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The growing threat of viral infections requires innovative therapeutic approaches to safeguard human health. Nanomaterials emerge as a promising solution to overcome the limitations associated with conventional therapies. The eco-friendly synthesis of silver nanoparticles (AgNPs) currently represents a method that guarantees antimicrobial efficacy, safety, and cost-effectiveness. This study explores the use of AgNPs derived from the peel (Lp-AgNPs) and juice (Lj-AgNPs) Citrus limon “Ovale di Sorrento”, cultivars of the Campania region. The antiviral potential was tested against viruses belonging to the Coronaviridae and Herpesviridae. AgNPs were synthesized by reduction method using silver nitrate solution mixed with aqueous extract of C. limon peel and juice. The formation of Lp-AgNPs and Lj-AgNPs was assessed using a UV–Vis spectrophotometer. The size, ζ-potential, concentration, and morphology of AgNPs were evaluated by dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and field emission-scanning electron microscopy (FE-SEM). Cytotoxicity was evaluated in a concentration range between 500 and 7.8 µg/mL on VERO-76 and HaCaT cells, with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium test bromide (MTT). Antiviral activity consisted of virus pre-treatment, co-treatment, cellular pre-treatment, and post-infection tests versus HSV-1 and SARS-CoV-2 at a multiplicity of infections (MOI) of 0.01. Plaque reduction assays and real-time PCR provided data on the antiviral potential of tested compounds. Lp-AgNPs and Lj-AgNPs exhibited spherical morphology with respective diameters of 60 and 92 nm with concentrations of 4.22 and 4.84 × 1010 particles/mL, respectively. The MTT data demonstrated minimal cytotoxicity, with 50 % cytotoxic concentrations (CC50) of Lp-AgNPs and Lj-AgNPs against VERO cells of 754.6 and 486.7 µg/mL. Similarly, CC50 values against HaCaT were 457.3 µg/mL for Lp-AgNPs and 339.6 µg/mL for Lj-AgNPs, respectively. In the virus pre-treatment assay, 90 % inhibitory concentrations of HSV-1 and SARS-CoV-2 were 8.54–135.04 µg/mL for Lp-AgNPs and 6.13–186.77 µg/mL for Lj-AgNPs, respectively. The molecular investigation confirmed the antiviral data, recording a reduction in the UL54 and UL27 genes for HSV-1 and in the Spike (S) gene for SARS-CoV-2, following AgNP exposure. The results of this study suggest that Lp-AgNPs and Lj-AgNPs derived from C. Limon could offer a valid ecological, natural, local and safe strategy against viral infections.

Abstract Efficient predictive biomarkers are needed for immune checkpoint inhibitor (ICI)-based immunotherapy in non-small cell lung cancer (NSCLC). Testing the predictive value of single nucleotide polymorphisms (SNPs) in programmed cell death 1 (PD-1) or its ligand 1 (PD-L1) has shown contrasting results. Here, we aim to validate the predictive value of PD-L1 SNPs in advanced NSCLC patients treated with ICIs as well as to define the molecular mechanisms underlying the role of the identified SNP candidate. rs822336 efficiently predicted response to anti-PD-1/PD-L1 immunotherapy in advanced non-oncogene addicted NSCLC patients as compared to rs2282055 and rs4143815. rs822336 mapped to the promoter/enhancer region of PD-L1, differentially affecting the induction of PD-L1 expression in human NSCLC cell lines as well as their susceptibility to HLA class I antigen matched PBMCs incubated with anti-PD-1 monoclonal antibody nivolumab. The induction of PD-L1 expression by rs822336 was mediated by a competitive allele-specificity binding of two identified transcription factors: C/EBPβ and NFIC. As a result, silencing of C/EBPβ and NFIC differentially regulated the induction of PD-L1 expression in human NSCLC cell lines carrying different rs822336 genotypes. Analysis by binding microarray further validated the competitive allele-specificity binding of C/EBPβ and NFIC to PD-L1 promoter/enhancer region based on rs822336 genotype in human NSCLC cell lines. These findings have high clinical relevance since identify rs822336 and induction of PD-L1 expression as novel biomarkers for predicting anti-PD-1/PD-L1-based immunotherapy in advanced NSCLC patients.

Background/Objectives: ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pose a serious public health threat as they are resistant to multiple antimicrobial agents. Bloodstream infections (BSIs) caused by ESKAPE bacteria have high mortality rates due to the limited availability of effective antimicrobials. This study aimed to evaluate the prevalence and susceptibility of ESKAPE pathogens causing BSIs over three years in a large tertiary hospital in Salerno. Methods: Conducted at the Clinical Microbiology Laboratory of San Giovanni di Dio e ‘‘Ruggi D’Aragona’’ Hospital from January 2020 to December 2022, blood culture samples from different departments were incubated in the BD BACTEC™ system for 5 days. Species identification was performed using MALDI-TOF MS, and antimicrobial resistance patterns were determined by the VITEK2 system. Results: Out of 3197 species isolated from positive blood cultures, 38.7% were ESKAPE bacteria. Of these, 59.9% were found in blood culture samples taken from men, and the most affected age group was those aged >60 years. (70.6%). Staphylococcus aureus was the main BSI pathogen (26.3%), followed by Klebsiella pneumoniae (15.8%). Significant resistance rates were found, including 35% of Staphylococcus aureus being resistant to oxacillin and over 90% of Acinetobacter baumannii being resistant to carbapenems. Conclusions: These results highlight the urgent need for antimicrobial stewardship programs to prevent incurable infections.

The programmed cell death pathways of apoptosis are important in mammalian cellular protection from infections. The activation of these pathways depends on the presence of membrane receptors that bind bacterial components to activate the transduction mechanism. In addition to bacteria, these mechanisms can be activated by outer membrane vesicles (OMVs). OMVs are spherical vesicles of 20–250 nm diameter, constitutively released by Gram-negative bacteria. They contain several bacterial determinants including proteins, DNA/RNA and proteins, that activate different cellular processes in host cells. This study focused on Klebsiella pneumoniae-OMVs in activating death mechanisms in human bronchial epithelial cells (BEAS-2B). Characterization of purified OMVs was achieved by scanning electron microscopy, nanoparticle tracking analysis and protein profiling. Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay while apoptotic induction was measured by flow cytometry and confirmed by western blotting. The OMVs produced showed a spherical morphology, with a diameter of 137.2 ± 41 nm and a vesicular density of 7.8 × 109 particles/mL Exposure of cell monolayers to 50 μg of K. pneumoniae-OMV for 14 h resulted in approximately 25 % cytotoxicity and 41.15–41.14 % of cells undergoing early and late apoptosis. Fluorescence microscopy revealed reduced cellular density, the presence of apoptotic bodies, chromatin condensation, and nuclear membrane blebbing in residual cells. Activation of caspases −3 and −9 and dysregulation of BAX, BIM and Bcl-xL indicated the activation of mitochondria-dependent apoptosis. Furthermore, a decrease in the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase involved endoplasmic reticulum stress with the potential formation of reactive oxygen species. These findings provide evidence for the role of OMVs in apoptosis and involvement in the pathogenesis of K. pneumoniae infections.

The nasopharyngeal tract contains a complex microbial community essential to maintaining host homeostasis. Recent studies have shown that SARS-CoV-2 infection changes the microbial composition of the nasopharynx. Still, little is known about how it affects the fungal microbiome, which could provide valuable insights into disease pathogenesis. Nasopharyngeal swabs were collected from 55 patients, during three distinct COVID-19 waves that occurred in the Campania Region (southern Italy). An RNA-seq-based analysis was performed to evaluate changes in mycobiota diversity, showing variations depending on the disease’s severity and the sample collection wave. The phyla Basidiomycota and Ascomycota were shown to have higher abundance in patients with severe symptoms. Furthermore, the diversity of the fungal population was greater in the second wave. Conclusion: According to our research, COVID-19 induces significant dysbiosis of the fungal microbiome, which may contribute to disease pathogenesis, and understanding its underlying mechanisms could contribute to developing effective treatments.

Drug repurposing is sparking considerable interest due to reduced costs and development times. The current study details the screening of teniposide, an antitumor drug, for its antibacterial activity against both Gram-positive and Gram-negative strains, with a focus on Staphylococcus epidermidis (S. epidermidis), the primary causative agent of nosocomial and transplant-related infections. The cytotoxicity was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and hemolysis assays on immortalized human keratinocyte (HaCaT) cells and human erythrocytes. After 20 h of treatment, the recorded concentrations causing 50% cytotoxicity (CC50) and hemolysis (HC50) were 33.63 and 121.55 μg/mL, respectively. The antibacterial screening employed disk diffusion, the broth microdilution method, LIVE/DEAD staining, and a time-killing test. The drug induced a growth inhibitory area in the 22–25 mm range for all Gram-positive strains. The minimum concentration that inhibited 90% of bacteria (MIC90) was 6.25 μg/mL against Staphylococcus aureus and S. epidermidis and 12.5 μg/mL versus Enterococcus faecalis, exhibiting bactericidal action. Treatment resulted in S. epidermidis cell morphology deformities and damage to the cell membrane, observed by scanning electron microscopy (SEM). Mechanism analysis revealed alterations in the selective permeability of the cell membrane, observed under the fluorescence microscope by the absorption of propidium iodide (PI). The synergistic effect of teniposide in combination with fosfomycin and gentamicin was documented by disk diffusion and checkboard assay, recording a fractional inhibitory concentration index (FICI) of 0.28 and 0.37, respectively. The drug’s action on S. epidermidis biofilm biomass was investigated using crystal violet (CV) and MTT. Teniposide affected biofilm viability in a dose-dependent manner, inducing, at a concentration of 3.12 μg/mL, a matrix inhibition of about 42% and 61%, with a sessile metabolic activity of 54% and 24% recorded after 2 and 24 h, respectively. Overall, this study suggests the potential repurposing of the anticancer drug teniposide as a therapeutic agent to counteract S. epidermidis infections.

The spread of antibiotic-resistant bacteria and the rise of emerging and re-emerging viruses in recent years constitute significant public health problems. Therefore, it is necessary to develop new antimicrobial strategies to overcome these challenges. Herein, we describe an innovative method to synthesize ligand-free silver nanoparticles by Pulsed Laser Ablation in Liquid (PLAL-AgNPs). Thus produced, nanoparticles were characterized by total X-ray fluorescence, zeta potential analysis, transmission electron microscopy (TEM), and nanoparticle tracking analysis (NTA). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to evaluate the nanoparticles’ cytotoxicity. Their potential was evaluated against the enveloped herpes simplex virus type 1 (HSV-1) and the naked poliovirus type 1 (PV-1) by plaque reduction assays and confirmed by real-time PCR and fluorescence microscopy, showing that nanoparticles interfered with the early stage of infection. Their action was also examined against different bacteria. We observed that the PLAL-AgNPs exerted a strong effect against both methicillin-resistant Staphylococcus aureus (S. aureus MRSA) and Escherichia coli (E. coli) producing extended-spectrum β-lactamase (ESBL). In detail, the PLAL-AgNPs exhibited a bacteriostatic action against S. aureus and a bactericidal activity against E. coli. Finally, we proved that the PLAL-AgNPs were able to inhibit/degrade the biofilm of S. aureus and E. coli.

Root canal treatment represents a significant challenge as current cleaning and disinfection methodologies fail to remove persistent bacterial biofilms within the intricate anatomical structures. Recently, the field of nanotechnology has emerged as a promising frontier with numerous biomedical applications. Among the most notable contributions of nanotechnology are nanoparticles, which possess antimicrobial, antifungal, and antiviral properties. Nanoparticles cause the destructuring of bacterial walls, increasing the permeability of the cell membrane, stimulating the generation of reactive oxygen species, and interrupting the replication of deoxyribonucleic acid through the controlled release of ions. Thus, they could revolutionize endodontics, obtaining superior results and guaranteeing a promising short- and long-term prognosis. Therefore, chitosan, silver, graphene, poly(lactic) co-glycolic acid, bioactive glass, mesoporous calcium silicate, hydroxyapatite, zirconia, glucose oxidase magnetic, copper, and zinc oxide nanoparticles in endodontic therapy have been investigated in the present review. The diversified antimicrobial mechanisms of action, the numerous applications, and the high degree of clinical safety could encourage the scientific community to adopt nanoparticles as potential drugs for the treatment of endodontic diseases, overcoming the limitations related to antibiotic resistance and eradication of the biofilm.

Abstract This study investigated the performance of 24 commercial disinfectants present on the market during last year according to the manufacturer’s instructions. Recently, national and international organizations of public health performed studies on disinfection products due to the increasing awareness of the potential and growing risks on human health, such as skin damage and reactions in the mucosal lining, especially for the healthcare workers in their frequent daily use. However, there are many limitations in the common cleaning/disinfection products on market as in the selection of effective disinfectants to decontaminate inanimate surfaces. We analyzed the disinfection power of hydrogen peroxide, quaternary ammonium compounds, alcohols, phenols and aldehydes used as active principles according to international guidelines. The antimicrobial properties were assessed by broth microdilution, and antibiofilm properties against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus); their virucidal efficacy was tested against Herpes simplex virus type 1 (HSV-1) and Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The quaternary ammonium compounds demonstrated better efficacy than others and in some cases ready to use products had also virucidal and antimicrobial activities after dilution at 0.125%. The scientific evidence indicates that many commercial products are used at high concentrations and high doses and this could have deleterious effects both on human health and the environment. A lower concentration of active ingredients would avoid the excessive release of chemicals into the environment and improve skin tolerance, ensuring the health and safety protection of workers, including the healthcare operators at their workplace.

The emergence of multidrug-resistant strains requires the urgent discovery of new antibacterial drugs. In this context, an antibacterial screening of a subset of anthelmintic avermectins against gram-positive and gram-negative strains was performed. Selamectin completely inhibited bacterial growth at 6.3 μg/mL concentrations against reference gram-positive strains, while no antibacterial activity was found against gram-negative strains up to the highest concentration tested of 50 μg/mL. Given its relevance as a community and hospital pathogen, further studies have been performed on selamectin activity against Staphylococcus aureus (S. aureus), using clinical isolates with different antibiotic resistance profiles and a reference biofilm-producing strain. Antibacterial studies have been extensive on clinical S. aureus isolates with different antibiotic resistance profiles. Mean MIC90 values of 6.2 μg/mL were reported for all tested S. aureus strains, except for the macrolide-resistant isolate with constitutive macrolide-lincosamide-streptogramin B resistance phenotype (MIC90 9.9 μg/mL). Scanning Electron Microscopy (SEM) showed that selamectin exposure caused relevant cell surface alterations. A synergistic effect was observed between ampicillin and selamectin, dictated by an FIC value of 0.5 against methicillin-resistant strain. Drug administration at MIC concentration reduced the intracellular bacterial load by 81.3%. The effect on preformed biofilm was investigated via crystal violet and confocal laser scanning microscopy. Selamectin reduced the biofilm biomass in a dose-dependent manner with minimal biofilm eradication concentrations inducing a 50% eradication (MBEC50) at 5.89 μg/mL. The cytotoxic tests indicated that selamectin exhibited no relevant hemolytic and cytotoxic activity at active concentrations. These data suggest that selamectin may represent a timely and promising macrocyclic lactone for the treatment of S. aureus infections.

Pseudomonas aeruginosa (PA) is a major Gram-negative opportunistic pathogen causing several serious acute and chronic infections in the nosocomial and community settings. PA eradication has become increasingly difficult due to its remarkable ability to evade antibiotics. Therefore, epidemiological studies are needed to limit the infection and aim for the correct treatment. The present retrospective study focused on PA presence among samples collected at the San Giovanni di Dio and Ruggi D’Aragona University Hospital in Salerno, Italy; its resistance profile and relative variations over the eight years were analyzed. Bacterial identification and antibiotic susceptibility tests were performed by VITEK® 2. In the 2015–2019 and 2020–2022 timeframes, respectively, 1739 and 1307 isolates of PA were obtained from respiratory samples, wound swabs, urine cultures, cultural swabs, blood, liquor, catheter cultures, vaginal swabs, and others. During 2015–2019, PA strains exhibited low resistance against amikacin (17.2%), gentamicin (25.2%), and cefepime (28.3%); moderate resistance against ceftazidime (34.4%), imipenem (34.6%), and piperacillin/tazobactam (37.7%); and high resistance against ciprofloxacin (42.4%) and levofloxacin (50.6%). Conversely, during the 2020–2022 era, PA showed 11.7, 21.1, 26.9, 32.6, 33.1, 38.7, and 39.8% resistance to amikacin, tobramycin, cefepime, imipenem, ceftazidime, ciprofloxacin, and piperacillin/tazobactam, respectively. An overall resistance-decreasing trend was observed for imipenem and gentamicin during 2015–2019. Instead, a significant increase in resistance was recorded for cefepime, ceftazidime, and imipenem in the second set of years investigated. Monitoring sentinel germs represents a key factor in optimizing empirical therapy to minimize the spread of antimicrobial resistance.

Nanotechnologies comprise approaches able to synthesize, manipulate and visualize matter at the nanometer scale offering numerous advantages, especially in the biomedical field. In particular, nanoparticles represent a promising tool in the medical field to overcome antimicrobial resistance, which represent current serious problem. Furthermore, nanoparticles use multiple mechanisms to target microorganisms, rendering difficult the development of antimicrobial resistance. In the present, we used a Samarium-Cobalt (SmCo) target, permanent magnet, to synthesize nanoparticles (Nps) by Pulsed Laser Ablation in Liquid (PLAL). To evaluate their possible antimicrobial effect against Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis and Streptococcus mutans, we performed minimal inhibitory concentration (MIC) essays. In addition we evaluated their ability to affect human keratinocyte cells viability. By time killing assay, we monitored bacterial growth after exposure to Nps-SmCo. PLAL is an innovative, cheap, fast and safe nanotechnology to allow the synthesis of small nanoparticles that could be exploited for applications as novel antimicrobials.

Due to the wide etiology of conjunctivitis, the expensive and time-consuming diagnosis requires new therapeutic strategies with broad-spectrum antimicrobial activity and nonselective mechanisms of action. In this context, eye drops could provide an alternative to conventional antimicrobial therapies. Here, we compare the antibacterial and antiviral activity of Oftasecur and Visuprime, commercially available ophthalmic solutions. Cytotoxicity assay was performed on Vero CCL-81 cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) test. Antibacterial efficacy was evaluated on Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae by disk diffusion, broth microdilution methods, and time-killing tests. Furthermore, the antiviral activity against HSV-1 was estimated by co-treatment, cell and viral pretreatment and post-treatment, via plaque reduction assay, fluorescence assessment (GFP-engineered HSV-1), and real-time PCR. After 24 h of exposure, Oftasecur and Visuprime showed a volume-inducing 50% of cytotoxicity of 125 and 15.8 μL, respectively Oftasecur and Visuprime induced 90% antibacterial activity in response to mean volume of 10.0 and 4.4 µL for Gram-positive and Gram-negative strains, respectively. Oftasecur exerted bactericidal action on both bacterial populations, while Visuprime was bacteriostatic on Gram-negative strains and slightly bactericidal on Gram-positive bacteria. A major impact on infectivity occurred by exposure of viral particles to the ophthalmic solutions. In detail, 50% of inhibition was verified by exposing the viral particles to 3.12 and 0.84 μL of Oftasecur and Visuprime, respectively, for 1 h. The reduction of the fluorescence and the expression of the viral genes confirmed the recorded antiviral activity. Due to their high antimicrobial efficiency, Oftasecur and Visuprime could represent a valid empirical strategy for the treatment of conjunctivitis.

Background: The disinfection process represents an important activity closely linked to the removal of micro-organisms in common processing systems. Traditional disinfectants are often not sufficient to avoid the spread of food pathogens; therefore, innovative strategies for decontamination are crucial to countering microbial transmission. This study aims to assess the antimicrobial efficiency of tetrapotassium iminodisuccinic acid salt (IDSK) against the most common pathogens present on surfaces, especially in food-borne environments. Methods: IDSK was synthesized from maleic anhydride and characterized through nuclear magnetic resonance (NMR) spectroscopy (both 1H-NMR and 13C-NMR), thermogravimetric analysis (TGA) and Fourier Transform Infrared (FTIR) spectroscopy. The antibacterial activity was performed via the broth microdilution method and time-killing assays against Escherichia coli, Staphylococcus aureus, Salmonella enterica, Enterococcus faecalis and Pseudomonas aeruginosa (IDSK concentration range: 0.5–0.002 M). The biofilm biomass eradicating activity was assessed via a crystal violet (CV) assay. Results: The minimum inhibitory concentration (MIC) of IDSK was 0.25 M for all tested strains, exerting bacteriostatic action. IDSK also reduced biofilm biomass in a dose-dependent manner, reaching rates of about 50% eradication at a dose of 0.25 M. The advantages of using this innovative compound are not limited to disinfecting efficiency but also include its high biodegradability and its sustainable synthesis. Conclusions: IDSK could represent an innovative and advantageous disinfectant for food processing and workers’ activities, leading to a better quality of food and safer working conditions for the operators.

Ficus rubiginosa plant extract showed antimicrobial activity, but no evidence concerning its antiviral properties was reported. The antiviral activity of the methanolic extract (MeOH) and its n-hexane (H) and ethyl acetate (EA) fractions against Herpes simplex virus-1 (HSV-1), Human coronavirus (HCoV) -229E, and Poliovirus-1 (PV-1) was investigated in the different phases of viral infection in the VERO CCL-81 cell line. To confirm the antiviral efficacy, a qPCR was conducted. The recorded cytotoxic concentration 50% was 513.1, 298.6, and 56.45 µg/mL for MeOH, H, and EA, respectively, assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay after 72 h of treatment. The Ficus rubiginosa leaf extract inhibited the replication of HSV-1 in the early stages of infection, showing a complete inhibition up to 0.62, 0.31, and 1.25 µg/mL. Against HCoV-229E, a total inhibition up to 1.25 µg/mL for MeOH and H as well as 5 µg/mL for EA was observed. Otherwise, no activity was recorded against PV-1. The leaf extract could act directly on the viral envelope, destructuring the lipid membrane and/or directly blocking the enriched proteins on the viral surface. The verified gene inhibition suggested that the treatments with M, H, and EA impaired HSV-1 and HCoV-229E replication, with a greater antiviral efficiency against HSV-1 compared to HCoV-229E, possibly due to a greater affinity of Ficus rubiginosa towards membrane glycoproteins and/or the different lipid envelopes.

Cutibacterium acnes (C. acnes) is the main causative agent of acne vulgaris. The study aims to evaluate the antimicrobial activity of a natural product, Arctostaphylos uva-ursi leaf extract, against C. acnes. Preliminary chemical–physical characterization of the extract was carried out by means of FT-IR, TGA and XPS analyses. Skin permeation kinetics of the extract conveyed by a toning lotion was studied in vitro by Franz diffusion cell, monitoring the permeated arbutin (as the target component of the extract) and the total phenols by HPLC and UV-visible spectrophotometry, respectively. Antimicrobial activity and time-killing assays were performed to evaluate the effects of Arctostaphylos uva-ursi leaf extract against planktonic C. acnes. The influence of different Arctostaphylos uva-ursi leaf extract concentrations on the biofilm biomass inhibition and degradation was evaluated by the crystal violet (CV) method. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was used to determine the viability of immortalized human keratinocytes (HaCaT) after exposure to Arctostaphylos uva-ursi leaf extract for 24 and 48 h. Levels of interleukin (IL)-1β, IL-6, IL-8 and tumour necrosis factor (TNF)-α were quantified after HaCaT cells cotreatment with Arctostaphylos uva-ursi leaf extract and heat-killed C. acnes. The minimum inhibitory concentration (MIC) which exerted a bacteriostatic action on 90% of planktonic C. acnes (MIC90) was 0.6 mg/mL. Furthermore, MIC and sub-MIC concentrations influenced the biofilm formation phases, recording a percentage of inhibition that exceeded 50 and 40% at 0.6 and 0.3 mg/mL. Arctostaphylos uva-ursi leaf extract disrupted biofilm biomass of 57 and 45% at the same concentrations mentioned above. Active Arctostaphylos uva-ursi leaf extract doses did not affect the viability of HaCaT cells. On the other hand, at 1.25 and 0.6 mg/mL, complete inhibition of the secretion of pro-inflammatory cytokines was recorded. Taken together, these results indicate that Arctostaphylos uva-ursi leaf extract could represent a natural product to counter the virulence of C. acnes, representing a new alternative therapeutic option for the treatment of acne vulgaris.

Staphylococcus aureus (S. aureus) represents an important pathogen of clinical relevance, causing a wide variety of symptoms. The broad distribution of multidrug-resistant strains necessarily demands new antibacterial agents for the treatment of S. aureus infections. The aim of this study was to assess the antibacterial activity of plant-derived compounds, pure 4,5″-dihydroxy-anthraquinone-2-carboxylic acid (Rhein), against standard and clinical isolated S. aureus strains. The hemolysis and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assays were used to determine the cytotoxicity on human erythrocytes and bronchial epithelial cells after treatment with Rhein. The antibacterial effect was assessed via disk diffusion test, broth microdilution methods, time-killing assays and live–dead evaluation (50–0.39 µg/mL). Rhein effect on the hemolytic activity of α-toxin and catalase were estimated. Moreover, crystal violet (CV) assay evaluated its impact on biofilm biomass. The compound exhibited 50% cytotoxic concentration (CC50) and 50% hemolysis concentration (EC50) of 43.6 and >50 µg/mL, respectively. The minimum inhibitory concentration (MIC) of Rhein was 12.5 µg/mL for all tested strains, exerting bacteriostatic action. MIC and sub-MIC concentrations of Rhein significantly reduced hemolytic and catalase activities, impairing the major virulence factors of S. aureus strains. Rhein also reduced biofilm biomass in a dose-dependent manner, reaching rates of about 50% eradication at a dose of 50 µg/mL. These findings suggest that Rhein could represent a promising therapeutic option for the treatment of S. aureus infections.

The ocular microbiome is of fundamental importance for immune eye homeostasis, and its alteration would lead to an impairment of ocular functionality. Little evidence is reported on the composition of the ocular microbiota of term infants and on the impact of antibiotic prophylaxis. Methods: A total of 20 conjunctival swabs were collected from newborns at birth and after antibiotic treatment. Samples were subjected to 16S rRNA sequencing via system MiSeq Illumina. The data were processed with the MicrobAT software and statistical analysis were performed using two-way ANOVA. Results: Antibiotic prophylaxis with gentamicin altered the composition of the microbiota. In detail, a 1.5- and 2.01-fold reduction was recorded for Cutibacterium acnes (C. acnes) and Massilia timonae (M. timonae), respectively, whereas an increase in Staphylococcus spp. of 6.5 times occurred after antibiotic exposure. Conclusions: Antibiotic prophylaxis altered the ocular microbiota whose understanding could avoid adverse effects on eye health.

Reproductive tract infections account for approximately 15% of male infertility cases. Escherichia coli (E. coli) represents the most frequently isolated bacterial strain in the semen of infertile men. All Gram-negative bacteria constitutively produce outer membrane vesicles (OMVs). The present study proved, for the first time, the involvement of OMVs in human sperm function. E. coli OMVs were isolated by ultracentrifugation and characterized via sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis. Human sperm was exposed to OMVs (8 µg/mL) for different times (30, 45, 60 and 90 min). The vitality, motility, morphology, ROS level and DNA fragmentation of spermatozoa were evaluated. OMVs reduced the progressive motility and increased the immobile spermatozoa amount after 30 min of treatment. In addition, a significant increase in the percentage of intracellular ROS and sperm DNA fragmentation was recorded for each vesicular exposure time. These preliminary findings prove that OMVs contribute to altering human sperm function via two mechanisms: (i) impaired motility and (ii) DNA fragmentation.

Microbial biofilms pose a serious threat to patients requiring medical devices (MDs). Prolonged periods of implantation carry a high risk of device-related infections (DRIs). Patients with DRIs often have negative outcomes following the failure of antibiotic treatment. Resistant DRIs are mainly due to the MDs contamination by bacteria producing biofilm. The present study aimed to detect biofilm formation among MD bacterial isolates and to explore their antibiotic resistance profile. The study was conducted on 76 MDs, collected at University Hospital of Campania “Luigi Vanvitelli,” between October 2019 and September 2020. Identification of isolates and antibiotic susceptibility testing were performed using Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and Phoenix Becton Dickinson, respectively. Biofilm-forming abilities were assessed using the tissue culture plate (TCP) method. Among the 94 MDs isolated strains, 42.7% were Gram-positive, 40.3% Gram-negative, and 17% Candida species. Among 78 bacterial strains, 43.6% were non-biofilm producers while 56.4% produced biofilms. All biofilm producing isolates were sensitive to a limited spectrum of antibiotic classes. All moderate and strong biofilm producers and 81% of weak biofilm producers were Multidrug Resistance (MDR) strains. In contrast, among non-biofilm producers, only 11.8% were classified as MDR strains. Our results highlighted that Sulfamides and Glycopeptides for the major Gram-positive strains and Fluoroquinolones, Carbapenems, and Aminoglycosides for the most represented Gram-negative isolates could be the most suitable therapeutic choice for most biofilm-DRIs.

Corynebacterium striatum (C. striatum) is an emerging multidrug-resistant (MDR) pathogen associated with nosocomial infections. In this scenario, we screened the antimicrobial activity of the anthelmintic drugs doramectin, moxidectin, selamectin and niclosamide against 20 C. striatum MDR clinical isolates. Among these, niclosamide was the best performing drug against C. striatum. Niclosamide cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay on immortalized human keratinocyte cells (HaCaT). After 20 h of treatment, the recorded 50% cytotoxic concentration (CC50) was 2.56 μg/mL. The antibacterial efficacy was determined via disc diffusion, broth microdilution method and time-killing. Against C. striatum, niclosamide induced a growth inhibitory area of 22 mm and the minimum inhibitory concentration that inhibits 90% of bacteria (MIC90) was 0.39 μg/mL, exhibiting bactericidal action. The biofilm biomass eradicating action was investigated through crystal violet (CV), MTT and confocal laser scanning microscopy (CLSM). Niclosamide affected the biofilm viability in a dose-dependent manner and degraded biomass by 55 and 49% at 0.39 μg/mL and 0.19 μg/mL. CLSM images confirmed the biofilm biomass degradation, showing a drastic reduction in cell viability. This study could promote the drug-repurposing of the anthelmintic FDA-approved niclosamide as a therapeutic agent to counteract the C. striatum MDR infections.

Aerobic vaginitis (AV) is a vaginal infectious condition, characterized by a high inflammatory response and/or signs of epithelial atrophy, a decrease in the amount of Lactobacillus spp. and an increase in enteric origin bacteria. AV, often misdiagnosed, is difficult to treat due to the emerging spread of multi-drug resistant bacterial strains. The present study aimed to define the prevalence of AV, to detect causative bacteria and their antimicrobial resistance pattern. Women 10–95 years old, admitted to San Giovanni di Dio e Ruggi d’Aragona Hospital, Salerno, Italy (in the years 2015–2019) are included in the study. Bacterial identification and antibiotic susceptibility tests were carried out by VITEK® 2. Among 2069 patients, 1176 tested positive for microbial growth. A higher incidence of infection was found in the 55–64 age group. Among the pathogenic strains, 50.4% were Gram-negative, and 49.6% were Gram-positive. Escherichia coli (E. coli) (32.5%) was the most representative strain, followed by Enterococcus faecalis (E. faecalis) (29.4%), Klebsiella pneumoniae (K. pneumoniae) (7.8%) and Enterococcus faecium (E. faecium) (7.7%). E. coli showed high sensitivity to carbapenems and amikacin. K. pneumoniae carbapenems resistance was fluctuating over time. Alarming resistance to vancomycin was not recorded for Enterococci. Both strains were sensitive to teicoplanin, linezolid and tigecycline. Proper diagnosis and an effective therapeutic approach are needed to improve AV management.

Lower respiratory tract infections (LRTIs) are the most common infections in humans. It is estimated that 2.74 million deaths worldwide occur each year due to LRTIs. The aim of the study was to determine the frequency and antibiotic susceptibility pattern of microorganisms isolated from respiratory samples of patients with LRTIs. Between January 2015 and December 2019, a total of 7038 sputum and bronchoaspirate samples from suspected LRTI patients were collected. Among them, 2753 samples (39.1%) showed significant microbial growth on culture media. The LRTI rate was higher in patients with male gender (67.1%) and with age between 40–59 years (48.6%). The microorganism identification and antibiotic susceptibility testing were performed with Vitek 2. Out of 4278 isolates species, 3102 (72.5%) were Gram-negative bacteria, 1048 (24.5%) were Gram-positive bacteria, and 128 (3.0%) were Candida spp. Major microorganisms isolated were Acinetobacter baumannii (18.6%), Staphylococcus aureus (15.2%), Pseudomonas aeruginosa (14.2%), and Klebsiella pneumoniae (10.9%). In antimicrobial susceptibility testing, Staphylococcus aureus isolates were mostly resistant to Penicillin G (84.1%) and Oxacillin (48.1%), whereas they demonstrated maximum sensitivity to Tigecycline (100%) and Linezolid (99.5%). Among Gram-negative isolates, Acinetobacter baumannii showed maximum sensitivity to Colistin but was resistant to other antibiotics (95–99%). Klebsiella pneumoniae isolates were mostly resistant to Cefotaxime (72.7%) and sensitive to Gentamicin (54.3%), and Pseudomonas aeruginosa was resistant to Ciprofloxacin (40.3%) and sensitive to Amikacin (85.9%). Gram-negative bacteria represented the species most commonly isolated. A high rate of antimicrobial resistance was observed in this study. In conclusion, the correct identification of causative microorganisms and their susceptibility patterns to antibiotics is crucial for choosing targeted and effective antibiotic therapy in LRTIs, and to prevent the emergence of multidrug-resistant bacteria.

Klebsiella pneumoniae is an opportunistic pathogen that causes nosocomial and community-acquired infections. The spread of resistant strains of K. pneumoniae represents a growing threat to human health, due to the exhaustion of effective treatments. K. pneumoniae releases outer membrane vesicles (OMVs). OMVs are a vehicle for the transport of virulence factors to host cells, causing cell injury. Previous studies have shown changes of gene expression in human bronchial epithelial cells after treatment with K. pneumoniae OMVs. These variations in gene expression could be regulated through microRNAs (miRNAs), which participate in several biological mechanisms. Thereafter, miRNA expression profiles in human bronchial epithelial cells were evaluated during infection with standard and clinical K. pneumoniae strains. Microarray analysis and RT-qPCR identified the dysregulation of miR-223, hsa-miR-21, hsa-miR-25 and hsa-let-7g miRNA sequences. Target gene prediction revealed the essential role of these miRNAs in the regulation of host immune responses involving NF-ĸB (miR-223), TLR4 (hsa-miR-21), cytokine (hsa-miR-25) and IL-6 (hsa-let-7g miRNA) signalling pathways. The current study provides the first large scale expression profile of miRNAs from lung cells and predicted gene targets, following exposure to K. pneumoniae OMVs. Our results suggest the importance of OMVs in the inflammatory response.