Antimicrobial efficacy of direct air gas soft jet plasma for the in vitro reduction of oral bacterial biofilms.

The aim of this study was to evaluate the antimicrobial efficacy of an air gas soft jet CAP for its potential use in removing oral biofilms, given that plasma-based technologies have emerged as promising methods in periodontology. Two types of biofilms were developed, one by Streptococcus mutans UA 159 bacterial strain and the other by a complex mixture of saliva microorganisms isolated from a patient with periodontitis. This latter biofilm was characterized via Next Generation Sequencing to determine the main bacterial phyla. The CAP source was applied at a distance of 6 mm for different time points. A statistically significant reduction of both CFU count and XTT was already detected after 60 s of CAP treatment. CLSM analysis supported CAP effectiveness in killing the microorganisms inside the biofilm and in reducing the thickness of the biofilm matrix. Cytotoxicity tests demonstrated the possible use of CAP without important side effects towards human gingival fibroblasts cell line. The current study showed that CAP treatment was able to significantly reduce preformed biofilms developed by both S. mutans and microorganisms isolated by a saliva sample. Further studies should be conducted on biofilms developed by additional saliva donors to support the potential of this innovative strategy to counteract oral pathogens responsible for periodontal diseases.

Biofilm and bacterial membrane vesicles: recent advances.

INTRODUCTION: Bacterial Membrane Vesicles (MVs) play important roles in cell-to-cell communication and transport of several molecules. Such structures are essential components of Extracellular Polymeric Substances (EPS) biofilm matrix of many bacterial species displaying a structural function and a role in virulence and pathogenesis. AREAS COVERED: In this review were included original articles from the last ten years by searching the keywords 'biofilm' and 'vesicles' on PUBMED and Scopus databases. The articles available in literature mainly describe a positive correlation between bacterial MVs and biofilms formation. The research on Espacenet and Google Patent databases underlines the available patents related to the application of both biofilm MVs and planktonic MVs in inhibiting biofilm formation. EXPERT OPINION: This review covers and analyzes recent advances in the study of the relationship between bacterial vesicles and biofilm. The huge number of papers discussing the role of MVs confirms the interest aimed at developing new applications in the medical field. The study of the MVs composition and biogenesis may contribute to the identification of components which could be (i) the target for the development of new drugs inhibiting the biofilm establishment; (ii) candidates for the development of vaccines; (iii) biomarkers for the diagnosis of bacterial infections.

The Immune System Response to Porphyromonas gingivalis in Neurological Diseases.

Previous studies have reported an association between oral microbial dysbiosis and the development and progression of pathologies in the central nervous system. Porphyromonas gingivalis (Pg), the keystone pathogen of the oral cavity, can induce a systemic antibody response measured in patients' sera using enzyme-linked immunosorbent assays. The present case-control study quantified the immune system's response to Pg abundance in the oral cavities of patients affected by different central nervous system pathologies. The study cohort included 87 participants: 23 healthy controls (HC), 17 patients with an acute neurological condition (N-AC), 19 patients with a chronic neurological condition (N-CH), and 28 patients with neurodegenerative disease (N-DEG). The results showed that the Pg abundance in the oral cavity was higher in the N-DEG patients than in the HC (p = 0.0001) and N-AC patients (p = 0.01). In addition, the Pg abundance was higher in the N-CH patients than the HCs (p = 0.005). Only the N-CH patients had more serum anti-Pg antibodies than the HC (p = 0.012). The inadequate response of the immune system of the N-DEG group in producing anti-Pg antibodies was also clearly indicated by an analysis of the ratio between the anti-Pg antibodies quantity and the Pg abundance. Indeed, this ratio was significantly lower between the N-DEG group than all other groups (p = 0.0001, p = 0.002, and p = 0.03 for HC, N-AC, and N-CH, respectively). The immune system's response to Pg abundance in the oral cavity showed a stepwise model: the response diminished progressively from the patients affected with an acute condition to the patients suffering from chronic nervous system disorders and finally to the patients affected by neurodegenerative diseases.

Erlotinib-containing benzenesulfonamides as anti-Helicobacter pylori agents through carbonic anhydrase inhibition.

Aim: Development of dual-acting antibacterial agents containing Erlotinib, a recognized EGFR inhibitor used as an anticancer agent, with differently spaced benzenesulfonamide moieties known to bind and inhibit Helicobacter pylori carbonic anhydrase (HpCA) or the antiviral Zidovudine. Methods & materials: Through rational design, ten derivatives were obtained via a straightforward synthesis including a click chemistry reaction. Inhibitory activity against a panel of pathogenic carbonic anhydrases and antibacterial susceptibility of H. pylori ATCC 43504 were assessed. Docking studies on α-carbonic anhydrase enzymes and EGFR were conducted to gain insight into the binding mode of these compounds. Results & conclusion: Some compounds proved to be strong inhibitors of HpCA and showed good anti-H. pylori activity. Computational studies on the targeted enzymes shed light on the interaction hotspots.

Antibiofilm activity and NMR-based metabolomic characterization of cell-free supernatant of Limosilactobacillus reuteri DSM 17938.

The microbial biofilm has been defined as a "key virulence factor" for a multitude of microorganisms associated with chronic infections. Its multifactorial nature and variability, as well as an increase in antimicrobial resistance, suggest the need to identify new compounds as alternatives to the commonly used antimicrobials. The aim of this study was to assess the antibiofilm activity of cell-free supernatant (CFS) and its sub-fractions (SurE 10 K with a molecular weight <10 kDa and SurE with a molecular weight <30 kDa), produced by Limosilactobacillus reuteri DSM 17938, vs. biofilm-producing bacterial species. The minimum inhibitory biofilm concentration (MBIC) and the minimum biofilm eradication concentration (MBEC) were determined via three different methods and an NMR metabolomic analysis of CFS and SurE 10K was performed to identify and quantify several compounds. Finally, the storage stability of these postbiotics was evaluated by a colorimetric assay by analyzing changes in the CIEL*a*b parameters. The CFS showed a promising antibiofilm activity against the biofilm developed by clinically relevant microorganisms. The NMR of CFS and SurE 10K identifies and quantifies several compounds, mainly organic acids and amino acids, with lactate being the most abundant metabolite in all the analyzed samples. The CFS and SurE 10 K were characterized by a similar qualitative profile, with the exception of formate and glycine detected only in the CFS. Finally, the CIEL*a*b parameters assess the better conditions to analyze and use these matrices for the correct preservation of bioactive compounds.

Antimicrobial and Antibiofilm Activities of Carvacrol, Amoxicillin and Salicylhydroxamic Acid Alone and in Combination vs. Helicobacter pylori: Towards a New Multi-Targeted Therapy.

The World Health Organization has indicated Helicobacter pylori as a high-priority pathogen whose infections urgently require an update of the antibacterial treatments pipeline. Recently, bacterial ureases and carbonic anhydrases (CAs) were found to represent valuable pharmacological targets to inhibit bacterial growth. Hence, we explored the underexploited possibility of developing a multiple-targeted anti-H. pylori therapy by assessing the antimicrobial and antibiofilm activities of a CA inhibitor, carvacrol (CAR), amoxicillin (AMX) and a urease inhibitor (SHA), alone and in combination. Minimal Inhibitory (MIC) and Minimal Bactericidal (MBC) Concentrations of their different combinations were evaluated by checkerboard assay and three different methods were employed to assess their capability to eradicate H. pylori biofilm. Through Transmission Electron Microscopy (TEM) analysis, the mechanism of action of the three compounds alone and together was determined. Interestingly, most combinations were found to strongly inhibit H. pylori growth, resulting in an additive FIC index for both CAR-AMX and CAR-SHA associations, while an indifferent value was recorded for the AMX-SHA association. Greater antimicrobial and antibiofilm efficacy of the combinations CAR-AMX, SHA-AMX and CAR-SHA against H. pylori were found with respect to the same compounds used alone, thereby representing an innovative and promising strategy to counteract H. pylori infections.

Preparation, Characterization, and Biological Evaluation of a Hydrophilic Peptide Loaded on PEG-PLGA Nanoparticles.

The encapsulation of peptides and proteins in nanosystems has been extensively investigated for masking unfavorable biopharmaceutical properties, including short half-life and poor permeation through biological membranes. Therefore, the aim of this work was to encapsulate a small antimicrobial hydrophilic peptide (H-Ser-Pro-Trp-Thr-NH2, FS10) in PEG-PLGA (polyethylene glycol-poly lactic acid-co-glycolic acid) nanoparticles (Nps) and thereby overcome the common limitations of hydrophilic drugs, which because they facilitate water absorption suffer from rapid degradation. FS10 is structurally related to the well-known RNAIII inhibiting peptide (RIP) and inhibits S. aureus biofilm formation. Various parameters, including different method (double emulsion and nanoprecipitation), pH of the aqueous phase and polymeric composition, were investigated to load FS10 into PEG-PLGA nanoparticles. The combination of different strategies resulted in an encapsulation efficiency of around 25% for both the double emulsion and the nanoprecipitation method. It was found that the most influential parameters were the pH­which tailors the peptides charge­and the polymeric composition. FS10-PEG-PLGA nanoparticles, obtained under optimized parameters, showed size lower than 180 nm with zeta potential values ranging from −11 to −21 mV. In vitro release studies showed that the Nps had an initial burst release of 48−63%, followed by a continuous drug release up to 21 h, probably caused by the porous character of the Nps. Furthermore, transmission electron microscopy (TEM) analysis revealed particles with a spherical morphology and size of around 100 nm. Antimicrobial assay showed that the minimum inhibitory concentration (MIC) of the FS10-loaded Nps, against S. aureus strains, was lower (>128 µg/mL) than that of the free FS10 (>256 µg/mL). The main goal of this work was to develop polymeric drug delivery systems aiming at protecting the peptide from a fast degradation, thus improving its accumulation in the target site and increasing the drug-bacterial membrane interactions.

Selective Inhibition of Helicobacter pylori Carbonic Anhydrases by Carvacrol and Thymol Could Impair Biofilm Production and the Release of Outer Membrane Vesicles.

Helicobacter pylori, a Gram-negative neutrophilic pathogen, is the cause of chronic gastritis, peptic ulcers, and gastric cancer in humans. Current therapeutic regimens suffer from an emerging bacterial resistance rate and poor patience compliance. To improve the discovery of compounds targeting bacterial alternative enzymes or essential pathways such as carbonic anhydrases (CAs), we assessed the anti-H. pylori activity of thymol and carvacrol in terms of CA inhibition, isoform selectivity, growth impairment, biofilm production, and release of associated outer membrane vesicles-eDNA. The microbiological results were correlated by the evaluation in vitro of H. pylori CA inhibition, in silico analysis of the structural requirements to display such isoform selectivity, and the assessment of their limited toxicity against three probiotic species with respect to amoxicillin. Carvacrol and thymol could thus be considered as new lead compounds as alternative H. pylori CA inhibitors or to be used in association with current drugs for the management of H. pylori infection and limiting the spread of antibiotic resistance.

Microbial Species Isolated from Infected Wounds and Antimicrobial Resistance Analysis: Data Emerging from a Three-Years Retrospective Study.

The antimicrobial resistance is a topic of global interest in the treatment of wound infections. The goal of this retrospective study was both the identification of the microorganisms responsible for wound infections and the determination of their drug susceptibility pattern. The study was performed from 2017 to 2019 and included 239 patients. Thirty-four species were isolated by culture methods and identified and analysed for their susceptibility patterns to antimicrobials through the Walk Away automated system. The presence of one species was the most frequent condition (75.3%), whereas a co-infection was detected in 24.7% of samples. The most common species were Gram-negative (57.9%), amongst which the most prevalent were Pseudomonas aeruginosa (40.2%), Escherichia coli (20.7%), Proteus mirabilis (11.2%), and Acinetobacter baumannii/haemolyticus (9.5%). Gram-positive bacteria were observed in 36.6%, Staphylococcus aureus (79.4%) being the most predominant species. At least one resistance to antibiotics was detected in 88.2% of isolates, while a multi-drug-resistance versus no less than 6 antimicrobials was detected in 29.2% of isolates. Although multi-drug resistant species and co-infections were observed, those were less frequently observed at the wound site. These conditions make the microorganisms eradication more difficult. The detection of a polymicrobial infection and multi-drug resistant microorganisms followed by a proper therapeutic treatment would lead to the resolution of the infection, promoting wound healing and the limitation of the spread of antibiotic resistance.

Correlation between the Antimicrobial Activity and Metabolic Profiles of Cell Free Supernatants and Membrane Vesicles Produced by Lactobacillus reuteri DSM 17938.

The aim of the work is to assess the antimicrobial activities of Cell Free Supernatants (CFS) and Membrane Vesicles (MVs), produced by Lactobacillus reuteri DSM 17938, versus Gram-positive and Gram-negative bacteria and investigate their metabolic profiles. The Minimum Inhibitory Concentration was determined through the broth microdilution method and cell proliferation assay and the Minimum Bactericidal Concentration was determined by Colony Forming Units counts. The characteristics of the antimicrobial compounds were evaluated by pH adjustments, proteinase treatment, and size fractionation of the CFS. The cytotoxicity of CFS was tested on two human cell lines. A detailed snapshot of the L. reuteri metabolism was attained through an untargeted metabolic profiling by means of high resolution Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS) coupled with Electrospray Ionization Source (ESI). The results showed (i) a greater efficacy of CFS and its fractions towards Gram-negative compared to Gram-positive bacteria; (ii) an antimicrobial effect related to pH-dependent compounds but not to MVs; (iii) a molecular weight < 3 KDa as well as an a non-proteinaceous nature of the antimicrobial compounds; and (iv) more than 200 and 500 putative metabolites annotated in MVs and supernatants, covering several classes of metabolites, including amino acids, lipids, fatty and organic acids, polyalcohols, nucleotides, and vitamins. Some putative compounds were proposed not only as characteristic of specific fractions, but also possibly involved in antimicrobial activity.

Antimicrobial and Antibiofilm Activities of New Synthesized Silver Ultra-NanoClusters (SUNCs) Against Helicobacter pylori.

Helicobacter pylori colonizes approximately 50% of the world's population, and it is the cause of chronic gastritis, peptic ulcer disease, and gastric cancer. The increase of antibiotic resistance is one of the biggest challenges of our century due to its constant increase. In order to identify an alternative or adjuvant strategy to the standard antibiotic therapy, the in vitro activity of newly synthesized Silver Ultra-NanoClusters (SUNCs), characterized by an average size inferior to 5 nm, against clinical strains of H. pylori, with different antibiotic susceptibilities, was evaluated in this study. MICs and MBCs were determined by the broth microdilution method, whereas the effect of drug combinations was determined by the checkerboard assay. The Minimum Biofilm Eradication Concentration (MBEC) was measured using AlamarBlue (AB) assay and colony-forming unit (CFU) counts. The cytotoxicity was evaluated by performing the MTT assay on the AGS cell line. The inhibitory activity was expressed in terms of bacteriostatic and bactericidal potential, with MIC50, MIC90, and MBC50 of 0.33 mg/L against planktonic H. pylori strains. Using the fractional inhibitory concentration index (FICI), SUNCs showed potential synergism with metronidazole and clarithromycin. The biofilm eradication was obtained after treatment with 2×, 3×, and 4× MIC values. Moreover, SUNCs showed low toxicity on human cells and were effective in eradicating a mature biofilm produced by H. pylori. The data presented in this study demonstrate that SUNCs could represent a novel strategy for the treatment of H. pylori infections either alone or in combination with metronidazole.

Detection and Quantification of eDNA-Associated Bacterial Membrane Vesicles by Flow Cytometry.

Bacteria generate membrane vesicles, which are structures known as extracellular vesicles (EVs), reported to be involved in different pathogenic mechanisms, as it has been demonstrated that EVs participate in biofilm formation, cell-to-cell communication, bacteria-host interactions, and nutrients supply. EVs deliver nucleic acids, proteins, and polysaccharides. It has been reported that Helicobacter pylori (H. pylori) and Lactobacillus reuteri (L. reuteri), of both planktonic and biofilm phenotypes, produce EVs carrying extracellular DNA (eDNA). Here, we used polychromatic flow cytometry (PFC) to identify, enumerate, and characterize EVs as well as the eDNA-delivering EV compartment in the biofilm and planktonic phenotypes of H.pylori ATCC 43629 and L. reuteri DSM 17938. Biofilm formation was demonstrated and analyzed by fluorescence microscopy, using a classical live/dead staining protocol. The enumeration of EVs and the detection of eDNA-associated EVs were performed by PFC, analyzing both whole samples (cells plus vesicles) and EVs isolated by ultracentrifugation confirm EVs isolated by ultracentrifugation. PFC analysis was performed relying on a known-size beaded system and a mix of three different fluorescent tracers. In detail, the whole EV compartment was stained by a lipophilic cationic dye (LCD), which was combined to PKH26 and PicoGreen that selectively stain lipids and DNA, respectively. Fluorescence microscopy results displayed that both H. pylori and L. reuteri produced well-structured biofilms. PFC data highlighted that, in both detected bacterial species, biofilms produced higher EVs counts when paralleled to the related planktonic phenotypes. Furthermore, the staining with PicoGreen showed that most of the generated vesicles were associated with eDNA. These data suggest that the use of PFC, set according to the parameters here described, allows for the study of the production of eDNA-associated EVs in different microbial species in the same or several phases of growth, thus opening new perspectives in the study of microbial derived EVs in clinical samples.

The Antibiofilm Effect of a Medical Device Containing TIAB on Microorganisms Associated with Surgical Site Infection.

Surgical site infections (SSIs) represent the most common nosocomial infections, and surgical sutures are optimal surfaces for bacterial adhesion and biofilm formation. Staphylococcus spp., Enterococcus spp., and Escherichia coli are the most commonly isolated microorganisms. The aim of this research was to evaluate the antibiofilm activity of a medical device (MD) containing TIAB, which is a silver-nanotech patented product. The antibacterial effect was evaluated against Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212, and E. coli ATCC 25922 by assessing the minimum inhibitory concentration (MIC) by the Alamar Blue® (AB) assay. The antibiofilm effect was determined by evaluation of the minimum biofilm inhibitory concentration (MBIC) and colony-forming unit (CFU) count. Subsequently, the MD was applied on sutures exposed to the bacterial species. The antimicrobial and antibiofilm effects were evaluated by the agar diffusion test method, confocal laser scanning microscopy (CLSM), and scanning electron microscopy (SEM). The MIC was determined for S. aureus and E. faecalis at 2 mg/mL, while the MBIC was 1.5 mg/mL for S. aureus and 1 mg/mL for E. faecalis. The formation of an inhibition zone around three different treated sutures confirmed the antimicrobial activity, while the SEM and CLSM analysis performed on the MD-treated sutures underlined the presence of a few adhesive cells, which were for the most part dead. The MD showed antimicrobial and antibiofilm activities versus S. aureus and E. faecalis, but a lower efficacy against E. coli. Surgical sutures coated with the MD have the potential to reduce SSIs as well as the risk of biofilm formation post-surgery.

Identification and characterization of the α-CA in the outer membrane vesicles produced by Helicobacter pylori.

The genome of Helicobacter pylori encodes for carbonic anhydrases (CAs, EC 4.2.1.1) belonging to the α- and ß-CA classes, which together with urease, have a pivotal role in the acid acclimation of the microorganism within the human stomach. Recently, in the exoproteome of H. pylori, a CA with no indication of the corresponding class was identified. Here, using the protonography and the mass spectrometry, a CA belonging to the α-class was detected in the outer membrane vesicles (OMVs) generated by planktonic and biofilm phenotypes of four H. pylori strains. The amount of this metalloenzyme was higher in the planktonic OMVs (pOMVs) than in the biofilm OMVs (bOMVs). Furthermore, the content of α-CA increases over time in the pOMVs. The identification of the α-CA in pOMVs and bOMVs might shed new light on the role of this enzyme in the colonization, survival, persistence, and pathogenesis of H. pylori.

Chromatographic Analyses, In Vitro Biological Activities, and Cytotoxicity of Cannabis sativa L. Essential Oil: A Multidisciplinary Study.

Due to renewed interest in the cultivation and production of Italian Cannabis sativa L., we proposed a multi-methodological approach to explore chemically and biologically both the essential oil and the aromatic water of this plant. We reported the chemical composition in terms of cannabinoid content, volatile component, phenolic and flavonoid pattern, and color characteristics. Then, we demonstrated the ethnopharmacological relevance of this plant cultivated in Italy as a source of antioxidant compounds toward a large panel of enzymes (pancreatic lipase, α-amylase, α-glucosidase, and cholinesterases) and selected clinically relevant, multidrug-sensible, and multidrug-resistant microbial strains (Staphylococcus aureus, Helicobacter pylori, Candida, and Malassezia spp.), evaluating the cytotoxic effects against normal and malignant cell lines. Preliminary in vivo cytotoxicity was also performed on Galleria mellonella larvae. The results corroborate the use of this natural product as a rich source of important biologically active molecules with particular emphasis on the role exerted by naringenin, one of the most important secondary metabolites.

Detection and Physicochemical Characterization of Membrane Vesicles (MVs) of Lactobacillus reuteri DSM 17938.

Membrane vesicles (MVs) are bilayer structures which bleb from bacteria, and are important in trafficking biomolecules to other bacteria or host cells. There are few data about MVs produced by the Gram-positive commensal-derived probiotic Lactobacillus reuteri; however, MVs from this species may have potential therapeutic benefit. The aim of this study was to detect and characterize MVs produced from biofilm (bMVs), and planktonic (pMVs) phenotypes of L. reuteri DSM 17938. MVs were analyzed for structure and physicochemical characterization by Scanning Electron Microscope (SEM) and Dynamic Light Scattering (DLS). Their composition was interrogated using various digestive enzyme treatments and subsequent Transmission Electron Microscopy (TEM) analysis. eDNA (extracellular DNA) was detected and quantified using PicoGreen. We found that planktonic and biofilm of L. reuteri cultures generated MVs with a broad size distribution. Our data also showed that eDNA was associated with pMVs and bMVs (eMVsDNA). DNase I treatment demonstrated no modifications of MVs, suggesting that an eDNA-MVs complex protected the eMVsDNA. Proteinase K and Phospholipase C treatments modified the structure of MVs, showing that lipids and proteins are important structural components of L. reuteri MVs. The biological composition and the physicochemical characterization of MVs generated by the probiotic L. reuteri may represent a starting point for future applications in the development of vesicles-based therapeutic systems.

Variants of G protein-coupled receptors: a reappraisal of their role in receptor regulation.

Truncated or shorter forms of G protein-coupled receptors (GPCRs), originating by alternative splicing, have been considered physiologically irrelevant for a rather long time. Nevertheless, it is now recognized that alternative splicing variants of GPCRs greatly increase the total number of receptor isoforms and can regulate receptor trafficking and signalling. Furthermore, dimerization of these truncated variants with other receptors concurs to expand receptor diversity. Highly truncated variants of GPCRs, typically, are retained in the endoplasmic reticulum (ER) and by heteromerization prevent the wild-type receptor to reach the plasma membrane, exerting a dominant-negative effect on its function. This can be responsible for some pathological conditions but in some other cases, it can offer protection from a disease because the expression of the receptor, that is necessary for binding an infectious agent, is attenuated. Here, we propose a possible new mechanism of creation of truncated GPCR variants through an internal ribosome entry site (IRES), a nucleotide sequence that allows cap independent translation of proteins by recruiting the ribosome in proximity of an internal initiation codon. We suggest that an IRES, situated in the third cytoplasmic loop, could be responsible for the translation of the last two transmembrane (TM) regions of the muscarinic M2receptor. IRES driven expression of this C-terminal part of the muscarinic M2receptor could represent a novel and additional mechanism of receptor regulation.