Carriage of Staphylococcus aureus among food handlers: An ongoing challenge in public health

Staphylococcus aureus is a commensal bacterium known to colonize the skin, nares, and gastrointestinal tract of humans. Asymptomatic workers can contaminate food via manual contact or through respiratory secretions thus becoming the source of staphylococcal food poisoning. This gastrointestinal intoxication occurs after the ingestion of food contaminated by enterotoxin-producing Staphylococcus aureus. Although most individuals overcome the infection without medical assistance and make a full recovery, in rare cases the infection can be life-threatening. Hence, Staphylococcus aureus food contamination represents a serious problem for both the food industry and healthcare systems. In the last few decades, many studies have investigated the prevalence of carriers among food handlers. We present an overview of all investigations carried out on nasal carriers working in different food industry settings highlighting the risk associated with cross-contamination

Assessment of hygienic conditions of recreational facility restrooms: an integrated approach

Introduction. Microbiological quality of recreational environ- ments included restrooms, is generally assessed by water and surface monitoring. In this study, an environmental monitoring, conducted in spring, of swimming pool restrooms of a recreation center located in the Marche region has been carried out. Seven water samples and seven surface swabs were collected. Moreover, six air samples have been included. The aim of this study was to evaluate if air microbiological monitoring, along with molecular detection in real-time PCR, could give additional useful information about the hygienic conditions of the facility. Methods. Heterotrophic Plate Count (HPC) both at 22°C (psy- chrophilic) and 37°C (mesophilic) was determined by separate cultures in all samples. The presence of Legionella pneumophila and Pseudomonas aeruginosa was evaluated by both culture and real-time PCR. Results. The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women’s one (> 100 vs < 10 CFU/ml), while in air samples was between < 100 and > 500. Concerning pathogen presence, both species Legionella pneumophila and Pseudomonas aeruginosa were detected only in men restroom, but in different sample types by using different methods (culture and real-time PCR). Conclusions. Air sampling may offer the advantage of giving more representative data about microbial presence in restrooms, including bacterial species transmitted through aerosol, like Legionella. Moreover, the concurrent use of molecular and micro- biological detection in an integrated approach could offer the advantage of greater sensitivity

Prevalence, Antibiotic-Resistance, and Replicon-Typing of Salmonella Strains among Serovars Mainly Isolated from Food Chain in Marche Region, Italy

Nontyphoidal salmonellosis (NTS) is the second most commonly reported gastrointesti nal infection in humans and an important cause of food-borne outbreaks in Europe. The use of antimicrobial agents for animals, plants, and food production contributes to the development of antibiotic-resistant Salmonella strains that are transmissible to humans through food. The aim of this study was to investigate the presence and the potential dissemination of multidrug-resistant (MDR) Salmonella strains isolated in the Marche Region (Central Italy) via the food chain. Strains were isolated from different sources: food, human, food animal/livestock, and the food-processing environment. Among them, we selected MDR strains to perform their further characterization in terms of resistance to tetracycline agent, carriage of tet genes, and plasmid profiles. Tetracycline resistance genes were detected by PCR and plasmid replicons by PCR-based replicon typing (PBRT). A total of 102 MDR Salmonella strains were selected among the most prevalent serovars: S. Infantis (n = 36/102), S. Derby (n = 20/102), S. Typhimurium (n = 18/102), and a monophasic variant of S. Typhimurium (MVST, n = 28/102). Resistance to sulfisoxazole (86%) and tetracycline (81%) were the most common, followed by ampicillin (76%). FIIS was the most predominant replicon (17%), followed by FII (11%) and FIB (11%) belonging to the IncF incompatibility group. Concerning the characterization of tet genes, tetB was the most frequently detected (27/89), followed by tetA (10/89), tetG (5/89), and tetM (1/89). This study showed the potential risk associated with the MDR Salmonella strains circulating along the food chain. Hence, epidemiological surveillance supported by molecular typing could be a very useful tool to prevent transmission of resistant Salmonella from food to humans, in line with the One Health approach

Prevalence of Coxiella burnetii in cows' and ewes' bulk tank milk samples from selected dairy farms of Central Italy

The prevalence of Coxiella burnetii, the causative agent of Q fever, in cattle and sheep raw milk farms was determined in Central Italy, an area in which dairy production plays an important economic role. Milk samples (n. 189), collected from 66 dairy farms in 2012–2013, were tested by a commercial real-time PCR assay. Seventeen dairy farms had at least one positive milk sample; percent positive was higher for cattle (50%) than sheep (21%) farms. Concerning milk, 15% of samples tested overall gave a positive result, with the highest percentage of positivity observed for bovine milk compared with sheep milk (41% and 12%, respectively). In the only bovine farm repeatedly sampled during the study, C. burnetii contamination was persistently found for almost a year. The prevalence calculated for the sheep farms showed a discontinuous trend with a maximum peak in February. The results obtained underline the widespread presence of the pathogen in the considered geographical area, giving new epidemiological information. Since the milk route of elimination is a potential vehicle of infection for farmers, veterinarians and for dairy stakeholders in general, BTM screening by real-time PCR can be applied as a useful surveillance tool both for the identification of infected flocks and implementation of control programmes

Comparison of UV, Peracetic Acid and Sodium Hypochlorite Treatment in the Disinfection of Urban Wastewater

One source of water contamination is the release of wastewater that has not undergone efficient treatment. The aim of this study was to evaluate the reduction obtained with sodium hypochlorite (NaClO), UV and peracetic acid disinfection treatment of Salmonella spp., pathogenic Campylobacter, STEC and bacterial indicators in three full-scale municipal wastewater plants. A general reduction in Salmonella was observed after disinfection, but these bacteria were detected in one UV-treated sample (culture method) and in 33%, 50% and 17% of samples collected after NaClO, UV and PAA disinfection treatments, respectively (PCR method). A better reduction was also observed under NaClO disinfection for the microbial indicators. Independent of the disinfection treatment, E. coli O157:H7 was not detected in the disinfected samples, whereas some samples treated with UV and PAA showed the presence of the stx1 gene. No reduction in the presence of stx2 genes was verified for any of the disinfection treatments. Campylobacter was not detected in any of the analysed samples. The overall results highlight a better reduction in microbiological parameters with a NaClO disinfection treatment in a full-scale municipal wastewater plant compared with UV and PAA. However, the results indicate that a complete and specific monitoring program is necessary to prevent a possible risk to public health

Validation and application of a quantitative real-time PCR assay to detect common wheat adulteration of durum wheat for pasta production

Pasta is the Italian product par excellence and it is now popular worldwide. Pasta of a superior quality is made with pure durum wheat. In Italy, addition of Triticum aestivum (common wheat) during manufacturing is not allowed and, without adequate labeling, its presence is considered an adulteration. PCRrelated techniques can be employed for the detection of common wheat contaminations. In this work, we demonstrated that a previously published method for the detection of T. aestivum, based on the gliadin gene, is inadequate. Moreover, a new molecular method, based on DNA extraction from semolina and real-time PCR determination of T. aestivum in Triticum spp., was validated. This multiplex real-time PCR, based on the dual-labeled probe strategy, guarantees target detection specificity and sensitivity in a short period of time. Moreover, the molecular analysis of common wheat contamination in commercial wheat and flours is described for the first time

Redox homeostasis as a target for new antimycobacterial agents

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and β-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1β), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment

none13sìListeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.openGiuditta Fiorella Schiavano * , Collins Njie Ateba , Annalisa Petruzzelli , Veronica Mele , Giulia Amagliani , Fabrizia Guidi , Mauro De Santi , Francesco Pomilio , Giuliana Blasi , Antonietta Gattuso , Stefania Di Lullo , Elena Rocchegiani, Giorgio BrandiSchiavano, GIUDITTA FIORELLA; Njie Ateba, Collins; Petruzzelli, Annalisa; Mele, Veronica; Amagliani, Giulia; Guidi, Fabrizia; DE SANTI, Mauro; Pomilio, Francesco; Blasi, Giuliana; Gattuso, Antonietta; Di Lullo, Stefania; Rocchegiani, Elena; Brandi, Giorgi

Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm