due to environmental conditions for L. monocytogenes (~1 log CFU/cm2) and for S. aureus (~2 log CFU/cm2). In this context, the use of REW can be justified for routine cleaning procedures of the surfaces, with the added value of being safe to handle, not containing environmental pollutants, and making it unnecessary to rinse surfaces due to its instability.

Loggerhead Sea Turtle as Possible Source of Transmission for Zoonotic Listeriosis in the Marine Environment.

Listeria monocytogenes is an ubiquitous pathogen isolated from different host species including fish, crustaceans, and molluscs, but it is rarely a pathogenic microorganism to marine reptiles. In particular, only two cases of fatal disseminated listeriosis have been described in the loggerhead sea turtle (Caretta caretta). In this study, we describe a lethal case of L. monocytogenes infection in a loggerhead sea turtle. The turtle was found alive, stranded on a beach in North-eastern Italy, but perished soon after being rescued. The autoptic examination revealed that heart, lung, liver, spleen, and urinary bladder were disseminated with multiple, firm, 0.1-0.5 mm sized, nodular, white-green lesions. Microscopically, these lesions corresponded with heterophilic granulomas with Gram+ bacteria within the necrotic center. Furthermore, the Ziehl-Neelsen stain was negative for acid-fast organisms. Colonies isolated from heart and liver were tested through MALDI-TOF for species identification, revealing the presence of L. monocytogenes. Whole Genome Sequencing on L. monocytogenes isolates was performed and the subsequent in silico genotyping revealed the belonging to Sequence Type 6 (ST 6); the virulence profile was evaluated, showing the presence of pathogenicity islands commonly observed in ST 6. Our results further confirm that L. monocytogenes should be posed in differential diagnosis in case of nodular lesions of loggerhead sea turtles; thus, given the zoonotic potential of the microorganism, animals should be treated with particular caution. In addition, wildlife animals can play an active role as carriers of possibly pathogenetic and virulent strains and contribute to the distribution of L. monocytogenes in the environment.

Influence of Fermentation Container Type on Chemical and Microbiological Parameters of Spontaneously Fermented Cow and Goat Milk.

Fermented goat milk is an artisanal beverage with excellent nutritional properties. There are limited data on its physicochemical properties, fatty acids, phenolic acids, and on any insight on microbiota. The aim of this research was to conduct a pilot study to compare these parameters in raw cow and goat milk before and after spontaneous fermentation in a clay pot and glass container at 37 °C for 24 h. Both types of milk and fermentation containers significantly affected the pH, acidity, proximate composition, viscosity, and whiteness index of fermented milks. A total of 17 fatty acids were identified in fermented milks, where palmitic, stearic, and myristic were the main saturated acids, and oleic and linoleic acids were the main unsaturated ones. These profiles were primarily influenced by the type of raw milk used. Three to five phenolic acids were identified in fermented milks, where quinic acid was the major phenolic compound, and salviolinic acid was identified only in raw goat milk. Preliminary metataxonomic sequencing analysis showed that the genera Escherichia spp. and Streptococcus spp. were part of the microbiota of both fermented milks, with the first genus being the most abundant in fermented goat milk, and Streptococcus in cow's milk. Moreover, Escherichia abundance was negatively correlated with the abundance of many genera, including Lactobacillus. Overall, the results of this pilot study showed significant variations between the physicochemical properties, the fatty and phenolic acids, and the microbial communities of goat and cow fermented milk, showing the opportunity to further investigate the tested parameters in fermented goat milk to promote its production.

Investigation of a Staphylococcus aureus sequence type 72 food poisoning outbreak associated with food-handler contamination in Italy.

On August 2019 a staphylococcal food poisoning outbreak occurred in an elderly home in Piedmont, Italy. The epidemiological investigation performed among the persons that consumed the meal identified chicken salad as the most likely source of the outbreak. Staphylococcus aureus was isolated from a total of seven samples, namely one vomit sample from a guest of the nursing home, two food samples (chicken salad with and without mayonnaise) and nasal swabs collected from a total of four persons working in the kitchen of the nursing home. The maximum likelihood tree obtained using single nucleotide polymorphisms analysis revealed that the isolates from the aforementioned samples clustered together. Multilocus sequence typing revealed that they belonged to Sequence Type 72. Fourier transform infrared spectroscopy (FTIR) was used in parallel to single nucleotide polymorphisms and whole genome sequencing for the determination of the degree of relatedness of the isolates. The results of the FTIR showed the same clustering obtained with single nucleotide polymorphisms and whole genome sequencing and revealed the source of infection. This study underlines the importance of both laboratory evidence and epidemiological data for outbreak investigation and further confirms that FTIR is a suitable support for the short-term epidemiological investigation on source attribution in case of a S. aureus infection.

Bovine paratuberculosis: results of a control plan in 64 dairy farms in a 4-year period.

Paratuberculosis is considered one of the most economically devastating infectious diseases of domestic livestock, and the most effective control strategy is a combination of 'test-and-cull' and on-farm biosecurity measures. In Italy, a Voluntary National Control Plan (VNCP) and guidelines have been introduced to reduce the impact of the disease, and farmers can voluntarily enroll in the control plan. The main aims of this study were: i) the description of the trend over a 4-year period on total, within-herd (WH) and between herd (BH) apparent seroprevalences observed in 64 dairy herds members of a mutual company located in Italy after the introduction of a proposed "Customized Control Plan" (CCP); ii) the evaluation of its effectiveness in terms of percentage of participating farms that decided to join the VNCP. Analyses on serum samples were performed with Enzyme-Linked Immuno Sorbent Assay (ELISA) method and revealed a general decrease in both total, WH and BH apparent seroprevalence. Total average apparent seroprevalence decreased from 2.39% in 2017 to 1% in 2020. Negative herds raised from 51.9% in 2017 to 71.1% in 2020, while farms with WH apparent seroprevalence > 5% decreased from 17.3% in 2017 to 4.4% in 2020. BH apparent seroprevalence decreased from 51.2% in 2017 to 29.2% in 2020. Among the 52 out of 64 herds that accepted to continue the proposed CCP after the first year, 41 (78.8%) joined in 2020 the VNCP, that assessed the health ranking of the herds. The results provide evidence that a control plan based on a farm-specific strategy and a subsidized testing process can effectively reduce the impact of paratuberculosis in dairy herds, especially in convincing farmers to continue in paratuberculosis control by joining the VNCP, including them in a National context and increasing their awareness of the disease.

Prevalence and Patterns of Antimicrobial Resistance among Escherichia coli and Staphylococcus spp. in a Veterinary University Hospital.

The occurrence of antimicrobial resistance in commensal strains of Escherichia coli and Staphylococcus spp. was investigated in 320 samples collected from patients and the environment of a veterinary university hospital-specifically, the consultation area (CA) and intensive care unit (ICU). E. coli was isolated in 70/160 samples (44%), while Staphylococcus spp. were isolated in 110/160 (69%) samples. The occurrence of multidrug-resistant (MDR) isolates from CA and ICU admission were similar for E. coli (1/12 (8%) versus 4/27 (15%), respectively) and Staphylococcus spp. (10/19 (53%) versus 26/50 (52%), respectively). MDR E. coli isolates increased significantly at hospital discharge (18/31; 58%; p = 0.008). Antimicrobial treatment administered during hospitalization was a risk factor for carriage of MDR E. coli (OR, 23.9; 95% CI: 1.18-484.19; p = 0.04) and MDR Staphylococcus spp. (OR, 19.5; 95% CI 1.30-292.76; p = 0.02), respectively. The odds ratio for MDR E. coli was 41.4 (95% CI 2.13-806.03; p = 0.01), if the administration of fluoroquinolones was evaluated. The mecA gene was detected in 19/24 (79%) coagulase-positive Staphylococcus spp. isolates resistant to oxacillin. High rates of MDR Staphylococcus spp. were reported. Hospitalization in the ICU and antimicrobial treatment were risk factors for colonization by MDR commensal bacteria.

Effectiveness of alkaline electrolyzed water in reducing bacterial load on surfaces intended to come into contact with food.

Alkaline electrolyzed water (REW) is known for its cleaning action. The aim of this work was to assess REW effectiveness in reducing microbial load on surfaces intended for contact with food. Stainlesssteel surfaces were experimentally contaminated, bacterial inactivation was tested before and after treatment with REW. Treatment with REW was operated spraying it on the contaminated plates until drying. Tests were conducted for Salmonella spp., Listeria spp., Staphylococcus aureus and Escherichia coli. The treatment revealed different degrees of sanitizing activity of REW on different bacterial species, with higher efficacy on E. coli and Salmonella spp. than S. aureus, Listeria spp.. Statistical analysis revealed a significant microbial load reduction (p<0.01) after treatment with REW, suggesting that it has a good disinfectant activity which, along with its easy and safe use, makes it a good alternative to many other more widely used disinfectants.

Assessment of the Impact on Human Health of the Presence of Norovirus in Bivalve Molluscs: What Data Do We Miss?

In the latest One Health ECDC EFSA technical report, Norovirus in fish and fishery products have been listed as the agent/food pair causing the highest number of strong-evidence outbreaks in the EU in 2019. This review aims to identify data gaps that must be filled in order to increase knowledge on Norovirus in bivalve molluscs, perform a risk assessment and rank the key mitigation strategies for this biological hazard, which is relevant to public health. Virologic determinations are not included in any of the food safety and process hygiene microbiologic criteria reflected in the current European regulations. In addition, the Escherichia coli-based indices of acceptable faecal contamination for primary production, as well as the food safety criteria, do not appear sufficient to indicate the extent of Norovirus contamination. The qualitative risk assessment data collected in this review suggests that bivalve molluscs present a high risk to human health for Norovirus only when consumed raw or when insufficiently cooked. On the contrary, the risk can be considered negligible when they are cooked at a high temperature, while information is still scarce for non-thermal treatments.

Antimicrobials and Food-Related Stresses as Selective Factors for Antibiotic Resistance along the Farm to Fork Continuum.

Antimicrobial resistance (AMR) is a global problem and there has been growing concern associated with its widespread along the animal-human-environment interface. The farm-to-fork continuum was highlighted as a possible reservoir of AMR, and a hotspot for the emergence and spread of AMR. However, the extent of the role of non-antibiotic antimicrobials and other food-related stresses as selective factors is still in need of clarification. This review addresses the use of non-antibiotic stressors, such as antimicrobials, food-processing treatments, or even novel approaches to ensure food safety, as potential drivers for resistance to clinically relevant antibiotics. The co-selection and cross-adaptation events are covered, which may induce a decreased susceptibility of foodborne bacteria to antibiotics. Although the available studies address the complexity involved in these phenomena, further studies are needed to help better understand the real risk of using food-chain-related stressors, and possibly to allow the establishment of early warnings of potential resistance mechanisms.

Epidemiologic case investigation on the zoonotic transmission of Staphylococcus aureus infection from goat to veterinarians.

Staphylococcus aureus infection led to a case of goat abortion, and four veterinarians contracted S. aureus infection from the goat during and after the abortion. Three veterinarians assisted a doe during the dystocic delivery of a dead foetus. Seventy-two hours after the dystocia, which ended with the goat's death, the veterinarians who assisted during the kidding and the veterinarian who performed the necropsy showed the presence of multiple, isolated, painful pustules 1-5 mm in diameter located along their forearms and knees. S. aureus was isolated from the pustules of the veterinarians, the placenta and uterus of the goat, the organs (brain, thymus gland, abomasum, liver and spleen) of the foetus, the scrotum and eye swabs of the buck, and mammary pustules of another goat from the same herd. Histological analysis revealed purulent metritis and inflammation of the placental cotyledons. Additional investigations eliminated the chances of other infections. S. aureus isolates recovered from the veterinarians, goats, foetus and buck were sensitive to the tested anti-microbials and did not encode staphylococcal enterotoxin genes (sea, ser, sep, see, seg and sei). The isolates were closely related, as indicated by the results of Fourier-transform infrared spectroscopy and comparative whole-genome sequencing analysis. The results of this study clearly support the hypothesis that an episode of professional zoonosis was caused by S. aureus infection during the abortion and also highlight the need for bacterial subtyping in epidemiological surveys.

Antimicrobial Resistance Profile and ExPEC Virulence Potential in Commensal Escherichia coli of Multiple Sources.

We recently described the genetic antimicrobial resistance and virulence profile of a collection of 279 commensal E. coli of food-producing animal (FPA), pet, wildlife and human origin. Phenotypic antimicrobial resistance (AMR) and the role of commensal E. coli as reservoir of extra-intestinal pathogenic Escherichia coli (ExPEC) virulence-associated genes (VAGs) or as potential ExPEC pathogens were evaluated. The most common phenotypic resistance was to tetracycline (76/279, 27.24%), sulfamethoxazole/trimethoprim (73/279, 26.16%), streptomycin and sulfisoxazole (71/279, 25.45% both) among the overall collection. Poultry and rabbit were the sources mostly associated to AMR, with a significant resistance rate (p > 0.01) to quinolones, streptomycin, sulphonamides, tetracycline and, only for poultry, to ampicillin and chloramphenicol. Finally, rabbit was the source mostly associated to colistin resistance. Different pandemic (ST69/69*, ST95, ST131) and emerging (ST10/ST10*, ST23, ST58, ST117, ST405, ST648) ExPEC sequence types (STs) were identified among the collection, especially in poultry source. Both ST groups carried high number of ExPEC VAGs (pandemic ExPEC STs, mean = 8.92; emerging ExPEC STs, mean = 6.43) and showed phenotypic resistance to different antimicrobials (pandemic ExPEC STs, mean = 2.23; emerging ExPEC STs, mean = 2.43), suggesting their role as potential ExPEC pathogens. Variable phenotypic resistance and ExPEC VAG distribution was also observed in uncommon ExPEC lineages, suggesting commensal flora as a potential reservoir of virulence (mean = 3.80) and antimicrobial resistance (mean = 1.69) determinants.

First isolation of Klebsiella variicola from a horse pleural effusion.

BACKGROUND: Respiratory diseases are the second most common cause of illnesses in horses, their etiology can be viral, bacterial, immune-mediated, or mechanical (Racklyeft and Love DN, Aust Vet J 78:549-59, 2000; Austin et al., J Am Vet Med Assoc 207:325-328, 1995; Arroyo et al., J Vet Intern Med 31:894-900, 2017). Klebsiella variicola is a Gram-negative bacterium that was initially identified as an endophyte in soil and plants such as bananas, rice, sugar cane and maize but recent studies have identified this microorganism as an emerging pathogen in humans (Rodríguez-Medina et al., Emerg Microbes Infect 8:973-988, 2019; Fontana et al., J Clin Microbiol 57:e00825-18, 2019; Rosenblueth et al., Syst Appl Microbiol 27:27-35, 2004). This paper describes, for the first time to our knowledge, the isolation of K. variicola from pleural effusion in a male adult horse. CASE PRESENTATION: 17-years Italian Saddle Horse with respiratory distress and fever was admitted to the Veterinary Teaching Hospital of the Department of Veterinary Medical Sciences, University of Bologna. At home, the patient had undergone antibiotic therapy without clinical improvement. Vital signs on admission revealed an increased respiratory rate, tachycardia, pyrexia and weight loss. The animal was submitted for collateral examination including thoracic radiology and ultrasound and thoracoscopy that showed bilateral pleural effusion associated with multifocal pulmonary atelectasis. During the thoracoscopic examination, that confirmed the presence of a seropurulent pleural effusion, a sample of pleural fluid was collected and Gram-negative bacteria were isolated and subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) that allowed the identification of K. variicola. The isolate was sensitive to amikacin, cefazolin, enrofloxacin, marbofloxacin, tetracycline, and trimethoprim-sulfamethoxazole;the horse was treated with Oxytetracycline and amikacin. Despite a general health improvement of the subject, the pleural effusion did not resolve after treatment. CONCLUSIONS: This paper describes, for the first time, the isolation of K. variicola in a horse with respiratory disease. The misidentification between K. variicola and K. pneumoniae has caused unawareness about significant aspects of this bacterial species. In fact, even though in animals the role of this bacterium is not clear, in humans it has been recognized as an emerging pathogen. The use of new methods for bacterial identification will probably lead to the isolation of a greater number of strains which will have to be studied to acquire knowledge that will be useful to clarify the clinical importance and relevance of K. variicola also in animals.

Distribution, virulence, genotypic characteristics and antibiotic resistance of Listeria monocytogenes isolated over one-year monitoring from two pig slaughterhouses and processing plants and their fresh hams.

Listeria monocytogenes contamination in raw pork and ready to eat foods is an important food safety concern, also for the increasing detection of antimicrobial-resistant isolates. Data on L. monocytogenes occurrence, persistence, distribution and genetic characterization in two different plants, namely in continuum from slaughtered pigs, environment and unfinished products (fresh hams) were observed by one-year monitoring and were integrated with their antimicrobial resistance patterns. A total of 98 samples out of the overall 1131 (8.7%) were positive for L. monocytogenes, respectively 2.6% and 13.2% in plants A and B: only three serotypes were identified, 1/2c (50%), 1/2b (36.7%) and 1/2a (13.27%), and strains were classified in 35 pulsotypes and 16 clusters by PFGE; a unique P-type was highlighted according to the detection of virulence genes. The contamination flow of L. monocytogenes has a low occurrence in slaughterhouse (Plant A = 1.1%, Plant B: 3.1%; p > 0.05) and increased throughout the processing chain with trimming area as the most contaminated (Plant A: 25%, Plant B: 57%; (p < 0.05)), both in the environment and in unfinished products (80% in hams before trimming in plant B). The dominant role of environmental contamination in post-slaughter processing is confirmed to be a significant cause of meat contamination by L. monocytogenes. Very high levels of resistance were observed for clindamycin (57%) and high resistance levels (>20-50%) to ciprofloxacin, oxacillin, levofloxacin and daptomycin, confirming the L. monocytogenes resistance trend to a wide range of antimicrobial agents. A total of 11 L. monocytogenes isolates were multidrug resistant and 7 out of them were isolated from slaughtered pigs. An interesting significant (p < 0.05) statistical correlation has been found between resistance to some antimicrobial agents and lineage/serotypes. Microbiological sampling of food and environments after sanitization are commonly used as verification procedure for the absence of L. monocytogenes in food plants and to give assurance of food safety, but strains characterization is necessary for industries to target specific control measures, like the enforcement of the hygiene program and of the control of operator activities, at least for permanent strains. The only presence of L. monocytogenes could not be considered as the conclusive assessment of a potential risk for public health, also in terms of emerging and emerged antimicrobial resistances.