Time is running out. No excuses to delay implementation of antimicrobial stewardship programmes: impact, sustainability, resilience and efficiency through an interrupted time series analysis (2017-2022).

Introduction: The WHO declared antimicrobial resistance (AMR) a significant concern in 2014, sparking initiatives to ensure responsible antibiotic use. In human medicine, Antimicrobial Stewardship Programmes (ASPs) in hospitals play a pivotal role in combating AMR. Although evidence supports the effectiveness of ASPs in optimizing antimicrobial use, often the lack of resources becomes an excuse to limit their dissemination and use. This paper provides a comprehensive report on a 6-year analysis of an ASP implemented in a healthcare region in north-east Italy. Methods: A retrospective data collection was conducted to assess the programme's impact on antibiotic consumption expressed as DDDs/100 patient-days, its sustainability over time, resilience during the COVID-19 pandemic and the efficiency of the ASP (relationship between workload and human resources). Results: A substantial overall reduction in antibiotic consumption (-14%), particularly in fluoroquinolones (-64%) and carbapenems (-68%), was demonstrated, showcasing the programme's impact. Sustainability was confirmed through enduring trends in antibiotic consumption and ecological analysis over time. The ASP demonstrates resilience by maintaining positive trends even amid the challenging COVID-19 pandemic. Efficiency was underscored by an increase in on-site consultations despite consistent human resources until 2021. Conclusions: This study offers insights into the prolonged success of a resource-efficient ASP, emphasizing the crucial role of long-term commitment in fostering responsible antibiotic use in the context of global health challenges such as AMR.

Dalbavancin in catheter-related bloodstream infections: a pilot study.

Background: Catheter-related bloodstream infections (CRBSI) represent a frequent complication of vascular catheterization, with high morbidity, mortality, and associated costs. Most infections are caused by Gram-positive bacteria; thus dalbavancin, a new long-acting lipoglicopeptide, may have a role in early patient discharge strategies optimizing treatment and reducing overall costs. Methods: In this small pilot feasibility study, we assessed the efficacy and safety of a "single step" treatment strategy combining dalbavancin administration (1500 mg IV single dose), catheter removal, and early discharge in adult patients admitted to medical wards in a three-year period. Results: We enrolled sixteen patients with confirmed Gram-positive CRBSI, with a mean age of 68 years and relevant comorbidities (median Charlson Comorbidity index=7). The most frequent causative agents were staphylococci, with 25% of methicillin-resistant strains, and the majority of infected devices were short term central venous catheter (CVC) and peripherally inserted central catheter (PICC). Ten out of sixteen patients had been treated empirically before dalbavancin administration. The mean time from dalbavancin administration to discharge was 2 days; none of the patients had adverse drug-related reactions; at 30- and 90-day follow-up, no patients have been readmitted to the hospital due to bacteraemia recurrence. Conclusions: Our results indicate that single-dose dalbavancin is highly effective, well-tolerated, and cost-saving for Gram-positive CRBSI.

Antimicrobial Stewardship in the Covid-19 Pandemic.

During COVID-19 pandemic, implementing and maintaining an antimicrobial stewardship protocol obtained both low rates of MDR microorganisms and low antimicrobial use in an 800-bed hospital network in northern Italy. Infectious diseases specialist consulting was crucial to maintain this protocol active.

Ceftobiprole and pneumonia in adults admitted to the emergency department is it time to assess a new therapeutic algorithm?

OBJECTIVE: Ceftobiprole is an advance generation cephalosporin which has broad-spectrum bacterial activity (both against Gram-positive and negative pathogens) and was approved for the treatment of community-acquired pneumonia (CAP) and non-ventilated hospital-acquired pneumonia (HAP) in most European countries. We aimed to evaluate the efficacy and safety of ceftobiprole in the treatment of pneumonia in a cohort of severely ill patients admitted to the emergency department (ED). METHODS: 1-year observational retrospective mono-centric study. Were defined two primary endpoints: first, to evaluate the clinical cure at the test-of-cure (TOC); the second, to evaluate the early improvement, defined as a reduction of symptoms and inflammatory parameters 72 hours after the start of treatment. The secondary endpoint is to evaluate the reduction of antibiotic "burden" using ceftobiprole despite standard of care in severe hospital-acquired pneumonia. RESULTS: During the study period, a total of 48 patients with severe pneumonia received ceftobiprole: twenty-two patients (45.8%) as empiric therapy, 9 (18.5%) as a de-escalation option from previous combination therapies, 13 patients (27.1%) as an escalation therapy from ceftriaxone or amoxicillin/clavulanate and four patients (8.3%) as a targeted therapy based on microbiological results. Ceftobiprole mean duration therapy was 10.2 days. Forty-six patients with severe pneumonia had an early clinical improvement 72 hours after the start of treatment (95.8%). In general, ceftobiprole was well tolerated; only one patient suspended the drug because of poor tolerability. The clinical cure at TOC was 85.4% and 30-days crude mortality was 10.4%. CONCLUSIONS: This study confirms that ceftobiprole is effective in severely ill patients with pneumonia at risk of poor outcomes.

Response of spontaneous plants from an ex-mining site of Elba island (Tuscany, Italy) to metal(loid) contamination.

The release of large amounts of toxic metals in the neighboring sites of abandoned mine areas represents an important environmental risk for the ecosystem, because it adversely affects soil, water, and plant growth. The aim of the present study was to investigate the metal(loid) (As, Cr, Cu, Ni, Pb, and Zn) contents of native Mediterranean plants grown on the ex-mining area of Elba island (Italy), with the prospective of its recovery by further phytoremediation technology. Soil samples were collected and characterized for metal(loid) content in total and potentially available (EDTA-extractable) fractions. Arsenic was particularly high, being 338 and 2.1 mg kg-1 as total and available fractions, respectively. Predominant native species, namely Dittrichia viscosa L. Greuter, Cistus salviifolius L., Lavandula stoechas L., and Bituminaria bituminosa L., were analyzed for metal content in the different plant organs. D. viscosa exhibited the highest metal(loid) content in the leaves and the singular behavior of translocating arsenic to the leaves (transfer factor about 2.06 and mean bioconcentration factor about 12.48). To assess the healthy status of D. viscosa plants, the leaves were investigated further. The activities of the main antioxidant enzymes and the levels of secondary metabolites linked to oxidative stress in plants from the ex-mining area were not significantly different from those of control plants, except for a lower content of carotenoids, indicating that native plants were adapted to grow in these polluted soils. These results indicate that D. viscosa can be suitable for the revegetation of highly metal-contaminated areas.

A SELDI-TOF approach to ecotoxicology: comparative profiling of low molecular weight proteins from a marine diatom exposed to CdSe/ZnS quantum dots.

Quantum dots (QDs), namely semiconductor nanocrystals, due to their particular optical and electronic properties, have growing applications in device technology, biotechnology and biomedical fields. Nevertheless, the possible threat to human health and the environment have attracted increasing attention as the production and applications of QDs increases rapidly while standard evaluation of safety lags. In the present study we performed proteomic analyses, by means of 2D gel electrophoresis and Surface Enhanced Laser Desorption Ionization-Time of Flight-Mass Spectrometry (SELDI-TOF-MS). We aimed to identify potential biomarkers of exposure to CdSe/ZnS quantum dots. The marine diatom Phaeodactylum tricornutum exposed to 2.5nM QDs was used as a model system. Both 2DE and SELDI showed the presence of differentially expressed proteins. By Principal Component Analysis (PCA) we were able to show that the differentially expressed proteins can discriminate between exposed and not exposed cells. Furthermore, a protein profile specific for exposed cells was obtained by SELDI analysis. To our knowledge, this is the first example of the application of SELDI technology to the analysis of microorganisms used as biological sentinel model of marine environmental pollution.

The response of Phaeodactylum tricornutum to quantum dot exposure: Acclimation and changes in protein expression.

Nanotechnology has a great potential to improve life and environmental quality, however the fate of nanomaterials in the ecosystems, their bioavailability and potential toxicity on living organisms are still largely unknown, mainly in the marine environment. Genomics and proteomics are powerful tools for understanding molecular mechanisms triggered by nanoparticle exposure. In this work we investigated the effect of exposure to CdSe/ZnS quantum dots (QDs) in the marine diatom Phaeodactylum tricornutum, using different physiological, biochemical and molecular approaches. The results show that acclimation to QDs reduced the growth inhibition induced by nanoparticles in P. tricornutum cultures. The increase of glutathione observed at the end of the lag phase pointed to cellular stress. Transcriptional expression of selected stress responsive genes showed up-regulation in the QD-exposed algae. A comparison of the proteomes of exposed and unexposed cells highlighted a large number of differentially expressed proteins. To our knowledge, this is the first report on proteome analysis of a marine microalga exposed to nanoparticles.

The puzzle of Italian rice origin and evolution: determining genetic divergence and affinity of rice germplasm from Italy and Asia.

The characterization of genetic divergence and relationships of a set of germplasm is essential for its efficient applications in crop breeding and understanding of the origin/evolution of crop varieties from a given geographical region. As the largest rice producing country in Europe, Italy holds rice germplasm with abundant genetic diversity. Although Italian rice varieties and the traditional ones in particular have played important roles in rice production and breeding, knowledge concerning the origin and evolution of Italian traditional varieties is still limited. To solve the puzzle of Italian rice origin, we characterized genetic divergence and relationships of 348 rice varieties from Italy and Asia based on the polymorphisms of microsatellite fingerprints. We also included common wild rice O. rufipogon as a reference in the characterization. Results indicated relatively rich genetic diversity (H(e) = 0.63-0.65) in Italian rice varieties. Further analyses revealed a close genetic relationship of the Italian traditional varieties with those from northern China, which provides strong genetic evidence for tracing the possible origin of early established rice varieties in Italy. These findings have significant implications for the rice breeding programs, in which appropriate germplasm can be selected from a given region and utilized for transferring unique genetic traits based on its genetic diversity and evolutionary relationships.

Staphylococcus aureus Efb protein expression in Nicotiana tabacum and immune response to oral administration.

Staphylococcus aureus (S. aureus) is one of the most widespread agent of diseases in humans and animals. In dairy cows, S. aureus is the most frequently isolated contagious pathogens in mastitis cases and vaccines are one of the potential tools to control the infections, thus decreasing the use of antibiotics. Among all the virulence factors produced by S. aureus, extra cellular fibrinogen binding protein (Efb) is an important one in the pathogenesis of mastitis. Plants are useful bioreactors to produce antigens and the aim of the study was the production of Efb in two cultivars of Nicotiana tabacum as a mean to produce vaccine against S. aureus in plants. A matrix attachment region (MAR) sequence was inserted near the two borders of transfer-DNA in the transformation vector in the two possible orientations. The presence of MAR elements in the transformation system significantly improved transformation efficiency and Efb protein yield up to a 2% level on total soluble protein (TSP). Mice orally immunized with transgenic lyophilized leaves produced an antigen-specific immune response.

The Arabidopsis central vacuole as an expression system for intracellular transporters: functional characterization of the Cl-/H+ exchanger CLC-7.

Functional characterization of intracellular transporters is hampered by the inaccessibility of animal endomembranes to standard electrophysiological techniques. Here, we used Arabidopsis mesophyll protoplasts as a novel heterologous expression system for the lysosomal chloride­proton exchanger CLC-7 from rat. Following transient expression of a rCLC-7:EGFP construct in isolated protoplasts, the fusion protein efficiently targeted to the membrane of the large central vacuole, the lytic compartment of plant cells. Membrane currents recorded from EGFP-positive vacuoles were almost voltage independent and showed time-dependent activation at elevated positive membrane potentials as a hallmark. The shift in the reversal potential of the current induced by a decrease of cytosolic pH was compatible with a 2Cl(-)/1H(+) exchange stoichiometry. Mutating the so-called gating glutamate into alanine (E245A) uncoupled chloride fluxes from the movement of protons, transforming the transporter into a chloride channel-like protein. Importantly, CLC-7 transport activity in the vacuolar expression system was recorded in the absence of the auxiliary subunit Ostm1, differently to recent data obtained in Xenopus oocytes using a CLC-7 mutant with partial plasma membrane expression. We also show that plasma membrane-targeted CLC-7(E245A) is non-functional in Xenopus oocytes when expressed without Ostm1. In summary, our data suggest the existence of an alternative CLC-7 operating mode, which is active when the protein is not in complex with Ostm1. The vacuolar expression system has the potential to become a valuable tool for functional studies on intracellular ion channels and transporters from animal cells.

Introgression from cultivated rice influences genetic differentiation of weedy rice populations at a local spatial scale.

Hybridization and introgression can play an important role in genetic differentiation and adaptive evolution of plant species. For example, a conspecific feral species may frequently acquire new alleles from its coexisting crops via introgression. However, little is known about this process. We analyzed 24 weedy rice (Oryza sativa f. spontanea) populations and their coexisting rice cultivars from northern Italy to study their genetic differentiation, outcrossing, and introgression based on microsatellite polymorphisms. A total of 576 maternal plants representing 24 weedy populations were used to estimate their genetic differentiation, and 5,395 progeny (seedlings) derived from 299 families of 15 selected populations were included to measure outcrossing rates. Considerable genetic differentiation (F (st) = 0.26) was detected among weedy rice populations, although the differentiation was not associated with the spatial pattern of the populations. Private alleles (28%) were identified in most populations that exhibited a multiple cluster assignments, indicating stronger genetic affinities of some weedy populations. Outcrossing rates were greatly variable and positively correlated (R (2) = 0.34, P = 0.02) with the private alleles of the corresponding populations. Paternity analysis suggested that ~15% of paternal specific alleles, a considerable portion of which was found to be crop-specific, were acquired from the introgression of the coexisting rice cultivars. Frequent allelic introgression into weedy populations resulting from outcrossing with nearby cultivars determines the private alleles of local feral populations, possibly leading to their genetic differentiation. Introgression from a crop may play an important role in the adaptive evolution of feral populations.

Thoracic radiation therapy and concomitant low-dose daily paclitaxel in non-small cell lung cancer: a phase I study.

The primary objective of the current study was to determine the maximum tolerated dose of paclitaxel (PTX) that could be added daily to radiation therapy (RT) in patients with non-small cell lung cancer (NSCLC). The secondary objective was to achieve a plasma concentration of PTX estimated to exert a radiosensitizing effect. Eighteen patients with locally advanced NSCLC were treated. 60 Gy of RT were given in 2 Gy fractions, 5 days per week. A daily dose of PTX was delivered by 4-h i.v. infusion before RT. The initial dose level of PTX was 9 mg/m2/day, escalated by 1 mg at each additional patient triplet. Two out of 6 patients experienced acute dose limiting toxicity (DLT) at the 12 mg/m2/day PTX dose level. PTX continuously greater than 10 nM, the estimated radiosensitizing condition, was achieved at the PTX dose level of 12 mg/m2/day. PTX at doses up to 11 mg/m2/day may be safely added to a conventional conformal RT course. Both DLT and the estimated radiosensitizing plasma exposure to PTX were encountered at the 12 mg/m(2)/day dose level.

Pharmacokinetic optimisation of treatment with oral etoposide.

Etoposide is a derivative of podophyllotoxin widely used in the treatment of several neoplasms, including small cell lung cancer, germ cell tumours and non-Hodgkin's lymphomas. Prolonged administration of etoposide aims for continuous inhibition of topoisomerase II, the intracellular target of etoposide, thus preventing tumour cells from repairing DNA breaks. However, the clinical advantages of extended schedules as compared with conventional short-term infusions remain unclear. Oral administration of etoposide represents the most feasible and economic strategy to maintain effective concentrations of drug for extended times. Nevertheless, the efficacy of oral etoposide therapy is contingent on circumventing pharmacokinetic limitations, mainly low and variable bioavailability. Inhibition of small bowel and hepatic metabolism of etoposide with specific cytochrome P450 inhibitors or inhibition of the intestinal P-glycoprotein efflux pump have been attempted to increase the bioavailability of oral etoposide, but the best results were obtained with daily oral administration of low etoposide doses (50-100 mg/day for 14-21 days). Saturable absorption of etoposide was reported for doses greater than 200 mg/day, whereas lower doses were associated with increased bioavailability, although they were characterised by high inter- and intrapatient variability. Pharmacokinetic parameters such as plasma trough concentration between two oral administrations (C(24,trough)), drug exposure time above a threshold value and area under the plasma concentration-time curve have been correlated with the pharmacodynamic effect of oral etoposide. Pharmacokinetic-pharmacodynamic relationships indicate that severe toxicity is avoided when peak plasma concentrations do not exceed 3-5 mg/L and C(24,trough) is under the threshold limit of 0.3 mg/L. To maintain effective etoposide plasma concentrations during prolonged oral administration, pharmacokinetic variability must be monitored in each patient, taking account of factors from many pharmacokinetic studies of etoposide, including absorption, distribution, protein binding, metabolism and elimination. Dosage reduction is generally useful to avoid haematological toxicity in patients with renal dysfunction (creatinine clearance <50 mL/min). The need for dosage adjustment based on liver function in patients with liver dysfunction is not completely defined, but generally is not indicated in patients with minor liver dysfunction. Adaptive dosage adjustment based on individual pharmacokinetic parameters, estimated using limited sampling strategies and population pharmacokinetic models, is more appropriate. This approach has been used with success in different clinical trials to increase the etoposide dosage, without significantly increasing toxicity. Various pharmacodynamic models have been proposed to guide etoposide oral dosage. However, they lack precision and accuracy and need to be refined by considering other predictor variables in order to extend their application in current clinical practice.

Vaccine antigen production in transgenic plants: strategies, gene constructs and perspectives.

Stable integration of a gene into the plant nuclear or chloroplast genome can transform higher plants (e.g. tobacco, potato, tomato, banana) into bioreactors for the production of subunit vaccines for oral or parental administration. This can also be achieved by using recombinant plant viruses as transient expression vectors in infected plants. The use of plant-derived vaccines may overcome some of the major problems encountered with traditional vaccination against infectious diseases, autoimmune diseases and tumours. They also offer a convenient tool against the threat of bio-terrorism. State of the art, experimental strategies, safety and perspectives are discussed in this article.