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The microbiological monitoring of mineral bottled waters results is crucial for the prevention of outbreaks in consumers. European and International regulations establish the quality of water intended for human consumption in order to preserve human health from the negative effects deriving from water contamination. Advanced methods targeting the faster detection of potential pathogens in drinking water may consent to the creation of an early warning system, enhancing water quality management. This study aimed to suggest the implementation of standard water quality evaluations, based on the characterization of the microbial composition of mineral bottled water brands, contributing to the periodic control of the water’s microbiological stability along with the shelf life, and, consequently, the stability of the supplying sources. Bottled water microbiota analysis was combined with the qualitative and quantitative evaluation of microbial loads in time, and the monitoring was performed in two seasons and two different storage conditions for a total of sixty days. The employment of molecular microbiology techniques (NGS and Sanger sequencing), compared to standardized cultural methods and integrated with metagenomic analysis, combining chemical and physical indicators for each sample, allowing for the generation of specific fingerprints for mineral bottled waters, pointing at simplifying and improving the foreseen risk assessment strategies to ensure the adequate traceability, quality and safety management of drinking water.

OBJECTIVE: This study was made to determine the incidence of nosocomial viral gastroenteritis in all children aged 0-4 years, admitted in the Pediatric Hospital over a 3-year period. METHODS: Astrovirus was detected by reverse transcriptase-PCR; routine diagnostic tests for Rotavirus, Adenovirus, and common bacterial pathogens were carried out on all samples. RESULTS: Of the 460 children with nosocomial diarrhea, 23 harbored Astrovirus (5%). Most cases occurred during the coldest months of the year. Children under 1 year of age were the most susceptible population. CONCLUSION: The collected data confirms the importance of viral etiology in nosocomial gastroenteritis. The reported rate of detection stresses the importance of Astrovirus in pediatric diarrhea. The authors recommend screening for this virus on a routine basis.

Ferric iron is an essential nutrient for bacterial growth. Pathogenic bacteria synthesize iron-chelating entities known as siderophores to sequestrate ferric iron from host organisms in order to colonize and replicate. The development of antimicrobial peptides (AMPs) conjugated to iron chelators represents a promising strategy for reducing iron availability, inducing bacterial death, and enhancing simultaneously the efficacy of AMPs. Here we designed, synthesized, and characterized three hydroxamate-based peptides Pep-cyc1, Pep-cyc2, and Pep-cyc3, derived from a cyclic temporin L peptide (Pep-cyc) developed previously by some of us. The Fe3+ complex formation of each ligand was characterized by UVvisible spectroscopy, mass spectrometry, IR, and NMR spectroscopies. In addition, the effect of Fe3+ on the stabilization of -helix conformation of hydroxamate-based peptides and the cotton effect were examined by CD spectroscopy. Moreover, the antimicrobial results obtained in vitro on some Gram-negative strains (K. Pneumoniae and E. coli) showed the ability of each peptide to chelate efficaciously Fe3+ obtaining a reduction of MIC values in comparison to their parent peptide Pepcyc. Our results demonstrated that siderophore conjugation could increase the efficacy and selectivity of AMPs used for the treatment of infectious diseases caused by Gram-negative pathogens.

8 Nanomaterials have revolutionized many scientific fields and are widely applied to address environmental problems and to 9 develop novel health care strategies. However, their mechanism of action is still poorly understood. Several nanomaterials 10 for medical applications are based on quantum dots (QDs). Despite their amazing physico-chemical properties, quantum dots 11 display significant adverse effects. In the present study, the effects of QDs on the motor nervous system of nematodes 12 Caenorhabditis elegans have been investigated as a non-mammalian alternative model. We also explored the possibility of 13 modifying the toxicity of QDs by coating with a cell-penetrating peptide gH625 and thus we analysed the effects determined 14 by QDs-gH625 complexes on the nematodes. With this work, we have demonstrated, by in vivo experiments, that the 15 peptide gH625 is able to reduce the side effects of metallic nanoparticle making them more suitable for medical applications.

Candida tropicalis is an emerging pathogen with a high mortality rate due to its virulence factors, including biofilm formation, that has important repercussions on the public health system. The ability of C. tropicalis to form biofilms, which are potentially more resistant to antifungal drugs and the consequent increasing antimicrobial resistance, highlights an urgent need for the development of novel antifungal. The present study analyzed the antibiofilm capacity of the arylamidine T-2307 on two strains of Candida tropicalis. Antimicrobial activity and time-killing assays were performed to evaluate the anticandidal effects of T-2307, the antibiofilm ability on biomass inhibition and eradication was evaluated by the crystal violet (CV) method. Furthermore, in Galleria mellonella infected larvae an increased survival after pre—and post- treatment with T-2307 was observed. The MTT test was used to determine the viability of immortalized human prostate epithelial cells (PNT1A) after exposure to different concentrations of T-2307. Levels of interleukin IL-4, IL-8, IL-10 were quantified after Candida infection of PNT1A cells and treatment. Active doses of T-2307 did not affect the viability of PNT1A cells, and drug concentrations of 0.005 or 0.01 µg mL−1 inhibited the secretion of inflammatory cytokines. Taken together, these results provide new information on T-2307, indicating this drug as a new and promising alternative therapeutic option for the treatment of Candida infections.

The increased incidence of mixed infections requires that the scientific community develop novel antimicrobial molecules. Essential oils and their bioactive pure compounds have been found to exhibit a wide range of remarkable biological activities and are attracting more and more attention. Therefore, the aim of this study was to evaluate myrtenol (MYR), one of the constituents commonly found in some essential oils, for its potential to inhibit biofilms alone and in combination with antimicrobial drugs against Candida auris/Klebsiella pneumoniae single and mixed biofilms. The antimicrobial activity of MYR was evaluated by determining bactericidal/fungicidal concentrations (MIC), and biofilm formation at sub-MICs was analyzed in a 96-well microtiter plate by crystal violet, XTT reduction assay, and CFU counts. The synergistic interaction between MYR and antimicrobial drugs was evaluated by the checkerboard method. The study found that MYR exhibited antimicrobial activity at high concentrations while showing efficient antibiofilm activity against single and dual biofilms. To understand the underlying mechanism by which MYR promotes single/mixed-species biofilm inhibition, we observed a significant downregulation in the expression of mrkA, FKS1, ERG11, and ALS5 genes, which are associated with bacterial motility, adhesion, and biofilm formation as well as increased ROS production, which can play an important role in the inhibition of biofilm formation. In addition, the checkerboard microdilution assay showed that MYR was strongly synergistic with both caspofungin (CAS) and meropenem (MEM) in inhibiting the growth of Candida auris/Klebsiella pneumoniae-mixed biofilms. Furthermore, the tested concentrations showed an absence of toxicity for both mammalian cells in the in vitro and in vivo Galleria mellonella models. Thus, MYR could be considered as a potential agent for the management of polymicrobial biofilms.

Temporins are one of the largest families of antimicrobial peptides with both anti-inflammatory and antimicrobial activity. Herein, for a panel of cyclic temporin L isoform analogues, the antifungal and antibiofilm activities were determined against representative Candida strains, including C. albicans, C. glabrata, C. auris, C. parapsilosis and C. tropicalis. The outcomes indicated a significant anti-candida activity against planktonic and biofilm growth for four peptides (3, 7, 15 and 16). The absence of toxicity up to high concentrations and survival after infection were assessed in vivo by using Galleria mellonella larvae, and the correlation between conformation and cytotoxicity was investigated by fluorescence assays and circular dichroism (CD). By combining fluorescence spectroscopy, CD, dynamic light scattering, confocal and atomic force microscopy, the mode of action of four analogues was hypothesized. The results pinpointed that peptide 3 emerged as a non-toxic compound showing a potent antibiofilm activity and represents a promising compound for biomedical applications.

The photochemical and ecotoxicological fate of acyclovir (ACY) through UV254 direct photolysis and in the presence of hydroxyl radicals (UV254/H2O2 process) were investigated in a microcapillary film (MCF) array photoreactor, which provided ultrarapid and accurate photochemical reaction kinetics. The UVC phototransformation of ACY was found to be unaffected by pH in the range from 4.5 to 8.0 and resembled an apparent autocatalytic reaction. The proposed mechanism included the formation of a photochemical intermediate (ϕACY = (1.62 ± 0.07)·10−3 mol ein−1) that further reacted with ACY to form by-products (k’ = (5.64 ± 0.03)·10−3 M−1 s−1). The photolysis of ACY in the presence of hydrogen peroxide accelerated the removal of ACY as a result of formation of hydroxyl radicals. The kinetic constant for the reaction of OH radicals with ACY (kOH/ACY) determined with the kinetic modeling method was (1.23 ± 0.07)·109 M−1 s−1 and with the competition kinetics method was (2.30 ± 0.11)·109 M−1 s−1 with competition kinetics. The acute and chronic effects of the treated aqueous mixtures on different living organisms (Vibrio fischeri, Raphidocelis subcapitata, D. magna) revealed significantly lower toxicity for the samples treated with UV254/H2O2 in comparison to those collected during UV254 treatment. This result suggests that the addition of moderate quantity of hydrogen peroxide (30–150 mg L−1) might be a useful strategy to reduce the ecotoxicity of UV254 based sanitary engineered systems for water reclamation.

Two natural mixtures, Allium sativum fermented extract (BGE) and cannabinol oil extract (CBD), were assessed for their ability to inhibit and remove Pseudomonas aeruginosa biofilms on soft contact lenses in comparison to a multipurpose Soft Contact Lens-care solution present on the Italian market. Pseudomonas aeruginosa (ATCC 9027 strain) and Pseudomonas aeruginosa clinical strains isolated from ocular swabs were tested. Quantification of the biofilm was done using the microtiter plate assay and the fractional inhibitory concentration index was calculated. Both forms of Pseudomonas aeruginosa generated biofilms. BGE at minimal inhibitory concentration (MIC) showed inhibition percentages higher than 55% for both strains, and CBD inhibited biofilm formation by about 70%. The care solution at MIC inhibited biofilm formation by about 50% for both strains tested. The effect of BGE on the eradication of the microbial biofilm on soft contact lenses at MIC was 45% eradication for P. aeruginosa ATCC 9027 and 36% for P. aeruginosa clinical strain. For CBD, we observed 24% biofilm eradication for both strains. For the care solution, the eradication MICs were 43% eradication for P. aeruginosa ATCC 9027 and 41% for P. aeruginosa clinical strain. It was observed that both the test soft contact lenses solution/BGE (fractional inhibitory concentration index: 0.450) and the test soft contact lenses solution/CBD (fractional inhibitory concentration index: 0.153) combinations exhibited synergistic antibiofilm activity against most of the studied bacteria. The study showed that BGE and CBD have good effect on inhibition of biofilm formation and removal of preformed biofilms, which makes them promising agents that could be exploited to develop more effective care solutions.

In recent years, many studies have highlighted the consistent finding of tramadol (TRA) in the effluents from wastewater treatment plants (WTPs) and also in some rivers and lakes in both Europe and North America, suggesting that TRA is removed by no more than 36% by specific disinfection treatments. The extensive use of this drug has led to environmental pollution of both water and soil, up to its detection in growing plants. In order to expand the knowledge about TRA toxicity as well as the nature of its disinfection by-products (DBPs), a simulation of the waste treatment chlorination step has been reported herein. In particular, we found seven new by-products, that together with TRA, have been assayed on different living organisms (Aliivibrio fischeri, Raphidocelis subcapitata and Daphnia magna), to test their acute and chronic toxicity. The results reported that TRA may be classified as a harmful compound to some aquatic organisms whereas its chlorinated product mixture showed no effects on any of the organisms tested. All data suggest however that TRA chlorination treatment produces a variety of DBPs which can be more harmful than TRA and a risk for the aquatic environment and human health.

A group of transcription factors, termed signal transducers and activators of transcription (STATs), appears to orchestrate the downstream events propagated by cytokine/growth factor interactions with their cognate receptors. Similarly, cytoplasmic Janus kinases (JAKs) seem to play a critical role in diverse signal transduction pathways that govern cellular survival, proliferation, differentiation and apoptosis. In this work, we analysed the effects of the Salmonella enterica serovar Typhimurium porins on signaling by the JAK/STAT pathway and IL-6 release in U937 cells. Porins and LPS of membrane from Gram-negative bacteria are factors implicated in septic shock. In our assays porins induce interleukin-6 (IL-6) release (110+/-2.6pg/ml) 24h after stimulation and STAT1/STAT3 tyrosine (Tyr701/Tyr705) and serine (Ser727) phosphorylation after 15min. By using several selective inhibitors we demonstrate that porins modulate the activation of STAT1/STAT3 through mitogen activated protein kinases (MAPKs) and not JAKs. Furthermore, we demonstrated that STAT1 and STAT3 are not involved in the modulation of IL-6 release in U937 cells stimulated with porins. Inhibition of tyrosine/serine phosphorylation mediated by MAPKs of STAT1 and STAT3 decrease the IL-6 secretion following porin stimulation. Therefore, suggesting a key role of this pathway in phosphorylation of Ser 727 in STAT1 and STAT3. These results are confirmed by porin or LPS-induced nuclear translocation of STAT1 and STAT3 in U937 cells.

The aim of this study was to evaluate the effect of the Heat Shock Proteins GroES, GroEL and DnaK on the expression of the costimulatory molecules CD80/CD86 in B cells and macrophages. The interactions among these molecules are able to highly influence the immune response through the regulation of cytokine liberation which, on their own, are able to regulate the immunological response by a feedback mechanism. Our results showed that, on B cells, GroES and GroEL stimulated the expression of CD86 but did not induce the increase of the CD80 expression. CD86 peak expression showed a peak after 24–48 h of culture and decreaseed 60h after the stimulation. GroES and GroEL also stimulated the expression of CD80 and CD86 on macrophages. The same HSPs did not modify the expression of CD80 and CD86 on cells having characteristics of activated macrophages, the A-THP-1 cell line. DnaK did not induce any increase in the expression of CD80 and CD86 on lymphocytes or macrophages.

We investigated the association and the invasion of a bovine aortic endothelial cell (BAEC) line by Pasteurella multocida to study the potential role of internalized bacteria and possible intracellular survival during Pasteurella infections. Our data indicate that P. multocida is able to adhere to and to invade BAECs. The density of the bacterial population plays a defined role for an optimal mechanism of interaction between bacteria and cells, an does the incubation period of association and invasion. The optimal bacteria/cells ratio was found to be 100/1, while the optimal infection time was approximately 4 h of incubation. Bacterial internalization was: dependent on microfilament and microtubule stability. The invasion ability of P. multocida in the presence of cytochalasin D was reduced by 60%; in the presence of colchicine it was reduced by 97% and in the presence of nocodazole it was reduced by 95%. Our data show that internalized P. multocida did not induce mortality of invaded endothelial cells. Some Pasteurella cells were able to survive and undergo exocytosis.

Candida albicans yeast cells suspended in sterilized sea water and cultivated in Brain Heart Infusion broth were compared. Viability, chemical composition, surface hydrophobicity and ultrastructural characteristics showed variations after incubation in sea water. The yeast cells developed some ultrastructural changes after about a month in sea water. The surface hydrophobicity of the yeast cells was gradually reduced, starting from day 16, and continued to decline throughout the 32 days in sea water. A decrease in total carbohydrate, lipid and protein contents was also observed and corresponded with ultrastructural modifications.

The response of Salmonella typhimurium to low nutrient levels was determined by measuring the concentrations of lipids, carbohydrates, DNA, RNA, and proteins over a 32-day starvation period. Ultrastructural integrity was observed by transmission electron microscopy. Lipid and carbohydrate content of bacterial cells rapidly declined within the first 16 days, while DNA and proteins exhibited a more gradual decline over the 32 days of starvation. In contrast, RNA content did not decrease appreciably upon nutrient starvation. Structural damage occurred especially after 16 days of starvation. After 32 days of nutrient deprivation, we recorded degenerative cellular forms, a coccoidal cell shape, a decrease in cellular volume, and the loss of the three-layered outer membrane. The morphological and structural alterations correlated with virulence in infected animals. We observed a decrease in virulence of S. typhimurium after 9, 16, and 32 days of starvation, reaching a maximal decrease after 32 days of nutrient deprivation. The decrease in virulence correlated to surface hydrophobicity alterations, adherence to eukaryotic cells, and phagocytosis. © 1994 Springer-Verlag.

The aim of this research was to evaluate the persistence of virulence characteristics of Streptococcus pyogenes cells after prolonged starvation in sea water. Studies were carried out on changes in viability, alterations in the chemical composition and surface hydrophobicity and the interaction of S. pyogenes with human polymorphonuclear leukocytes (PMN) after starvation. Results showed that surface hydrophobicity decreased progressively starting after three days of starvation and was correlated with the decrease in total carbohydrate, lipid and protein content. These values correlated with a better interaction of S. pyogenes cells with the PMN, as shown by a chemiluminescence increase that reached a peak after 32 days of starvation. Furthermore, bacterial cells became more easily phagocytized and killed by human PMN.

The aim of this study is to evaluate the effect of lipoteichoic acid (LTA) and muramic acid (MA) on costimulatory molecules CD80/CD86 on THP-1 cells and CD28/CD152 on Jurkat cells. The interactions between these molecules strongly influence the immune response through the regulation of cytokine release which, on its own, is able to regulate the immunological response by a feedback mechanism. Our results show that LTA and MA regulate expression of CD86 on macrophages while the expression of CD80 remains unmodified. LTA and MA increase the expression of CD86 on THP-1 cells, a macrophage cell line. MA increased Jurkat T cells CD152 expression.