Is Silver Addition to Scaffolds Based on Polycaprolactone Blended with Calcium Phosphates Able to Inhibit Candida albicans and Candida auris Adhesion and Biofilm Formation?

Candida spp. periprosthetic joint infections are rare but difficult-to-treat events, with a slow onset, unspecific symptoms or signs, and a significant relapse risk. Treatment with antifungals meets with little success, whereas prosthesis removal improves the outcome. In fact, Candida spp. adhere to orthopedic devices and grow forming biofilms that contribute to the persistence of this infection and relapse, and there is insufficient evidence that the use of antifungals has additional benefits for anti-biofilm activity. To date, studies on the direct antifungal activity of silver against Candida spp. are still scanty. Additionally, polycaprolactone (PCL), either pure or blended with calcium phosphate, could be a good candidate for the design of 3D scaffolds as engineered bone graft substitutes. Thus, the present research aimed to assess the antifungal and anti-biofilm activity of PCL-based constructs by the addition of antimicrobials, for instance, silver, against C. albicans and C. auris. The appearance of an inhibition halo around silver-functionalized PCL scaffolds for both C. albicans and C. auris was revealed, and a significant decrease in both adherent and planktonic yeasts further demonstrated the release of Ag+ from the 3D constructs. Due to the combined antifungal, osteoproliferative, and biodegradable properties, PCL-based 3D scaffolds enriched with silver showed good potential for bone tissue engineering and offer a promising strategy as an ideal anti-adhesive and anti-biofilm tool for the reduction in prosthetic joints of infections caused by Candida spp. by using antimicrobial molecule-targeted delivery.

In Vitro Antifungal Activity of Selected Essential Oils against Drug-Resistant Clinical Aspergillus spp. Strains.

BACKGROUND: Treatment options for aspergillosis include amphotericin B (AMB) and azole compounds, such as itraconazole (ITZ). However, serious side effects related to these antifungal agents are increasingly evident, and resistance continues to increase. Currently, a new trend in drug discovery to overcome this problem is represented by natural products from plants, or their extracts. Particularly, there is a great interest in essential oils (EOs) recognized for their antimicrobial role towards bacteria, fungi and viruses. METHODS: In this study, we evaluated the antifungal activity of eleven commercial EOs-clove, eucalyptus, geranium, hybrid lavender, lavender, lemon, lemongrass, neroli, oregano, tea tree and red red thyme-in comparison with AMB and ITZ against Aspergillus flavus, A. fumigatus and A. niger clinical isolates. Antifungal activity was determined by broth microdilution method, agar diffusion technique, fungistatic and fungicidal activities and vapor contact assay. RESULTS: Gas chromatography-mass spectrometry analysis displayed two groups of distinct biosynthetical origin: monoterpenes dominated the chemical composition of the most oils. Only two aromatic compounds (eugenol 78.91% and eugenyl acetate 11.64%) have been identified as major components in clove EO. Lemongrass EO exhibits the strongest antimicrobial activity with a minimum inhibitory concentration of 0.56 mg/mL and a minimum fungicidal concentration of 2.25-4.5 mg/mL against Aspergillus spp. strains. Clove and geranium EOs were fairly effective in inhibiting Aspergillus spp. growth. CONCLUSIONS: These results demonstrate the antimicrobial potential of some EOs and support the research of new alternatives or complementary therapies based on EOs.

Tuning of Silver Content on the Antibacterial and Biological Properties of Poly(ɛ-caprolactone)/Biphasic Calcium Phosphate 3D-Scaffolds for Bone Tissue Engineering.

There is a growing interest in tissue engineering, in which biomaterials play a pivotal role in promoting bone regeneration. Furthermore, smart functionalization can provide biomaterials with the additional role of preventing orthopedic infections. Due to the growing microbial resistance to antimicrobials used to treat those infections, metal ions, such as silver, thanks to their known wide range of bactericidal properties, are believed to be promising additives in developing antibacterial biomaterials. In this work, novel poly(ε-caprolactone) (PCL)-based 3D scaffolds have been designed and developed, where the polymer matrix was modified with both silver (Ag), to supply antibacterial behavior, and calcium phosphates (biphasic calcium phosphate, BCP) particles to impart bioactive/bioresorbable properties. The microstructural analysis showed that constructs were characterized by square-shaped macropores, in line with the morphology and size of the templating salts used as pore formers. Degradation tests demonstrated the important role of calcium phosphates in improving PCL hydrophilicity, leading to a higher degradation degree for BCP/PCL composites compared to the neat polymer after 18 days of soaking. The appearance of an inhibition halo around the silver-functionalized PCL scaffolds for assayed microorganisms and a significant (p < 0.05) decrease in both adherent and planktonic bacteria demonstrate the Ag+ release from the 3D constructs. Furthermore, the PCL scaffolds enriched with the lowest silver percentages did not hamper the viability and proliferation of Saos-2 cells. A synergic combination of antimicrobial, osteoproliferative and biodegradable features provided to 3D scaffolds the required potential for bone tissue engineering, beside anti-microbial properties for reduction in prosthetic joints infections.

Facile One-Step Electrospinning Process to Prepare AgNPs-Loaded PLA and PLA/PEO Mats with Antibacterial Activity.

Nanofibers can play an important role in developing new kinds of medical applications. Poly(lactic acid) (PLA) and PLA/poly(ethylene oxide) (PEO) antibacterial mats containing silver nanoparticles (AgNPs) were prepared by a simple one-step electrospinning method that allows AgNPs to be synthesized simultaneously with the preparation of the electrospinning solution. The electrospun nanofibers were characterized by scanning electron microscopy, transmission electron microscopy and thermogravimetry, while silver release over time was monitored by inductively coupled plasma/optical emission spectroscopy. The antibacterial activity was tested against Staphylococcus epidermidis and Escherichia coli by colony forming unit (CFU) count on agar after 15, 24 and 48 h of incubation. AgNPs were found to be mainly concentrated in the PLA nanofiber core, and the mats showed steady but slow Ag release in the short term; in contrast, AgNPs were uniformly distributed in the PLA/PEO nanofibers, which released up to 20% of their initial silver content in 12 h. A significant (p < 0.05) antimicrobial effect towards both tested bacteria, highlighted by a reduction in the CFU/mL counts, was observed for the nanofibers of PLA and PLA/PEO embedded with AgNPs, with a stronger effect exerted by the latter, confirming the more efficient silver release from these samples. The prepared electrospun mats may have good potential for use in the biomedical field, particularly in wound dressing applications, where a targeted delivery of the antimicrobial agent is highly desirable to avoid infections.

Design, Characterization, and Biological Activities of Erythromycin-Loaded Nanodroplets to Counteract Infected Chronic Wounds Due to Streptococcus pyogenes.

Streptococcus pyogenes causes a wide spectrum of diseases varying from mild to life threatening, despite antibiotic treatment. Nanoparticle application could facilitate the foreign pathogen fight by increasing the antimicrobial effectiveness and reducing their adverse effects. Here, we designed and produced erythromycin-loaded chitosan nanodroplets (Ery-NDs), both oxygen-free and oxygen-loaded. All ND formulations were characterized for physico-chemical parameters, drug release kinetics, and tested for biocompatibility with human keratinocytes and for their antibacterial properties or interactions with S. pyogenes. All tested NDs possessed spherical shape, small average diameter, and positive Z potential. A prolonged Ery release kinetic from Ery-NDs was demonstrated, as well as a favorable biocompatibility on human keratinocytes. Confocal microscopy images showed ND uptake and internalization by S. pyogenes starting from 3 h of incubation up to 24 h. According to cell counts, NDs displayed long-term antimicrobial efficacy against streptococci significantly counteracting their proliferation up to 24 h, thanks to the known chitosan antimicrobial properties. Intriguingly, Ery-NDs were generally more effective (104-103 log10 CFU/mL), than free-erythromycin (105 log10 CFU/mL), in the direct killing of streptococci, probably due to Ery-NDs adsorption by bacteria and prolonged release kinetics of erythromycin inside S. pyogenes cells. Based on these findings, NDs and proper Ery-NDs appear to be the most promising and skin-friendly approaches for the topical treatment of streptococcal skin infections allowing wound healing during hypoxia.

Dalbavancin Boosts the Ability of Neutrophils to Fight Methicillin-Resistant Staphylococcus aureus.

Polymorphonuclear leukocytes (PMNs) are the most important cell type involved in the early nonspecific host response to bacterial pathogens. Staphylococcus aureus has evolved mechanisms to evade immune responses that contribute to its persistence in PMNs, and acquired resistance to several antimicrobials. Additionally, methicillin-resistant S. aureus (MRSA) is one of the most common causes of acute bacterial skin and skin-structure infections (ABSSSIs). Dalbavancin (DBV), a lipoglycopeptide, is indicated for the treatment of ABSSSIs, and has a broad spectrum of action against most microorganisms. Here, we sought to determine the effect of DBV on the neutrophil killing of MRSA and its potential immunomodulating activity. Our results revealed that DBV boosts MRSA killing by acting on both bacteria and PMNs. DBV pre-treatment of PMNs did not change the respiratory burst or degranulation, while an increased trend in neutrophil extracellular traps-associated elastase and in the production of TNFα and CXCL8 was revealed. In parallel, DBV caused a delay in the apoptosis of MRSA-infected neutrophils. In conclusion, we demonstrated a cooperative effect between the antimicrobial properties of PMNs and DBV, thus owing to their immunomodulatory activity. In the choice of the treatment management of serious S. aureus infections, DBV should be considered as an outstanding option since it reinforces PMNs pathogen clearance capability by exerting its effect directly, not only on MRSA but also on neutrophils.

A Rapid and Specific Real-Time PCR Assay for the Detection of Clinically Relevant Mucorales Species.

Infections triggered by filamentous fungi placed in the order Mucorales, phylum Zygomycota, can cause serious harm to immunocompromised patients. Since there is lack of a standardized PCR (polymerase chain reaction) assay for early diagnosis of this fungal infection, this work was aimed to develop a new PCR assay able to detect the presence of Mucorales genera in clinical specimens. Here, we describe a novel diagnostic TaqMan MGB probe assay for precise and rapid detection of the most common clinical species of Mucorales. Zygomycete-specific oligonucleotides were designed to specifically amplify and bind highly conserved sequences of fungal 28S rRNA gene. Additionally, we succeeded in differentiating Mucorales species (i.e., Rhizopus, Lichtheimia, Mucor, and Rhizomucor) in artificially infected serum samples, suggesting that the quantitative capability of this real-time PCR assay could potentially optimize the diagnosis of mucormycosis.

Combination of Poly(ε-Caprolactone) Biomaterials and Essential Oils to Achieve Anti-Bacterial and Osteo-Proliferative Properties for 3D-Scaffolds in Regenerative Medicine.

Biomedical implants, an essential part of the medical treatments, still suffer from bacterial infections that hamper patients' recovery and lives. Antibiotics are widely used to cure those infections but brought antibiotic resistance. Essential oils (EOs) demonstrate excellent antimicrobial activity and low resistance development risk. However, EO application in medicine is still quite scarce and almost no research work considers its use in combination with bioresorbable biomaterials, such as the poly(ε-caprolactone) (PCL) polymer. This work aimed to combine the antibacterial properties of EOs and their components, particularly eugenol and cinnamon oil, against Staphylococcus aureus, S. epidermidis and Escherichia coli, with those of PCL for medical applications in which good tissue regeneration and antimicrobial effects are required. The PCL porous scaffolds, added with increasing (from 30% to 50%) concentrations of eugenol and cinnamon oil, were characterized by square-shaped macropores. Saos-2 cells' cell viability/proliferation was hampered by 40 and 50% EO-enriched PCL, whereas no cytotoxic effect was recorded for both 30% EO-added PCL and pure-PCL. The antibacterial tests revealed the presence of a small inhibition halo around the 30% eugenol and cinnamon oil-functionalized PCL scaffolds only for staphylococci, whereas a significant decrease on both adherent and planktonic bacteria was recorded for all the three microorganisms, thus proving that, even if the EOs are only in part released by the EO-added PCL scaffolds, an anti-adhesive feature is anyway achieved. The scaffold will have the ability to support new tissue formation and simultaneously will be able to prevent post-surgical infection. This research shows the great potential in the use of EOs or their single components, at low concentrations, for biomaterial functionalization with enhanced anti-bacterial and biointegration properties.

Evaluation of a diagnostic algorithm for rapid identification of Gram-negative species and detection of extended-spectrum ß-lactamase and carbapenemase directly from blood cultures.

OBJECTIVES: To evaluate a rapid diagnostic algorithm based on MALDI-TOF MS, lateral flow immunoassays (LFIAs) and molecular testing performed directly from positive blood cultures (BCs) for Gram-negative species identification and detection of CTX-M extended-spectrum ß-lactamases and main carbapenemases. METHODS: Non-duplicate BCs positive to Gram-negative bacteria at microscope examination were subjected to species identification by direct MALDI-TOF MS following recovery of bacterial pellet by Rapid MBT Sepsityper® kit. Subsequently, NG-Test® CARBA 5 and NG-Test® CTX-M MULTI LFIAs were performed according to identified microbial species. Eazyplex® SuperBug CRE molecular assay was performed in cases of NG-Test® CARBA 5 negative results in patients with documented carbapenemase-producers carriage. Results of rapid diagnostic workflow were compared with those obtained by conventional diagnostic routine. RESULTS: Overall, the direct MALDI-TOF MS protocol allowed reliable identification to the species level of 92.1% of the 2133 monomicrobial BCs. Rate of matched identification was significantly higher for Enterobacterales (97.3%) in comparison to non-fermenting Gram-negative species (80.2%), obligate anaerobic bacteria (42.1%) and fastidious Gram-negative species (41.5%). The overall sensitivity of NG-Test® CARBA 5 and NG-Test® CTX-M MULTI was 92.2% and 91.6%, respectively. Integration of Easyplex® SuperBug CRE allowed the detection of blaKPC mutants associated with ceftazidime/avibactam resistance, reaching 100% sensitivity in carbapenemase detection. Both LFIAs and molecular testing showed no false-positive results. CONCLUSIONS: Algorithms based on MALDI-TOF MS, LFIAs and molecular testing may represent a cost-effective tool to timely identify Gram-negative species and detect resistance markers directly from BCs. According to local epidemiology, these results may allow antimicrobial stewardship interventions including prompt use of new approved drugs.

Antibacterial and Antifungal Efficacy of Medium and Low Weight Chitosan-Shelled Nanodroplets for the Treatment of Infected Chronic Wounds.

Purpose: Medium versus low weight (MW vs LW) chitosan-shelled oxygen-loaded nanodroplets (cOLNDs) and oxygen-free nanodroplets (cOFNDs) were comparatively challenged for biocompatibility on human keratinocytes, for antimicrobial activity against four common infectious agents of chronic wounds (CWs) - methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, Candida albicans and C. glabrata - and for their physical interaction with cell walls/membranes. Methods: cNDs were characterized for morphology and physico-chemical properties by microscopy and dynamic light scattering. In vitro oxygen release from cOLNDs was measured through an oximeter. ND biocompatibility and ability to promote wound healing in human normoxic/hypoxic skin cells were challenged by LDH and MTT assays using keratinocytes. ND antimicrobial activity was investigated by monitoring upon incubation with/without MW or LW cOLNDs/cOFNDs either bacteria or yeast growth over time. The mechanical interaction between NDs and microorganisms was also assessed by confocal microscopy. Results: LW cNDs appeared less toxic to keratinocytes than MW cNDs. Based on cell counts, either MW or LW cOLNDs and cOFNDs displayed long-term antimicrobial efficacy against S. pyogenes, C. albicans, and C. glabrata (up to 24 h), whereas a short-term cytostatic effects against MRSA (up to 6 h) was revealed. The internalization of all ND formulations by all four microorganisms, already after 3 h of incubation, was showed, with the only exception to MW cOLNDs/cOFNDs that adhered to MRSA walls without being internalized even after 24 h. Conclusion: cNDs exerted bacteriostatic and fungistatic effects, due to the presence of chitosan in the outer shell and independently of oxygen addition in the inner core. The duration of such effects strictly depends on the characteristics of each microbial species, and not on the molecular weight of chitosan in ND shells. However, LW chitosan was better tolerated by human keratinocytes than MW. For these reasons, the use of LW NDs should be recommended in future research to assess cOLND efficacy for the treatment of infected CWs.

Current Knowledge on Biomaterials for Orthopedic Applications Modified to Reduce Bacterial Adhesive Ability.

A significant challenge in orthopedics is the design of biomaterial devices that are able to perform biological functions by substituting or repairing various tissues and controlling bone repair when required. This review presents an overview of the current state of our recent research into biomaterial modifications to reduce bacterial adhesive ability, compared with previous reviews and excellent research papers, but it is not intended to be exhaustive. In particular, we investigated biomaterials for replacement, such as metallic materials (titanium and titanium alloys) and polymers (ultra-high-molecular-weight polyethylene), and biomaterials for regeneration, such as poly(ε-caprolactone) and calcium phosphates as composites. Biomaterials have been designed, developed, and characterized to define surface/bulk features; they have also been subjected to bacterial adhesion assays to verify their potential capability to counteract infections. The addition of metal ions (e.g., silver), natural antimicrobial compounds (e.g., essential oils), or antioxidant agents (e.g., vitamin E) to different biomaterials conferred strong antibacterial properties and anti-adhesive features, improving their capability to counteract prosthetic joint infections and biofilm formation, which are important issues in orthopedic surgery. The complexity of biological materials is still far from being reached by materials science through the development of sophisticated biomaterials. However, close interdisciplinary work by materials scientists, engineers, microbiologists, chemists, physicists, and orthopedic surgeons is indeed necessary to modify the structures of biomaterials in order to achieve implant integration and tissue regeneration while avoiding microbial contamination.

Antimicrobial oxygen-loaded nanobubbles as promising tools to promote wound healing in hypoxic human keratinocytes.

Chronic wounds (CWs) are typically characterized by persistent hypoxia, exacerbated inflammation, and impaired skin tissue remodeling. Additionally, CWs are often worsened by microbial infections. Oxygen-loaded nanobubbles (OLNBs), displaying a peculiar structure based on oxygen-solving perfluorocarbons such as perfluoropentane in the inner core and polysaccharydes including chitosan in the outer shell, have proven effective in delivering oxygen to hypoxic tissues. Antimicrobial properties have been largely reported for chitosan. In the present work chitosan/perfluoropentane OLNBs were challenged for biocompatibility with human skin cells and ability to promote wound healing processes, as well as for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA) and Candida albicans. After cellular internalization, OLNBs were not toxic to human keratinocytes (HaCaT), whereas oxygen-free NBs (OFNBs) slightly affected their viability. Hypoxia-dependent inhibition of keratinocyte migratory ability after scratch was fully reversed by OLNBs, but not OFNBs. Both OLNBs and OFNBs exerted chitosan-induced short-term bacteriostatic activity against MRSA (up to 6 h) and long-term fungistatic activity against C. albicans (up to 24 h). Short-term antibacterial activity associated with NB prolonged adhesion to MRSA cell wall (up to 24 h) while long-term antifungal activity followed NB early internalization by C. albicans (already after 3 h of incubation). Taken altogether, these data support chitosan-shelled and perfluoropentane-cored OLNB potential as innovative, promising, non-toxic, and cost-effective antimicrobial devices promoting repair processes to be used for treatment of MRSA- and C. albicans-infected CWs.

Activity of ceftolozane-tazobactam, ceftazidime-avibactam, meropenem-vaborbactam, cefiderocol and comparators against Gram-negative organisms causing bloodstream infections in Northern Italy (2019-2021): emergence of complex resistance phenotypes.

Herein we assessed the frequency of Gram-negative organisms causing bloodstream infections and activity spectrum of ceftolozane-tazobactam (CTZ), ceftazidime-avibactam (CZA), meropenem-vaborbactam (MEV), cefiderocol (CFDC) and comparators. Overall, 1605 Gram-negative isolates were consecutively collected during 2019-2021. Enterobacterales represented more than 75% and exhibited >90% susceptibility to CZA (97%), amikacin (91.8%) and meropenem (90.6%). ESBL-producing Enterobacterales isolates showed high rates of susceptibility towards CZA (100%), carbapenems (89.1-100%) and CTZ (84.9-95.1%). MEV displayed the highest activity against KPC-producing Enterobacterales (MIC50/90, 0.75/4 mg/L; 92.9% susceptible) followed by CZA (MIC50/90, ≤2/>8 mg/L; 89.3% susceptible), CFDC (MIC50/90, 0.25/4 mg/L, 87.5% susceptible) and colistin (MIC50/90, ≤2/4 mg/L, 83.9% susceptible). High proportions of P. aeruginosa isolates were susceptible to colistin (97.8%), CZA (97.2%), CTZ (96.1%) and amikacin (94.5%). CFDC showed potent activity against Acinetobacter baumannii (MIC50/90, 0.5/1 mg/L; 97.2% susceptible), multi-drug resistant P. aeruginosa (MIC50/90, 0.25/1 mg/L; 96% susceptible), and Stenotrophomonas maltophilia (MIC50/90, 0.12/0.25 mg/L; 100% susceptible).

Exposure to the agricultural fungicide tebuconazole promotes Aspergillus fumigatus cross-resistance to clinical azoles.

Resistance to clinical triazoles in Aspergillus fumigatus is a growing concern for individuals at high risk of Aspergillus infection. Two triazole resistance selection routes are currently being investigated: one occurring in triazole-treated patients in healthcare settings, and the second taking place in the environment due to the widespread use of agricultural triazoles. This study aimed to assess the ability of agricultural azoles to promote cross-resistance to clinical azoles in A. fumigatus. Five A. fumigatus isolates susceptible to clinical azoles were exposed to the triazole 14α-demethylase inhibitor, tebuconazole (TBC), and then antifungal susceptibility tests for voriconazole, itraconazole, posaconazole and isavuconazole were performed. Under TBC selection pressure, all A. fumigatus isolates exhibited resistance to clinical triazoles. However, only two displayed a multiresistant phenotype to clinical azoles. TBC exposure was also associated with delayed conidia formation and progressive absence of conidiation. Noteworthy, no TBC-exposed clones harbored TR34/L98H mutation, as judged by real-time PCR assays. The observation that TBC exposure promotes cross-resistance to clinical triazoles warrants careful and thorough assessment of the human health risk associated with agricultural azoles. The absence of TR34/L98H mutation in cross-resistant A. fumigatus isolates suggests that other cyp51A mutations may be involved in clinical azole cross-resistance.

Positive and Negative Ions Potently Inhibit the Viability of Airborne Gram-Positive and Gram-Negative Bacteria.

Positive and negative ions (PAIs and NAIs, respectively) generated by air ionizers curb indoor spread of airborne pathogens through cellular oxidative damage. Thus, here, we asked whether ion exposure of Staphylococcus aureus and Escherichia coli bacteria-either plated on agar or trapped in air filters-would affect their viability and whether this effect would be influenced by variations in bacterial type and load, action area, distance from the ion generator, exposure time, or filter type. We selected these two vegetative bacterium species because, besides being representative of Gram-positive and Gram-negative strains, respectively, they are widely recognized as the two most common airborne pathogens. We observed a robust ion inhibitory effect on the viability of free bacteria regardless of the experimental condition employed. Specifically, 12-h ion exposure of plated S. aureus and E. coli, at either 5 cm or 10 cm from the ion source, reduced bacterial viability by ∼95% and 70%, respectively. Furthermore, 3-h ion exposure was sufficient to reduce the viability of both bacterial species trapped in filters. Our results showing a strong antibacterial activity of PAI and NAI under all experimental conditions tested further support the use of air ionizers for preventing and/or containing airborne infection in domestic and nondomestic settings. IMPORTANCE Indoor air is a well-established vehicle for direct and indirect spread of a wide variety of human pathogens-as bioaerosols are composed of bacteria, viruses, fungi, and other types of organisms-that may trigger some pathologies. Plasmacluster ionizers are known for their ability to generate positively or negatively charged air ions (PAIs and NAIs, respectively) that can kill/inactivate indoor airborne pathogens, through oxidative stress-induced damage, in various environments. Given these premises, the aim of this study was to evaluate the viability of Gram-positive and Gram-negative bacteria exposed to PAI and NAI under different experimental variables such as bacterial type and load, action area, distance from the ion generator, ion exposure time, and filter type. Altogether, our findings, demonstrating a remarkable PAI and NAI antibacterial activity, stress the importance of using air ionizers to prevent indoor airborne infection.

Novel Silver-Functionalized Poly(ε-Caprolactone)/Biphasic Calcium Phosphate Scaffolds Designed to Counteract Post-Surgical Infections in Orthopedic Applications.

In this study, we designed and developed novel poly(ε-caprolactone) (PCL)-based biomaterials, for use as bone scaffolds, through modification with both biphasic calcium phosphate (BCP), to impart bioactive/bioresorbable properties, and with silver nitrate, to provide antibacterial protection against Staphylococcus aureus, a microorganism involved in prosthetic joint infections (PJIs). Field emission scanning electron microscopy (FESEM) showed that the samples were characterized by square-shaped macropores, and energy dispersive X-ray spectroscopy analysis confirmed the presence of PCL and BCP phases, while inductively coupled plasma-mass spectrometry (ICP-MS) established the release of Ag+ in the medium (~0.15-0.8 wt% of initial Ag content). Adhesion assays revealed a significant (p < 0.0001) reduction in both adherent and planktonic staphylococci on the Ag-functionalized biomaterials, and the presence of an inhibition halo confirmed Ag release from enriched samples. To assess the potential outcome in promoting bone integration, preliminary tests on sarcoma osteogenic-2 (Saos-2) cells indicated PCL and BCP/PCL biocompatibility, but a reduction in viability was observed for Ag-added biomaterials. Due to their combined biodegrading and antimicrobial properties, the silver-enriched BCP/PCL-based scaffolds showed good potential for engineering of bone tissue and for reducing PJIs as a microbial anti-adhesive tool used in the delivery of targeted antimicrobial molecules, even if the amount of silver needs to be tuned to improve osteointegration.

Inhibition of Human Neutrophil Functions In Vitro by Multiple Sclerosis Disease-Modifying Therapies.

There is a growing optimism about the potential of new disease-modifying therapies (DMTs) in the management of relapsing-remitting multiple sclerosis (RRMS) patients. However, this initial enthusiasm has been tempered by evidence indicating that multiple sclerosis (MS) patients undergoing DMT may be at higher risk of developing infections through incompletely understood mechanisms. As neutrophils provide the first line of defense against pathogens, here we have compared the effects of some of the commonly used MS DMTs (i.e., moderate-efficacy injective, first-line: interferonß-1b (IFNß-1b), glatiramer acetate (GA); and high-efficacy, second-line: fingolimod (FTY) and natalizumab (NAT)) on the in vitro viability and functions of neutrophils isolated from healthy subjects. All the DMTs tested impaired the ability of neutrophils to kill Klebsiella pneumoniae, whereas none of them affected the rate of neutrophil apoptosis or CD11b and CD62L cell surface expression. Intriguingly, only FTY exposure negatively affected K. pneumoniae-induced production of reactive oxygen species (ROS) in polymorphonuclear leukocytes (PMNs). Furthermore, neutrophils exposed to K. pneumoniae secreted enhanced amounts of CXCL8, IL-1ß and TNF-α, which were differentially regulated following DMT pretreatment. Altogether, these findings suggest that DMTs may increase the susceptibility of MS patients to microbial infections, in part, through inhibition of neutrophil functions. In light of these data, we recommend that the design of personalized therapies for RRMS patients should take into account not just the mechanism of action of the chosen DMT but also the potential risk of infection associated with the administration of such therapeutic compounds to this highly vulnerable population.

Serological and molecular detection of Bartonella henselae in specimens from patients with suspected cat scratch disease in Italy: A comparative study.

Cat scratch disease (CSD) is an infectious disease caused by Bartonella henselae, usually characterized by self-limiting regional lymphadenopathy and fever. Given the low clinical diagnostic sensitivity and specificity of conventional anti-B. henselae indirect immunofluorescence assays (IFAs), real-time polymerase chain reaction (PCR)-based detection of B. henselae is now being proposed as a more sensitive tool to diagnose CSD. Thus, here we have assessed the efficacy of real-time PCR in detecting B. henselae in different specimens from patients with suspected CSD and compared it to that of IFA. From March 2011 to May 2016, at the Microbiology and Virology Unit, Azienda Ospedaliera Universitaria Città della Salute e della Scienza di Torino, Turin, Italy, 115 clinical specimens (56 aspirated pus, 39 fresh lymph node biopsies, and 20 whole blood samples) and 99 sera from 115 patients with suspected CSD (62 females and 53 males between the ages of 3 months and 68 years) were analyzed by both real-time PCR, used in a qualitative way, and IFA (IgM and IgG) for the presence of B. henselae. For 16 patients, serological results were not available due to a clinical decision not to request the test. B. henselae DNA positivity was detected by real-time PCR in 37.39% of patients, while 62.61% of them were negative. Thus, patients were divided into two groups: real-time PCR+ (n = 43) and real-time PCR- (n = 72). Real-time PCR screening of whole blood, biopsies, and aspirated pus revealed B. henselae positivity in 40%, 38.46%, and 35.71% of patients, respectively. When we analyzed samples by IFA, we found the presence of B. henselae in 28 out of 99 (28.28%) patients, of which 11 (11.11%) belonged to the real-time PCR+ group and 17 (17.17%) to the real-time PCR- group. Among the 71 seronegative subjects, 16 (16.16%) were found positive for B. henselae by real-time PCR. Thus, by combining the results of both assays, we were able to increase the percentage of B. henselae positive specimens from 27.27% (real-time PCR) or 28.28% (IFA) to 44.44% (real-time PCR+IFA). Altogether, these findings indicate that the early detection of B. henselae in patients with suspicious CSD through combined real-time PCR and serological analyses can lead to a more accurate diagnosis of CSD, thereby allowing prompt and appropriate disease management.

Single versus Double Skin Preparation for Infection Prevention in Proximal Humeral Fracture Surgery.

Preoperative skin preparation plays a major role in preventing postoperative infections. This study aims to compare a single skin preparation (povidone iodine) with a double skin preparation (chlorhexidine gluconate followed by povidone iodine). Forty patients affected by proximal humeral fracture were included in the study. The day of surgery the two skin preparation strategies were performed in the same shoulder, divided into two areas, at the level of the deltopectoral approach. Skin swabs were collected from each area and subjected to microbiological analysis. Both skin preparations significantly reduced the positive culture rate. Coagulase-negative staphylococci (CoNS) dropped from 92.5% to 40% and to 7.5% after the single and double skin preparation (p<0.001), respectively. The positivity rate was reduced from 50% to 17.5% (p=0.002) and from 27.5% to 0% (p= 0.001) for Propionibacterium acnes and Staphylococcus aureus, respectively, with no difference between the two preparations. The double skin preparation had a more significant effect on bacterial load against CoNS compared to the single skin preparation (p<0.001 versus p= 0.015). In conclusion, both the approaches reduced S. aureus and P. acnes skin load, whereas the double skin preparation is more effective than the single one against CoNS. In light of our findings, preoperative strategies able to reduce bacterial load could potentially increase the final efficacy of perioperative traditional skin preparations.

Unexpected Listeria monocytogenes detection with a dithiothreitol-based device during an aseptic hip revision.

Prosthetic joint infection diagnosis is often difficult since biofilm-embedded microorganisms attach well to the prosthetic surfaces and resist their detection by conventional methods. DL-dithiothreitol has been described as a valid method for biofilm detachment on orthopedic devices. We report the case of an occasional detection of Listeria monocytogenes in a non immuno-compromised patient with a preoperative diagnosis of aseptic loosening. The infection diagnosis due to such rare bacteria was made postoperatively, thanks to a DL-dithiothreitol-based device. This may be considered a feasible approach for the microbiological analysis of prosthetic joint infection, considering that a prompt diagnosis of such biofilm-associated infections could bring some advantages, such as an early and appropriate antibiotic therapy administration and a reduction of undiagnosed infections.