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.

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.

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.

Characterization of Vegetative Bacillus cereus and Bacillus subtilis Strains Isolated from Processed Cheese Products in an Italian Dairy Plant.

Processed cheese is a commercial product characterized by high microbiological stability and extended shelf life obtained through the application of severe heat treatment. However, spore-forming bacteria can survive through thermal processes. Among them, microorganisms belonging to Bacillus genus have been reported. In this study, we examined the microbiological population of the first hours' production of processed cheeses in an Italian dairy plant during two seasons, between June and October 2020. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to identify bacteria colonies, allowing the isolation of Bacillus cereus and Bacillussubtilis strains. These results were further confirmed by amplification and sequencing of 16 rRNA bacterial region. A multi-locus sequence type (MLST) analysis was performed to assess the genetic similarity among a selection of isolates. The fourteen B. cereus strains showed two sequence types: ST-32 was observed in only one strain and the ST-371 in the remaining thirteen isolates. On the contrary, all twenty-one B. subtlis strains, included in the study, showed a new allelic profile for the pycA gene, resulting in a new sequence type: ST-249. For B. cereus strains, analysis of toxin genes was performed. All isolates were positive for nheABC, entFM, and cytK, while hblABCD, bceT, and ces were not detected. Moreover, the biofilm-forming ability of B. cereus and B. subtilis strains was assessed, and all selected isolates proved to be biofilm formers (most of them were stronger producers). Considering the genetical similarity between isolates, jointly with the capacity to produce biofilm, the presence of a recurring Bacillus population could be hypothesized.

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.

S. aureus Biofilm Protein Expression Linked to Antimicrobial Resistance: A Proteomic Study.

Antimicrobial resistance (AMR) represents one of the most critical challenges that humanity will face in the following years. In this context, a "One Health" approach with an integrated multidisciplinary effort involving humans, animals and their surrounding environment is needed to tackle the spread of AMR. One of the most common ways for bacteria to live is to adhere to surfaces and form biofilms. Staphylococcus aureus (S. aureus) can form biofilm on most surfaces and in a wide heterogeneity of environmental conditions. The biofilm guarantees the survival of the S. aureus in harsh environmental conditions and represents an issue for the food industry and animal production. The identification and characterization of biofilm-related proteins may provide interesting insights into biofilm formation mechanisms in S. aureus. In this regard, the aims of this study were: (i) to use proteomics to compare proteomes of S. aureus growing in planktonic and biofilm forms in order to investigate the common features of biofilm formation properties of different strains; (ii) to identify specific biofilm mechanisms that may be involved in AMR. The proteomic analysis showed 14 differentially expressed proteins among biofilm and planktonic forms of S. aureus. Moreover, three proteins, such as alcohol dehydrogenase, ATP-dependent 6-phosphofructokinase, and fructose-bisphosphate aldolase, were only differentially expressed in strains classified as high biofilm producers. Differentially regulated catabolites metabolisms and the switch to lower oxygen-related metabolisms were related to the sessile conformation analyzed.

Antimicrobial-Resistance of Food-Borne Pathogens.

The antimicrobial-resistance (AMR) in bacteria represents a major challenge for public health [...].

Assessment of the Antibiotic Resistance Profile, Genetic Heterogeneity and Biofilm Production of Methicillin-Resistant Staphylococcus aureus (MRSA) Isolated from The Italian Swine Production Chain.

The main aim of the present study was to evaluate the level of antibiotic resistance, prevalence and virulence features of methicillin-resistant Staphylococcus aureus (MRSA) isolated from heavy swine at abattoir level and farming environments in Lombardy (Northern Italy). With this scope, 88 different heavy swine farms were surveyed, obtaining a total of n = 440 animal swabs and n = 150 environmental swabs. A total of n = 87 MRSA isolates were obtained, with an overall MRSA incidence of 17.50% (n = 77) among animal samples and a 6.67% (n = 10) among environmental. Molecular characterisation using multilocus sequence typing (MLST) plus spa-typing showed that sequence type ST398/t899 and ST398/t011 were the most commonly isolated genotypes, although other relevant sequence types such as ST1 or ST97 were also found. A lack of susceptibility to penicillins, tetracycline and ceftiofur was detected in >91.95, 85.05 and 48.28% of the isolates, respectively. Resistance to doxycycline (32.18%), enrofloxacin (27.59%) and gentamicin (25.29%) was also observed. Additionally, a remarkable level of antibiotic multiresistance (AMR) was observed representing a 77.01% (n = 67) of the obtained isolates. Genetic analysis revealed that 97.70% and 77.01% of the isolates harboured at least one antibiotic resistance or enterotoxin gene, respectively, pointing out a high isolate virulence potential. Lastly, 55.17% (n = 48) were able to produce measurable amounts of biofilm after 24 h. In spite of the current programmes for antibiotic reduction in intensively farming, a still on-going high level of AMR and virulence potential in MRSA was demonstrated, making this pathogen a serious risk in swine production chain, highlighting once more the need to develop efficient, pathogen-specific control strategies.

Development and test of a visual-only meat inspection system for heavy pigs in Northern Italy.

BACKGROUND: There is a general consensus in recognizing that traditional meat inspection is no longer able to address the hazards related to meat consumption. Moreover, it has been shown that invasive procedures, such as palpation and incision, can increase microbial contamination in carcasses. For these reasons, legislations all over the world are changing meat inspection techniques, moving towards visual-only techniques. Hence, there was also the need to test visual-only inspection in pigs in Italy. RESULTS: A protocol for visual-only post-mortem inspection was produced together with a 24-class scheme used to record pathological lesions. A list of guidelines needed for univocal interpretation and classification of lesions was developed. To record lesions at the slaughtering line, a light instrument that is resistant to the slaughter environment was designed and then produced in collaboration with an electro-medical company. Six contracted veterinarians were chosen and trained. They performed visual-only post-mortem inspections on 231.673 heavy pigs in three different slaughterhouses of Northern Italy. Visual-only inspection was compared to traditional inspection on 38.819 pig carcasses. No relevant differences were found between the two systems. CONCLUSIONS: The comparison between traditional and visual-only inspection showed that visual-only inspection can be adopted in pig slaughterhouse. The analysis of the performance of the veterinarians stressed the importance of standardization and continuous education for veterinarians working in this field.

Listeria monocytogenes Biofilms in the Wonderland of Food Industry.

The foodborne pathogen Listeria monocytogenes is a concern in food safety because of its ability to form biofilm and to persist in food industry. In this mini-review, the issue represented by this pathogen and some of the latest efforts performed in order to investigate the composition of biofilms formed by L. monocytogenes are summarized.

Metabolic profiling by (1)H NMR of ground beef irradiated at different irradiation doses.

This work describes a metabolic profiling study of non-irradiated and irradiated beef (at 2.5, 4.5 and 8 kGy) using (1)H NMR and chemometrics. The assignment of all major NMR signals of the aqueous/methanolic extracts was performed. A comprehensive multivariate data analysis proved the ability to distinguish between the irradiated and non-irradiated beef. Classification trees revealed that three metabolites (glycerol, lactic acid esters and tyramine or a p-substituted phenolic compound) are important biomarkers for classification of the irradiated and non-irradiated beef samples. Overall, the achieved metabolomic results show that the changes in the metabolic profile of meat provide a valuable insight to be used in detecting irradiated beef. The use of the NMR-based approach simplifies sample preparation and decrease the time required for analysis, compared to available official analytical procedures.