Quantitative Microbial Risk Assessment as support for bathing waters profiling

Profiling bathing waters supported by Quantitative Microbial Risk Assessment (QMRA) is key to the WHO's recommendations for the 2020/2021 revision of the European Bathing Water Directive. We developed an areaspecific QMRA model on four pathogens, using fecal indicator concentrations (E. coil, enterococci) for calculating pathogen loads. The predominance of illness was found to be attributable to Human Adenovirus, followed by Salmonella, Vibrio, and Norovirus. Overall, the cumulative illness risk showed a median of around 1 case/10000 exposures. The risk estimates were strongly influenced by the indicators that were used, suggesting the need for a more detailed investigation of the different sources of fecal contamination. Area-specific threshold values for fecal indicators were estimated on a risk-basis by modelling the cumulative risk against E. coll. and enterococci concentrations. To improve bathing waters assessment, we suggest considering source apportionment locally estimating of pathogen/indicator ratios, and calculating site-specific indicators thresholds based on risk assessment

Skin marks in bottlenose dolphins (Tursiops truncatus) interacting with artisanal fishery in the central Mediterranean Sea.

Skin marks occur frequently in many cetacean species across the globe revealing a broad spectrum of causes, including social interactions, infectious diseases and injuries produced by anthropogenic factors. The current study used photo-id data from 2005-2014 to estimate the skin mark pattern on resident bottlenose dolphins (Tursiops truncatus) from the Aeolian Archipelago (Italy). Thirteen skin mark types were identified and their origin, prevalence and permanence time were examined. The pattern of skin marks was assessed for the abundance, richness, distribution and severity in six body regions and compared among age classes, sex and degree of dolphins' interaction with trammel nets (DIN). Our results showed higher prevalence, abundance, richness and distribution of skin marks in adults than in the younger age classes, with the exception of black marks and white ring lesions. The prevalence and abundance of skin marks were higher in males than females, with the exception of scratches and white patches. Moreover, gunshot wounds, mutilations and irregular dorsal fin edges were found only on adult males. Since males showed higher DIN than females and, in dolphins with higher DIN, skin marks were more abundant and frequently distributed in different body regions, the skin mark pattern in regard to DIN seems to be sex-related. The more severe marks were observed on adults, males and dolphins with higher DIN, namely skin disorder, tooth rake marks, small shallow indentations, deep indentations and mutilations. On the contrary, the severity of scratches, white patches and dark ring lesions was higher in females than males, but not significantly related to DIN and age of the individuals. Our results showed that photo-id data provide an efficient and cost-effective approach to document the occurrence of skin marks in free-ranging bottlenose dolphin populations, a critical step toward understanding the cause and supporting the conservation strategies

Detection of Monkeypox Virus DNA in Airport Wastewater, Rome, Italy

Environmental surveillance can be a complementary tool for detecting pathogens circulating in communities. We detected monkeypox virus DNA in wastewater from Italy’s largest airport by using real-time PCR assays targeting the G2R region and F3L and N3R genes and sequencing. Wastewater surveillance can be quickly adapted to investigate emerging threats

In vitro anticholinergic drugs affect CD8+ peripheral blood T-cells apoptosis in COPD.

Novel pharmacological strategies are aimed at the resolution of systemic inflammation in COPD potentiating peripheral blood T-cell (PBT-cell) apoptosis. Although muscarinic acetylcholine receptors (mAChRs) M(3) and choline-acetyltransferase (ChAT) participate in the airway inflammation of COPD, their role in PBT-cell apoptosis remains unexplained. We evaluated in PBT-cells from COPD patients, smoker (S) and control (C) subjects: (1) apoptosis (by annexin V binding), (2) mAChR M(3) and ChAT expression, acetylcholine (ACh)-binding; (3) choline levels in serum and PBT-cells extracts. We tested the effects of Tiotropium (Spiriva(®)) and hemicholinium-3 (HCh-3) on apoptosis, NFκB pathway, caspases 3 and 8 activity and choline levels, in PBT-cells from COPD patients. We showed that: (1) apoptosis, mAChR M(3) and ChAT expression and the CD3+ and CD8+ ACh-binding are increased in PBT-cells from COPD patients when compared to C subjects, while CD4+/CD8+ ratio of ACh-binding to PBT cells was reduced in COPD; (2) choline levels are higher in serum and PBT-cells extracts from COPD patients than in S and C; (3) Tiotropium and HCh-3 reduced CD4+ and increased CD8+ apoptosis via caspases 3 and 8 activities and via IκB mediated mechanisms in COPD patients. This study suggests the involvement of non-neuronal components of cholinergic system in the regulation of PBT-cell apoptosis in COPD and demonstrates that Tiotropium regulates CD4+ and CD8+ PBT-cell apoptosis. It provides novel putative pharmacological targets for the resolution of systemic inflammation in COPD

β2long-acting and anticholinergic drugs synergistically control TGFβ1-mediated neutrophilic inflammation in COPD: an “in vitro” model.

TGFβ1 is involved in airway inflammation in COPD and in the regulation of muscarinic receptors (mAChRs) expression. We quantified TGFβ1 and Acetylcholine (ACh) concentrations in induced sputum supernatants (ISs) from 10 Healthy Controls (HC), 14 Healthy Smokers (HS) and 16 COPDs. We tested: 1) ISs from HC, HS and COPD on neutrophil adhesion to human bronchial epithelial cells (16HBE) in the presence or absence of an anti-TGFβ1ab or Olodaterol (β2-adrenoceptor agonist) and Tiotropium (Spiriva®) alone or in combination; 2) ISs from COPD on mAChRs expression in 16HBE in the presence or absence of anti-TGFβ1-antibody and Olodaterol; 3) hrTGFβ1on neutrophil adhesion and mChRs and ChAT expression in 16HBE. We showed that: 1) TGFβ1 and ACh concentrations are increased in ISs from COPD in comparison with HC and HS; 2) ISs from COPD caused higher levels of neutrophil adhesion to 16HBE than ISs from HC and HS and this was significantly reduced by TGFβ1 depletion and by the addition of Olodaterol or Tiotropium alone with a synergistic effect when the drugs were used in combination; 3) mAChR2, mAChR3 and ChAT expression was increased in 16HBE stimulated with ISSs from COPD and are reduced by TGFβ1 depletion while not by Olodaterol; 4) in vitro experiments we confirmed that hrTGFβ1 increases mAChR2, mAChR3 and ChAT expression. These findings suggest that TGFβ1 and mAChRs promote neutrophil adhesion to bronchial epithelial cells during airway inflammation in COPD. Olodaterol and Tiotropium used in combination might synergistically control this proinflammatory event in COPD

Coliphages as indicators of fecal contamination in wastewater treatment (SCA.Re.S. Project)

Bacteriophages are fecal indicators of viral contamination, because they are more similar to human pathogenic viruses than the traditional fecal indicator bacteria, in terms of log-reduction during wastewater treatment and persistence in the aquatic environment. The aim of this study was to evaluate the presence of bacteriophages and enteric viruses: Enterovirus, Adenovirus, Norovirus, Hepatitis A and E, Rotavirus and Pepper mild mottle virus (PMMoV), in the context of SCA.Re.S. (Evaluation of sanitary risk related to the discharge of wastewater to the ground) project. The investigation focused on a wastewater treatment plant located in an area fractured by karst in the Salento peninsula (Apulia, Italy). In autumn (September-November) 2019, water samples were monthly collected from three sites (treated wastewater, infiltration trench and monitoring well). The somatic coliphage were analyzed by standardized culture-based methods, according to BS EN ISO 10705-2:2001. Coliphage density was enumerated using plaque assay method on appropriate host bacteria (E. coli Famp) and expressed as plaque forming units PFU/100mL. Nested RT-PCR assay was used for detection of enteric viruses. The median values of coliphages were 590 PFU/100mL in treated wastewater, 1000 PFU/100mL in infiltration trench while all samples from monitoring well were under the detection limit. All samples were positive for at least one viral pathogen. PMMoV was detected only in monitoring well. The results confirmed the role of coliphages as indicators of viral contamination. Overall, we observed a gradual reduction in the concentration/occurrence of coliphages and viruses across the karst-fissured soil, until the complete removal in the monitoring well. Different soil properties are probably involved in this phenomenon such as straining, soil pores, microorganism size, and adsorption onto soil particle. Moreover, we can also hypothesize natural degradation over time, phototoxicity or ingestion by multicellular organisms in the soil and/or monitoring well water

“Cysteinyl leukotriene-1 receptor activation in a human bronchial epithelial cell line leads to signal transducer and activator of transcription 1-mediated eosinophil adhesion.”

Abstract We studied the effect of leukotriene D(4) (LTD(4)) on a human bronchial epithelial cell line (16HBE) overexpressing the cysteinyl leukotriene (CysLT) (1) receptor (HBECysLT(1)R), looking at the associated signal transduction mechanisms as well as at effects on inflammatory cell adhesion. The results obtained showed that LTD(4) increases the phosphorylation of extracellular signal-regulated protein kinase (ERK) 1/2 and of the signal transducer and activator of transcription 1 (STAT-1) in serine 727 (STAT-1Ser727), resulting in increased eosinophil adhesion to HBECysLT(1)R, associated with enhanced surface expression of intercellular adhesion molecule (ICAM) 1. Pretreatment with a CysLT(1)R-selective antagonist or with a selective inhibitor of protein kinase C (PKC) or with a selective inhibitor of the mitogen-activated protein kinase kinase (MEK) successfully suppressed both LTD(4)-induced STAT-1Ser727 phosphorylation and the associated increase in eosinophil adhesion. The use of the MEK inhibitor and of the selective CysLT(1)R antagonist in electrophoretic mobility shift assay experiments showed that LTD(4) promotes the nuclear translocation of STAT-1 through the activation of ERK1/2 pathway. The key role of STAT-1 in leukotriene D(4) transduction signaling was confirmed by RNA interference experiments, where silencing of STAT-1 expression abolished the effect of leukotriene D(4) on eosinophil adhesion. In conclusion, for the first time, we provide evidence of the involvement of STAT-1 in the signal transduction mechanism of the CysLT(1) receptor; phosphorylation of STAT-1, through PKC and ERK1/2 activation, causes enhanced ICAM-1 surface expression and eosinophil adhesion. Effective CysLT(1)R antagonism may therefore contribute to the control of the chronic inflammatory condition that characterizes human airways in asthma

Cigarette smoke extract activates human bronchial epithelial cells affecting non-neuronal cholinergic system signalling in vitro.

AIMS: Acetylcholine (ACh) is synthesized by Choline Acetyl-Transferase (ChAT) that exerts its physiological effects in airway epithelial cells via muscarinic receptor (MR) activation. We evaluate the effect of ACh stimulation on human bronchial epithelial cells (16-HBE) and test whether cigarette smoke extract (CSE) can modify the basal cellular response to ACh affecting the non-neuronal cholinergic system signalling. MAIN METHODS: ACh stimulated 16-HBE were tested for ACh-binding, Leukotriene B(4) (LTB(4)) release and ERK1/2 and NFkB pathway activation. Additionally, we investigated all the aforementioned parameters as well as ChAT and MR proteins and mRNA expression and endogenous ACh production in CSE-treated 16-HBE. KEY FINDINGS: We showed that ACh induced in 16-HBE, in a concentration-dependent manner, LTB(4) release via the activation of ERK1/2 and NFkB pathways. The addition of Tiotropium (Spiriva®), Gallamine, Telenzepine and 4-DAMP (muscarinic receptor antagonists), as well as of PD 098059 (MAPKK inhibitor) and BAY117082 (inhibitor of IkBα phosphorilation), down-regulated the ACh-induced effects. Additionally, CSE treatment of 16-HBE increased the binding of ACh, and shifted the LTB4 release from the concentration ACh 1μM to 10nM. Finally, we observed that the treatment of 16-HBE with CSE increased the expression of ChAT, M(2) and M(3) and of endogenous ACh production in 16-HBE. Tiotropium regulated the LTB4 release and ACh production in CSE treated 16-HBE. SIGNIFICANCE: CSE increases the pro-inflammatory activity of human bronchial epithelial cells, and promotes the cellular response to lower concentrations of ACh, by affecting the expression of ChAT and MRs. Tiotropium might prevent pro-inflammatory events generated by ACh together with CSE