Alginate microparticles containing silver@hydroxyapatite functionalized calcium carbonate composites.

This paper focuses on the preparation and characterization of antibacterial alginate microparticles containing silver@hydroxyapatite functionalized calcium carbonate composites for tissue engineering. Microparticles were prepared by cross-linking a silver@composite sodium alginate dispersion with CaCl2. This method showed a very good silver efficiency loading and the presence of silver chloride nanoparticles was detected. Silver free microparticles, containing hydroxyapatite functionalized calcium carbonates and neat alginate microparticles were prepared as well. All microparticles were characterized for water absorption and for in vitro bioactivity by immersion in simulated body fluid (SBF). Finally, antimicrobial and antibiofilm activities as well as cytotoxicity were evaluated. Microparticles containing silver@composites exhibited good antimicrobial and antibiofilm activities against Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa and Candida albicans, but exerted a certain cytotoxicity against the tested cell models (fibroblasts and osteoblasts). Microparticles containing hydroxyapatite functionalized calcium carbonates were found to be always less cytotoxic, also in comparison to neat alginate microparticles, proving that the presence of the inorganic matrices exerts a protective effect on microparticle cytotoxicity.

A novel black poplar propolis extract with promising health-promoting properties: focus on its chemical composition, antioxidant, anti-inflammatory, and anti-genotoxic activities.

Propolis is a resinous mixture produced by honeybees which has been used since ancient times for its useful properties. However, its chemical composition and bioactivity may vary, depending on the geographical area of origin and the type of tree bees use for collecting pollen. In this context, this research aimed to investigate the total phenolic content (using the Folin-Ciocalteu assay) and the total antioxidant capacity (using the FRAP, DPPH, and ABTS assays) of three black poplar (Populus nigra L.) propolis (BPP) solutions (S1, S2, and S3), as well as the chemical composition (HPLC-ESI-MSn) and biological activities (effect on cell viability, genotoxic/antigenotoxic properties, and anti-inflammatory activity, and effect on ROS production) of the one which showed the highest antioxidant activity (S1). The hydroalcoholic BPP solution S1 was a prototype of an innovative, research-type product by an Italian nutraceutical manufacturer. In contrast, hydroalcoholic BPP solutions S2 and S3 were conventional products purchased from local pharmacy stores. For the three extracts, 50 phenolic compounds, encompassing phenolic acids and flavonoids, were identified. In summary, the results showed an interesting chemical profile and the remarkable antioxidant, antigenotoxic, anti-inflammatory and ROS-modulating activities of the innovative BPP extract S1, paving the way for future research. In vivo investigations will be a possible line to take, which may help corroborate the hypothesis of the potential health benefits of this product, and even stimulate further ameliorations of the new prototype.

Efficacy of Violet-Blue (405 nm) LED Lamps for Disinfection of High-Environmental-Contact Surfaces in Healthcare Facilities: Leading to the Inactivation of Microorganisms and Reduction of MRSA Contamination.

Effective disinfection procedures in healthcare facilities are essential to prevent transmission. Chemical disinfectants, hydrogen peroxide vapour (HPV) systems and ultraviolet (UV) light are commonly used methods. An emerging method, violet-blue light at 405 nm, has shown promise for surface disinfection. Its antimicrobial properties are based on producing reactive oxygen species (ROS) that lead to the inactivation of pathogens. Studies have shown significant efficacy in reducing bacterial levels on surfaces and in the air, reducing nosocomial infections. The aim of this study was to evaluate the antimicrobial effectiveness of violet-blue (405 nm) LED lamps on high-contact surfaces in a hospital infection-control laboratory. High-contact surfaces were sampled before and after 7 days of exposure to violet-blue light. In addition, the effect of violet-blue light on MRSA-contaminated surfaces was investigated. Exposure to violet-blue light significantly reduced the number of bacteria, yeasts and moulds on the sampled surfaces. The incubator handle showed a low microbial load and no growth after irradiation. The worktable and sink showed an inconsistent reduction due to shaded areas. In the second experiment, violet-blue light significantly reduced the microbial load of MRSA on surfaces, with a greater reduction on steel surfaces than on plastic surfaces. Violet-blue light at 405 nm has proven to be an effective tool for pathogen inactivation in healthcare settings Violet-blue light shows promise as an additional and integrated tool to reduce microbial contamination in hospital environments but must be used in combination with standard cleaning practices and infection control protocols. Further research is needed to optimise the violet-blue, 405 nm disinfection method.

Ag/Ag3PO4 Nanoparticle-Decorated Hydroxyapatite Functionalized Calcium Carbonate: Ultrasound-Assisted Sustainable Synthesis, Characterization, and Antimicrobial Activity.

Silver nanoparticles are usually prepared by the reduction of silver cations through chemical and non-sustainable procedures that involve the use of reducing chemical agents. Therefore, many efforts have been made in the search for sustainable alternative methods. Among them, an ultrasound-assisted procedure could be a suitable and sustainable method to afford well-dispersed and nanometric silver particles. This paper describes a sustainable, ultrasound-assisted method using citrate as a reducing agent to prepare silver@hydroxyapatite functionalized calcium carbonate composites. For comparison, an ultrasound-assisted reduction was performed in the presence of NaBH4. The composites obtained in the presence of these two different reducing agents were compared in terms of nanoparticle nature, antimicrobial activity, and cytotoxic activity. The nanoparticle nature was investigated by several techniques, including X-ray powder diffraction, field-emission scanning electron microscopy, transmission electron microscopy, UV-Vis spectroscopic measurements, and X-ray photoemission spectroscopy. Nanoparticles with a predominance of Ag or Ag3PO4 were obtained according to the type of reducing agent used. All composites were tested for antimicrobial and antibiofilm activities against Gram-positive and Gram-negative (Staphylococcus aureus and Pseudomonas aeruginosa, respectively) bacteria and for cytotoxicity towards human skin keratinocytes and human fibroblasts. The nature of the nanoparticles, Ag or Ag3PO4, and their predominance seemed to affect the in vitro silver release and the antimicrobial and antibiofilm activities. The composites obtained by the citrate-assisted reduction gave rise to the best results.

Alginate Ag/AgCl Nanoparticles Composite Films for Wound Dressings with Antibiofilm and Antimicrobial Activities.

Recently, silver-based nanoparticles have been proposed as components of wound dressings due to their antimicrobial activity. Unfortunately, they are cytotoxic for keratinocytes and fibroblasts, and this limits their use. Less consideration has been given to the use of AgCl nanoparticles in wound dressings. In this paper, a sustainable preparation of alginate AgCl nanoparticles composite films by simultaneous alginate gelation and AgCl nanoparticle formation in the presence of CaCl2 solution is proposed with the aim of obtaining films with antimicrobial and antibiofilm activities and low cytotoxicity. First, AgNO3 alginate films were prepared, and then, gelation and nanoparticle formation were induced by film immersion in CaCl2 solution. Films characterization revealed the presence of both AgCl and metallic silver nanoparticles, which resulted as quite homogeneously distributed, and good hydration properties. Finally, films were tested for their antimicrobial and antibiofilm activities against Staphylococcus epidermidis (ATCC 12228), Staphylococcus aureus (ATCC 29213), Pseudomonas aeruginosa (ATCC 15692), and the yeast Candida albicans. Composite films showed antibacterial and antibiofilm activities against the tested bacteria and resulted as less active towards Candida albicans. Film cytotoxicity was investigated towards human dermis fibroblasts (HuDe) and human skin keratinocytes (NCTC2544). Composite films showed low cytotoxicity, especially towards fibroblasts. Thus, the proposed sustainable approach allows to obtain composite films of Ag/AgCl alginate nanoparticles capable of preventing the onset of infections without showing high cytotoxicity for tissue cells.

Antimicrobial Oleogel Containing Sustainably Prepared Silver-Based Nanomaterials for Topical Application.

Oleogels containing silica-silver-based nanomaterials were prepared to be used as potential antimicrobial treatment for preventing and curing skin infections. Fumed silica was used as a bifunctional excipient able to offer support to silver-based nanoparticle growth and act as a gelling agent for oleogel formulation. First, silica-silver composites were prepared following a sustainable method by contact of fumed silica and silver nitrate in the presence of ethanol and successive UV irradiation. The composites were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), ATR FT-IR spectroscopy and UV-Vis spectrophotometry. The presence of 8-20 nm spherical nanoparticles, in addition to the silica aggregates and AgNO3 crystals, was detected. The composites showed good antimicrobial activity against the Gram-negative Pseudomonas aeruginosa and the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis. Thus, they were formulated in an oleogel, obtained using fumed silica as a gelling agent. For comparison, oleogels containing AgNO3 were prepared according to two different formulative techniques. The silica-silver-based oleogels showed good antimicrobial activity and did not show cytotoxic effects for fibroblasts and keratinocytes.

Bud-Poplar-Extract-Embedded Chitosan Films as Multifunctional Wound Healing Dressing.

Wounds represent a major global health challenge. Acute and chronic wounds are sensitive to bacterial infection. The wound environment facilitates the development of microbial biofilms, delays healing, and promotes chronic inflammation processes. The aim of the present work is the development of chitosan films embedded with bud poplar extract (BPE) to be used as wound dressing for avoiding biofilm formation and healing delay. Chitosan is a polymer with antimicrobial and hydrating properties used in wound dressing, while BPE has antibacterial, antioxidative, and anti-inflammatory properties. Chitosan-BPE films showed good antimicrobial and antibiofilm properties against Gram-positive bacteria and the yeast Candida albicans. BPE extract induced an immunomodulatory effect on human macrophages, increasing CD36 expression and TGFß production during M1/M2 polarization, as observed by means of cytofluorimetric analysis and ELISA assay. Significant antioxidant activity was revealed in a cell-free test and in a human neutrophil assay. Moreover, the chitosan-BPE films induced a good regenerative effect in human fibroblasts by in vitro cell migration assay. Our results suggest that chitosan-BPE films could be considered a valid plant-based antimicrobial material for advanced dressings focused on the acceleration of wound repair.

Drug efflux transporters in Staphylococcus pseudintermedius: in silico prediction and characterization of resistance.

OBJECTIVES: To perform an in silico prediction of drug efflux pumps (EPs) in Staphylococcus pseudintermedius and investigate their role in conferring resistance to antibiotic and biocidal agents and biofilm formation. METHODS: A S. pseudintermedius efflux mutant was obtained by stimulating an isogenic line (ATCC 49444) with increasing concentrations of an efflux system substrate. Changes in antimicrobial susceptibility and biofilm-forming capability were evaluated in the presence/absence of the EP inhibitors (EPIs) thioridazine and reserpine and the efflux activity was assayed by fluorometry. Homologues of EPs of Staphylococcus aureus and Staphylococcus epidermidis were searched by exploratory GenBank investigations. Gene expression analyses and sequencing were then conducted on selected genes. RESULTS: Susceptibility to chlorhexidine, gentamicin and ciprofloxacin, but not enrofloxacin, was affected by the increased efflux and it was variably restored by the EPIs. The efflux mutant showed much greater biofilm formation that the original strain, which was significantly inhibited by thioridazine and reserpine at MIC/2. A high expression of norA, which was mgrA-independent, was found in the S. pseudintermedius efflux mutant, apparently regulated by an 11 bp deletion in its promoter region, whilst lmrB was transitorily overexpressed. icaA, which encodes the polysaccharide intercellular adhesin forming the extracellular matrix of staphylococcal biofilm, was also up-regulated. CONCLUSIONS: EPs, particularly NorA, are supposed to have complex involvement in multiple stages of resistance development. Overexpression of EPs appears to be correlated with a remarkable increase of S. pseudintermedius biofilm production; however, the regulatory mechanisms remain to be explored.

Overcoming Antibiotic Resistance: Playing the 'Silver Nanobullet' Card.

Enhancing the antibacterial activity of old antibiotics by a multitarget approach, such as combining antibiotics with metal nanoparticles, is a valuable strategy to overcome antibacterial resistance. In this work, the synergistic antimicrobial effect of silver nanoparticles and antibiotics, immobilized on a solid support, was investigated. Nanometric layered double hydroxides (LDH) based on Zn(II) and Al(III) were prepared by the double microemulsion technique. The dual function of LDH as an anionic exchanger and support for metal nanoparticles was exploited to immobilize both silver and antibiotics. Cefazolin (CFZ), a ß-lactam, and nalidixic acid (NAL), a quinolone, were selected and intercalated into LDH obtaining ZnAl-CFZ and ZnAl-NAL samples. These samples were used for the growth of silver nanoparticles with dimension ranging from 2.5 to 8 nm. Silver and antibiotics release profiles, from LDH loaded with antibiotics and Ag/antibiotics, were evaluated in two different media: water and phosphate buffer. Interestingly, the release profiles are affected by both the acceptor media and the presence of silver. The synergistic antibacterial activity of LDH containing both silver and antibiotics were investigated on gram-positives (Staphylococcus aureus and Streptococcus pneumoniae) and gram-negatives (Pseudomonas aeruginosa) and compared with the plain antimicrobials and LDH containing only antibiotics or silver.

Effect of a UV-C Automatic Last-Generation Mobile Robotic System on Multi-Drug Resistant Pathogens.

BACKGROUND: Healthcare-associated infections caused by multi-drug resistant (MDR) pathogens are associated with increased mortality and morbidity among hospitalized patients. Inanimate surfaces, and in particular high-touch surfaces, have often been described as the source for outbreaks of nosocomial infections. The present work aimed to evaluate the efficacy of a last-generation mobile (robotic) irradiation UV-C light device R2S on MDR microorganisms in inanimate surfaces and its translation to hospital disinfection. METHODS: The efficacy of R2S system was evaluated in environmental high-touch surfaces of two separate outpatient rooms of Perugia Hospital in Italy. The static UV-C irradiation effect was investigated on both the bacterial growth of S. aureus, MRSA, P. aeruginosa, and K. pneumoniae KPC and photoreactivation. The antimicrobial activity was also tested on different surfaces, including glass, steel, and plastic. RESULTS: In the environmental tests, the R2S system decreased the number of bacteria, molds, and yeasts of each high-touch spot surface (HTSs) compared with manual sanitization. UV-C light irradiation significantly inhibits in vitro bacterial growth, also preventing photoreactivation. UV-C light bactericidal activity on MDR microorganisms is affected by the type of materials of inanimate surfaces. CONCLUSIONS: The last-generation mobile R2S system is a more reliable sanitizing procedure compared with its manual counterpart.

Solid State Photoreduction of Silver on Mesoporous Silica to Enhance Antifungal Activity.

A solid-state Ultraviolet-photoreduction process of silver cations to produce Ag0 nanostructures on a mesoporous silica is presented as an innovative method for the preparation of efficient environmental anti-fouling agents. Mesoporous silica powder, contacted with AgNO3, is irradiated at 366 nm, where silica surface defects absorb. The detailed characterization of the materials enables us to document the silica assisted photo-reduction. The appearance of a Visible (Vis) band centered at 470 nm in the extinction spectra, due to the surface plasmon resonance of Ag0 nanostructures, and the morphology changes observed in transmission electron microscopy (TEM) images, associated with the increase of Ag/O ratio in energy dispersive X-ray (EDX) analysis, indicate the photo-induced formation of Ag0. The data demonstrate that the photo-induced reduction of silver cation occurs in the solid state and takes place through the activation of silica defects. The activation of the materials after UV-processing is then tested, evaluating their antimicrobial activity using an environmental filamentous fungus, Aspergillus niger. The treatment doubled inhibitory capacity in terms of minimal inhibitory concentration (MIC) and biofilm growth. The antimicrobial properties of silver-silica nanocomposites are investigated when dispersed in a commercial sealant; the nanocomposites show excellent dispersion in the silicon and improve its anti-fouling capacity.

Ethidium bromide exposure unmasks an antibiotic efflux system in Rhodococcus equi.

BACKGROUND: This study introduces a newly created strain (Rhodococcus equiEtBr25) by exposing R. equi ATCC 33701 to ethidium bromide (EtBr), a substrate for MDR transporters. Such an approach allowed us to investigate the resulting phenotype and genetic mechanisms underlying the efflux-mediated resistance in R. equi. METHODS: R. equi ATCC 33701 was stimulated with increasing concentrations of EtBr. The antimicrobial susceptibility of the parental strain and R. equiEtBr25 was investigated in the presence/absence of efflux pump inhibitors (EPIs). EtBr efflux was evaluated by EtBr-agar method and flow cytometry. The presence of efflux pump genes was determined by conventional PCR before to quantify the expression of 30 genes coding for membrane transporters by qPCR. The presence of erm(46) and mutations in 23S rRNA, and gyrA/gyrB was assessed by PCR and DNA sequencing to exclude the occurrence of resistance mechanisms other than efflux. RESULTS: R. equi EtBr25 showed an increased EtBr efflux. Against this strain, the activity of EtBr, azithromycin and ciprofloxacin was more affected than that of rifampicin and azithromycin/rifampicin combinations. Resistances were reversed by combining the antimicrobials with EPIs. Gene expression analysis detected a marked up-regulation of REQ_RS13460 encoding for a Major Facilitator Superfamily (MFS) transporter. G→A transition occurred in the transcriptional repressor tetR/acrR adjacent to REQ_RS13460. CONCLUSIONS: Exposure of R. equi to EtBr unmasked an efflux-mediated defence against azithromycin and ciprofloxacin, which seemingly correlates with the overexpression of a specific MFS transporter. This genotype may mirror an insidious low-level resistance of clinically important isolates that could be countered by EPI-based therapies.

Evaluation of Lumipulse® G SARS-CoV-2 antigen assay automated test for detecting SARS-CoV-2 nucleocapsid protein (NP) in nasopharyngeal swabs for community and population screening.

OBJECTIVES: To compare the Lumipulse® SARS-CoV-2 antigen test with the gold standard real-time reverse transcription-polymerase chain reaction (RT-PCR) for diagnosis of SARS-CoV-2 infection and to evaluate its role in screening programs. METHODS: Lumipulse® SARS-CoV-2 antigen assay was compared with the gold standard RT-PCR test in a selected cohort of 226 subjects with suspected SARS-CoV-2 infection, and its accuracy was evaluated. Subsequently, the test was administered to a real-life screening cohort of 1738 cases. ROC analysis was performed to explore test features and cutoffs. All tests were performed in the regional reference laboratory in Umbria, Italy. RESULTS: A 42.0% positive result at RT-PCR was observed in the selected cohort. The Lumipulse® system showed 92.6% sensitivity (95% CI 85.4-97.0%) and 90.8% specificity (95% CI 84.5-95.2%) at 1.24 pg/mL optimal cutoff. In the screening cohort, characterized by 5.2% prevalence of infection, Lumipulse® assay showed 100% sensitivity (95% CI 96.0-100.0%) and 94.8% specificity (95% CI 93.6-95.8%) at 1.645 pg/mL optimal cutoff; the AUC was 97.4%, NPV was 100% (95% CI 99.8-100.0%) and PPV was 51.1% (95% CI 43.5-58.7%). CONCLUSIONS: The Lumipulse® SARS-CoV-2 antigen assay can be safely employed in the screening strategies in small and large communities and in the general population.

Bioinspired Reactive Interfaces Based on Layered Double Hydroxides-Zn Rich Hydroxyapatite with Antibacterial Activity.

This work is focused on the preparation and multi-technique characterization of potentially biocompatible reactive interfaces obtained by combining layered double hydroxides (LDHs) and hydroxyapatite (HA). Antimicrobial and osteoinductive metallic ions as Zn2+ and Ga3+ were chosen as intralayer constituents of LDH to obtain ZnAl and ZnAlGa systems. These LDHs, exchanged with dihydrogenphosphate anions, promoted the precipitation of HA on the LDH surface yielding HA@LDH composites. X-ray diffraction quantitative analysis, through the Rietveld refinement method, coupled with elemental analysis and micro-Raman spectroscopy showed the formation of a mixed Ca-Zn HA phase. Scanning electron microscopy revealed that HA, in the presence of LDH, grew preferentially along its a-axis, thus crystallizing mainly in the form of flake crystals. LDH and HA@LDH composites showed antibacterial activity against Staphylococcus aureus and Pseudomonas aeruginosa at not cytotoxic concentrations for human osteoblasts (hFob 1.19), especially when Ga cations were present in the LDH structure. The effect of the presence of HA in the composites on the bone-bonding ability and on human osteoblast proliferation was also investigated. The HA seemed to reduce the toxicity of the LDH toward human osteoblast while did not affect the bone-bonding ability. This multidisciplinary study provides the bio-chemical, structural characterization of new LDH and HA@LDH composites, evaluating also their bioactivity to be potentially applicable to titanium-based prostheses.

Biofunctionalization of Poly(lactide-co-glycolic acid) Using Potent NorA Efflux Pump Inhibitors Immobilized on Nanometric Alpha-Zirconium Phosphate to Reduce Biofilm Formation.

Polymeric composites, where bioactive species are immobilized on inorganic nanostructured matrix, have received considerable attention as surfaces able to reduce bacterial adherence, colonization, and biofilm formation in implanted medical devices. In this work, potent in-house S. aureus NorA efflux pump inhibitors (EPIs), belonging to the 2-phenylquinoline class, were immobilized on nanometric alpha-zirconium phosphate (ZrP) taking into advantage of acid-base or intercalation reactions. The ZrP/EPI were used as filler of poly(lactide-co-glycolic acid) (PLGA) to obtain film composites with a homogeneous distribution of the ZrP/EPI fillers. As reference, PLGA films loaded with ZrP intercalated with thioridazine (TZ), that is recognized as both a NorA and biofilm inhibitor, and with the antibiotic ciprofloxacin (CPX) were prepared. Composite films were characterized by X-ray diffraction, scanning electron microscopy, and thermogravimetric analysis. The ability of the composite films, containing ZrP/EPI, to inhibit biofilm formation was tested on Staphylococcus aureus ATCC 29213 and Staphylococcus epidermidis ATCC 12228, and it was compared with that of the composite loaded with ZrP/TZ. Finally, the antibacterial activity of CPX intercalated in ZrP was evaluated when used in combination with ZrP/EPI in the PLGA films.

Accelerate Pheno™ blood culture detection system: a literature review.

Accelerate Pheno™ (ACC) is a fully automated system providing rapid identification of a panel of bacteria and yeasts, and antimicrobial susceptibility testing of common bacterial pathogens responsible for bloodstream infections and sepsis. Diagnostic accuracy for identification ranges from 87.9 to 100%, and antimicrobial susceptibility testing categorical agreement is higher than 91%. The present review includes peer-reviewed studies on ACC published to date. Both interventional and hypothetical studies evidenced the potential positive clinical role of ACC in the management and therapy of patients with bloodstream infections and sepsis, due to the important reduction in time to report, suggesting a crucial impact on the therapeutic management of these patients, provided the presence of a hospital antimicrobial stewardship program, a 24/7 laboratory operating time and a strict collaboration between clinical microbiologist and clinician. Further prospective multicenter studies are necessary to explore the impact of this system on mortality, length of stay and spread of multidrug-resistant organisms.

Biocompatible alginate silica supported silver nanoparticles composite films for wound dressing with antibiofilm activity.

Alginate films containing pyrogenic silica supported silver nanoparticles were prepared as potential wound dressings. First silica supported silver nanoparticles (CAB-O-SIL-Ag) were prepared via solid state sintering route without the use of any solvent and reducing agent. The obtained composite was characterized by X-ray powder diffraction, transmission electron microscopy and UV-vis spectroscopy which evidenced the presence of 8-20 nm spherical silver nanoparticles uniformly distributed and grown on the silica surface. Then the CAB-O-SIL-Ag was used as filler to prepare alginate films by casting method and successive gelation. Films with two different silver concentrations were prepared. They showed good hydration properties and a very slow silver release. Films exhibited antimicrobial and antibiofilm activities against Staphylococcus aureus and Pseudomonas aeruginosa and showed no cytotoxicity towards human skin keratinocytes and human fibroblasts HuDe.

Glutathione S-transferase P influences the Nrf2-dependent response of cellular thiols to seleno-compounds.

Recent findings suggest a functional interaction of the drug resistance enzyme glutathione S-transferase P (GSTP) with the transcription factor Nrf2, a master regulator of the adaptive stress response to cellular electrophiles. The effect of this interaction on the metabolism and redox of cellular thiols was investigated in this study during the exposure to alkylating Se-compounds in murine embryonic fibroblasts (MEFs). GSTP1-1 gene ablation was confirmed to upregulate Nrf2 activity and to increase Cys uptake and the de novo biosynthesis of reduced glutathione (GSH) that was readily released in the extracellular medium together with other cellular thiols. This latter response was associated with a higher expression of the membrane transporter MRP1 and was markedly stimulated by the treatment with alkylating Se-compounds together with protein S-glutathionylation that was observed to be under the influence of  GSTP expression. The response of cellular thiols to Se-compounds was not altered by the transient (SiRNA-induced) or stable inactivation of NRF2 in GSTP competent or hGSTP1 transfected cells, while defects of GSH biosynthesis, efflux, and redox were observed after NRF2 silencing in GSTP-/- MEFs. In conclusion, GSTP is confirmed to functionally interact with Nrf2 and to have a prominent position in the pecking order of factors that control both the Nrf2-dependent and independent response of cellular thiols to alkylating agents.

C-2 phenyl replacements to obtain potent quinoline-based Staphylococcus aureus NorA inhibitors.

NorA is the most studied efflux pump of Staphylococcus aureus and is responsible for high level resistance towards fluoroquinolone drugs. Although along the years many NorA efflux pump inhibitors (EPIs) have been reported, poor information is available about structure-activity relationship (SAR) around their nuclei and reliability of data supported by robust assays proving NorA inhibition. In this regard, we focussed efforts on the 2-phenylquinoline as a promising chemotype to develop potent NorA EPIs. Herein, we report SAR studies about the introduction of different aryl moieties on the quinoline C-2 position. The new derivative 37a showed an improved EPI activity (16-fold) with respect to the starting hit 1. Moreover, compound 37a exhibited a high potential in time-kill curves when combined with ciprofloxacin against SA-1199B (norA+). Also, 37a exhibited poor non-specific effect on bacterial membrane polarisation and showed an improvement in terms of "selectivity index" in comparison to 1.

Phenotypic Characterization of Rhodococcus equi Biofilm Grown In Vitro and Inhibiting and Dissolving Activity of Azithromycin/Rifampicin Treatment.

Microbial biofilm has been implicated in a wide range of chronic infections. In spite of the fact that Rhodococcus equi is a recognized cause of chronic disease in animals and humans, few studies have focused on the sessile phenotype of R. equi. The aim of this research was to phenotypically characterize the biofilm development of R. equi and its answerability for hypo-responsiveness to macrolides and rifampicin. Biofilm formation is initiated by bacterial adhesion to the surface. In this work, the ability of R. equi to adhere to the surface of human lung epithelial cells was detected by a fluorometric adhesion test performed on 40 clinical isolates. Subsequently, the capability of R. equi to produce biofilm was investigated by colorimetric, fluorescence and scanning electron microscopy analysis, revealing a general slow growth of rhodococcal biofilm and different sessile phenotypes among field isolates, some also including filamented bacteria. Azithromycin treatment produced a higher long-term inhibition and dissolution of R. equi biofilms than rifampicin, while the two antibiotics combined boosted the anti-biofilm effect in a statistically significant manner, although this was not equally effective for all R. equi isolates. Increasing the MIC concentrations of drugs tenfold alone and in combination did not completely eradicate pre-formed R. equi biofilms, while a rifampicin-resistant isolate produced an exceptionally abundant extracellular matrix. These results have strengthened the hypothesis that biofilm production may occur as an antibiotic tolerance system in R. equi, potentially determining persistence and, eventually, chronic infection.