Repurposing the Antibacterial Activity of the Drug Teniposide Against Gram‐Positive Bacteria

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

Outer Membrane Vesicles Derived from Klebsiella pneumoniae Influence the miRNA Expression Profile in Human Bronchial Epithelial BEAS-2B Cells

: 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

Klebsiella pneumoniae-OMVs activate death-signaling pathways in Human Bronchial Epithelial Host Cells (BEAS-2B)

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

Comparative analysis of peracetic acid (PAA) and permaleic acid (PMA) in disinfection processes

The growing demand to reduce chlorine usage and control disinfection byproducts increased the development of new strategies in wastewater treatments. Organic peracids are increasingly attracting interest in disinfection activities as a promising alternative to chlorine and chlorine-based agents. In this study, we assessed the antimicrobial properties against escherichia coli(E. coli)and staphylococcus aureus(S. aureus) of a new organic peracid, permaleic acid (PMA) compared with the reference peracetic acid (PAA). Disinfectant properties were evaluated by i) disk diffusion agar, ii) broth microdilution, iii) antibiofilm properties. PMA demonstrated a 10- and 5-fold decrease inthe microbial inhibitory concentration (MIC) value against E. coli andS. aureus respectively,comparedto PAA. Results showed greater efficacy of PMA regarding wastewater (WW) and treated wastewater (TWW)disinfection at low concentrations. Furthermore, the biofilm degradation ability was only observed followingPMA treatment, for both strains. Bacterial regrowth from biofilm matrix after PAA and PMA disinfection, in theabsence and presence of organic matter, was evaluated. PMA was more efficient than PAA to prevent the regrowth of planktonic cells ofS. aureusandE. coli. After PAA and PMA treatment, in the presence of organic matter,the bacterial regrowth inhibition was maintained up to 10 and 5 g/L, respectively. Based on these results, PMA could be used as a valid alternative to the currently used disinfection methods

Niclosamide as a Repurposing Drug against Corynebacterium striatum Multidrug-Resistant Infections

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

Antimicrobial Resistance in Pseudomonas aeruginosa before and during the COVID-19 Pandemic

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 (R) 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

Rhein: A novel antibacterial compound against Streptococcus mutans infection

Streptococcus mutans (S. mutans) is considered the main causative agent of dental caries. The study aims to evaluate the antimicrobial activity of a natural plant product, pure 4,5''-dihydroxy-anthraquinone-2-carboxylic acid (Rhein) against S. mutans. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to determine the viability of immortalized human keratinocytes (HaCaT) following treatment with Rhein. Assay for antimicrobial activity and the time-killing test were performed to evaluate Rhein effects against planktonic S. mutans. The effect of different concentrations of Rhein on biofilm biomass and the metabolism of biofilm cells were evaluated through crystal violet and MTT assays. Further, Rhein-treated biofilms were viewed by confocal laser scanning microscopy. Rhein effects on acid production and acid environment tolerance were also assessed. The minimum inhibitory concentration (MIC) of Rhein, exerting bacteriostatic action on 90% of planktonic S. mutans (MIC90), was 5.69 µg/mL. MIC and sub-MIC concentrations of Rhein affected the metabolism of biofilm cells and disrupted biofilm biomass with minimal biofilm eradication concentrations (MBEC) inducing 50% (MBEC50) and 90% eradication (MBEC90) of 6.31 and > 50 µg/mL, respectively. Confocal images displayed a significant reduction in biofilm biomass following treatment with increasing concentrations of the compound. Rhein also reduced the virulence of the biofilm by affecting acid production and acid tolerance. Conversely, active concentrations of Rhein did not affect HaCaT cell viability. Together, these findings indicate that Rhein, a natural product that counteracts the virulence of S. mutans, may represent a novel therapeutic option for dental caries

Postmortem interval assessment by MALDI-TOF mass spectrometry analysis in murine cadavers

This study assessed the use of matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry as an alternative method to identify species associated with the thanatomicrobiota and epinecrotic communities

Dynamics of nasopharyngeal tract phageome and association with disease severity and age of patients during three waves of COVID-1

In December 2019, several patients were hospitalized and diagnosed with severeacute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) infection, whichsubsequently led to a global pandemic. To date, there are no studies evaluatingthe relationship between the respiratory phageome and the SARS‐CoV‐2infection. The current study investigated the phageome profiles in thenasopharyngeal swabs collected from 55 patients during the three differentwaves of coronavirus disease 2019 (COVID‐19) in the Campania Region(Southern Italy). Data obtained from the taxonomic profiling show that phagefamilies belonging to the orderCaudoviraleshave a high abundance in the patientsamples. Moreover, the severity of the COVID‐19 infection seems to becorrelated with the phage abundance