Modulation of Stat-1 in human macrophages infected with different species of intracellular pathogenic bacteria.

none6The infection of human macrophages by pathogenic bacteria induces different signaling pathways depending on the type of cellular receptors involved in the microorganism entry and on their mechanism(s) of survival and replication in the host cell. It was reported that Stat proteins play an important role in this process. In the present study, we investigate the changes in Stat-1 activation (phosphorylation in p-tyr(701)) after uptake of twoGram-positive (Listeria monocytogenes and Staphylococcus aureus) and two Gram-negative bacteria (Salmonella typhimurium and Legionella pneumophila) characterized by their varying abilities to enter, survive, and replicate in human macrophages. Comparing the results obtained with Gram-negative and Gram-positive bacteria, Stat-1 activation in macrophages does not seem to be related to LPS content. The p-tyr(701) Stat-1 expression levels were found to be independent of the internalized bacterial number and IFN-gamma release. On the contrary, Jak/Stat-1 pathway activation only occurs when an active infection has been established in the host macrophage, and it is plausible that the differences in the expression levels of p-tyr(701) Stat-1 could be due to different survival mechanisms or to differences in bacteria life cycles within macrophages.openSchiavano, Giuditta Fiorella; Dominici, Sabrina; Rinaldi, Laura; Cangiano, Alfonsina Mariarosaria; Brandi, Giorgio; Magnani, MauroSchiavano, GIUDITTA FIORELLA; Dominici, Sabrina; Rinaldi, Laura; Cangiano, ALFONSINA MARIAROSARIA; Brandi, Giorgio; Magnani, Maur

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

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

A new humanized antibody is effective against pathogenic fungi in vitro

Invasive fungal infections mainly affect patients undergoing transplantation, surgery, neoplastic disease, immunocompromised subjects and premature infants, and cause over 1.5 million deaths every year. The most common fungi isolated in invasive diseases are Candida spp., Cryptococcus spp., and Aspergillus spp. and even if four classes of antifungals are available (Azoles, Echinocandins, Polyenes and Pyrimidine analogues), the side effects of drugs and fungal acquired and innate resistance represent the major hurdles to be overcome. Monoclonal antibodies are powerful tools currently used as diagnostic and therapeutic agents in different clinical contexts but not yet developed for the treatment of invasive fungal infections. In this paper we report the development of the first humanized monoclonal antibody specific for β-1,3 glucans, a vital component of several pathogenic fungi. H5K1 has been tested on C. auris, one of the most urgent threats and resulted efficient both alone and in combination with Caspofungin and Amphotericin B showing an enhancement effect. Our results support further preclinical and clinical developments for the use of H5K1 in the treatment of patients in need

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

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics

Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-L-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice

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

La radiazione UV nella disinfezione dell’acqua. Acqua e salute per la popolazione

Riflessioni della Water Decade 2005-215 e Giornate Mondiali dell'acqu