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.

Immunophenotyping of hemocytes from infected Galleria mellonella larvae as an innovative tool for immune profiling, infection studies and drug screening.

In recent years, there has been a considerable increasing interest in the use of the greater wax moth Galleria mellonella as an animal model. In vivo pharmacological tests, concerning the efficacy and the toxicity of novel compounds are typically performed in mammalian models. However, the use of the latter is costly, laborious and requires ethical approval. In this context, G. mellonella larvae can be considered a valid option due to their greater ease of use and the absence of ethical rules. Furthermore, it has been demonstrated that the immune system of these invertebrates has similarity with the one of mammals, thus guaranteeing the reliability of this in vivo model, mainly in the microbiological field. To better develop the full potential of this model, we present a novel approach to characterize the hemocyte population from G. mellonella larvae and to highlight the immuno modulation upon infection and treatments. Our approach is based on the detection in isolated hemocytes from G. mellonella hemolymph of cell membrane markers typically expressed by human immune cells upon inflammation and infection, for instance CD14, CD44, CD80, CD163 and CD200. This method highlights the analogies between G. mellonella larvae and humans. Furthermore, we provide an innovative tool to perform pre-clinical evaluations of the efficacy of antimicrobial compounds in vivo to further proceed with clinical trials and support drug discovery campaigns.

Latest Update on Outer Membrane Vesicles and Their Role in Horizontal Gene Transfer: A Mini-Review.

Outer membrane vesicles (OMVs) are spherical, lipid-based nano-structures, which are released by Gram-negative bacteria in both in vitro and in vivo conditions. The size and composition of OMVs depend on not only the producer bacterial species but also cells belonging to the same strain. The mechanism of vesicles' biogenesis has a key role in determining their cargo and the pattern of macromolecules exposed on their surface. Thus, the content of proteins, lipids, nucleic acids, and other biomolecules defines the properties of OMVs and their beneficial or harmful effects on human health. Many studies have provided evidence that OMVs can be involved in a plethora of biological processes, including cell-to-cell communication and bacteria-host interactions. Moreover, there is a growing body of literature supporting their role in horizontal gene transfer (HGT). During this process, OMVs can facilitate the spreading of genes involved in metabolic pathways, virulence, and antibiotic resistance, guaranteeing bacterial proliferation and survival. For this reason, a deeper understanding of this new mechanism of genetic transfer could improve the development of more efficient strategies to counteract infections sustained by Gram-negative bacteria. In line with this, the main aim of this mini-review is to summarize the latest evidence concerning the involvement of OMVs in HGT.

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.

Nature-Inspired Compounds: Synthesis and Antibacterial Susceptibility Testing of Eugenol Derivatives against H. pylori Strains.

The antimicrobial properties of one of the most important secondary metabolites, Eugenol (EU), inspired us to design and synthesize three different series of derivatives enhancing its parent compound's anti-Helicobacter pylori activity. Thus, we prepared semisynthetic derivatives through (A) diazo aryl functionalization, (B) derivatization of the hydroxy group of EU, and (C) elongation of the allyl radical by incorporating a chalcogen atom. The antibacterial evaluation was performed on the reference NCTC 11637 strain and on three drug-resistant clinical isolates and the minimal inhibitory and bactericidal concentrations (MICs and MBCs) highlight the role of chalcogens in enhancing the antimicrobial activity (less than 4 µg/mL for some compounds) of the EU scaffold (32-64 µg/mL).

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.

Emerging compounds and therapeutic strategies to treat infections from Trypanosoma brucei: an overhaul of the last 5-years patents.

INTRODUCTION: Human African Trypanosomiasis is a neglected disease caused by infection from parasites belonging to the Trypanosoma brucei species. Only six drugs are currently available and employed depending on the stage of the infection: pentamidine, suramin, melarsoprol, eflornithine, nifurtimox, and fexinidazole. Joint research projects were launched in an attempt to find new therapeutic options for this severe and often lethal disease. AREAS COVERED: After a brief description of the recent literature on the parasite and the disease, we searched for patents dealing with the proposal of new antitrypanosomiasis agents and, following the PRISMA guidelines, we filtered the results to those published from 2018 onwards returning suitable entries, which represent the contemporary landscape of compounds/strategies against Trypanosoma brucei. In addition, some relevant publications from the overall scientific literature were also discussed. EXPERT OPINION: This review comprehensively covers and analyzes the most recent advances not only in the discovery of new inhibitors and their structure-activity relationships but also in the assessment of innovative biological targets opening new scenarios in the MedChem field. Finally, also new vaccines and formulations recently patented were described. However, natural and synthetic compounds were analyzed in terms of inhibitory activity and selective toxicity against human cells.

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.

Extracellular Vesicles of Probiotics: Shedding Light on the Biological Activity and Future Applications.

For many decades, the proper functioning of the human body has become a leading scientific topic. In the course of numerous experiments, a striking impact of probiotics on the human body has been documented, including maintaining the physiological balance of endogenous microorganisms, regulating the functioning of the immune system, enhancing the digestive properties of the host, and preventing or alleviating the course of many diseases. Recent research, especially from the last decade, shows that this health-benefiting activity of probiotics is largely conditioned by the production of extracellular vesicles. Although the importance of extracellular vesicles in the virulence of many live-threatening pathogens is widely described in the literature, much less is known with respect to the health-promoting effect of extracellular vesicles secreted by non-pathogenic microorganisms, including probiotics. Based on this, in the current review article, we decided to collect the latest literature data on the health-inducing properties of extracellular vesicles secreted by probiotics. The characteristics of probiotics' extracellular vesicles will be extended by the description of their physicochemical properties and the proteome in connection with the biological activities exhibited by these structures.

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.

In Vitro Activity of the Arylaminoartemisinin GC012 against Helicobacter pylori and Its Effects on Biofilm.

This study evaluated the in vitro activity of the arylaminoartemisinin GC012, readily obtained from dihydroartemisinin (DHA), against clinical strains of Helicobacter pylori (H. pylori) with different antibiotic susceptibilities in the planktonic and sessile state. The activity was assessed in terms of bacteriostatic and bactericidal potential. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by the broth microdilution method. After treatment with GC012, all bacterial strains showed significantly lower MIC and MBC values compared to those of DHA. The effect of combination of GC012 with antibiotics was examined using the checkerboard method. GC012 displayed synergistic interactions with metronidazole, clarithromycin, and amoxicillin in all the strains. The antibiofilm activity was evaluated via crystal violet staining, AlamarBlue® assay, colony-forming unit count, and fluorescence microscopy. At ½ MIC and » MIC concentration, both GC012 and DHA inhibited biofilm formation, but only GC012 showed a minimal biofilm eradication concentration (MBEC) on mature biofilm. Furthermore, both compounds induced structural changes in the bacterial membrane, as observed by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is thereby demonstrated that GC012 has the potential to be efficacious against H. pylori infection.

Biofilm Formation of Helicobacter pylori in Both Static and Microfluidic Conditions Is Associated With Resistance to Clarithromycin.

It is widely accepted that production of biofilm is a protective mechanism against various type of stressors, including exposure to antibiotics. However, the impact of this structure on the spread of antibiotic resistance in Helicobacter pylori is still poorly understood. Therefore, the aim of the current research was to determine the relationship between biofilm formation and antibiotic resistance of H. pylori. The study was carried out on 24 clinical strains with different resistance profiles (antibiotic-sensitive, mono-resistant, double-resistant and multidrug-resistant) against clarithromycin (CLR), metronidazole (MTZ) and levofloxacin (LEV). Using static conditions and a crystal violet staining method, a strong correlation was observed between biofilm formation and resistance to CLR but not MTZ or LEV. Based on the obtained results, three the strongest and three the weakest biofilm producers were selected and directed for a set of microfluidic experiments performed in the Bioflux system combined with fluorescence microscopy. Under continuous flow conditions, it was observed that strong biofilm producers formed twice as much of biofilm and created significantly more eDNA and in particular proteins within the biofilm matrix when compared to weak biofilm producers. Additionally, it was noticed that strong biofilm producers had higher tendency for autoaggregation and presented morphostructural differences (a greater cellular packing, shorter cells and a higher amount of both OMVs and flagella) in relation to weak biofilm counterparts. In conclusion, resistance to CLR in clinical H. pylori strains was associated with a broad array of phenotypical features translating to the ability of strong biofilm formation.

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.

Novel Biologically Active Molecules, Biomaterials, and Nanoparticles for Microbial Biofilm Control in Human Medicine.

The aim of the present special issue, proposed by two Co-Guest Editors with expertise in Clinical Microbiology and Medicinal Chemistry, is to collect and disseminate some of the most significant and innovative contributions focused on biofilm removal strategies, based on the use of natural or synthetic compounds/molecules/peptides or nanoparticles as well as biofilm formation inhibition aimed at both the control and monitoring of biofilm infections in medicine, food, industry, and natural environments [...].

Carbonic Anhydrases: New Perspectives on Protein Functional Role and Inhibition in Helicobacter pylori.

Our understanding of the function of bacterial carbonic anhydrases (CAs, EC 4.2.1.1) has increased significantly in the last years. CAs are metalloenzymes able to modulate CO2, HCO3 - and H+ concentration through their crucial role in catalysis of reversible CO2 hydration (CO2 + H2O ⇄ HCO3 - + H+). In all living organisms, CA activity is linked to physiological processes, such as those related to the transport and supply of CO2 or HCO3 -, pH homeostasis, secretion of electrolytes, biosynthetic processes and photosynthesis. These important processes cannot be ensured by the very low rate of the non-catalyzed reaction of CO2 hydration. It has been recently shown that CAs are important biomolecules for many bacteria involved in human infections, such as Vibrio cholerae, Brucella suis, Salmonella enterica, Pseudomonas aeruginosa, and Helicobacter pylori. In these species, CA activity promotes microorganism growth and adaptation in the host, or modulates bacterial toxin production and virulence. In this review, recent literature in this research field and some of the above-mentioned issues are discussed, namely: (i) the implication of CAs from bacterial pathogens in determining the microorganism growth and virulence; (ii) the druggability of these enzymes using classical CA inhibitors (CAIs) of the sulfonamide-type as examples; (iii) the role played by Helicobacter pylori CAs in the acid tolerance/adaptation of the microbe within the human abdomen; (iv) the role of CAs played in the outer membrane vesicles spawned by H. pylori in its planktonic and biofilm phenotypes; (v) the possibility of using H. pylori CAIs in combination with probiotic strains as a novel anti-ulcer treatment approach. The latter approach may represent an innovative and successful strategy to fight gastric infections in the era of increasing resistance of pathogenic bacteria to classical antibiotics.

The Potential of Colonic Tumor Tissue Fusobacterium nucleatum to Predict Staging and Its Interplay with Oral Abundance in Colon Cancer Patients.

BACKGROUND: Intestinal microbiota dysbiosis may enhance the carcinogenicity of colon cancer (CC) by the proliferation and differentiation of epithelial cells. Oral Fusobacterium nucleatum (Fn) and Porphyromonas gingivalis (Pg) have the ability to invade the gut epithelium, promoting tumor progression. The aim of the study was to assess whether the abundance of these odontopathogenic bacteria was associated with colon cancer. We also investigated how lifestyle factors could influence the oral Fn and Pg abundance and CC. METHODS: Thirty-six CC patients were included in the study to assess the Pg and Fn oral and colon tissue abundance by qPCR. Oral health data, food habits and lifestyles were also recorded. RESULTS: Patients had a greater quantity of Fn in the oral cavity than matched CC and adjacent non-neoplastic mucosa (adj t) tissues (p = 0.004 and p < 0.001). Instead, Pg was not significantly detected in colonic tissues. There was an association between the Fn quantity in the oral and CC tissue and a statistically significant relation between the Fn abundance in adenocarcinoma (ADK) and staging (p = 0.016). The statistical analysis revealed a tendency towards a greater Fn quantity in CC (p = 0.073, η2p = 0.12) for high-meat consumers. CONCLUSION: In our study, Pg was absent in colon tissues but was correlated with the oral inflammation gingival and plaque indices. For the first time, there was evidence that the Fn oral concentration can influence colon tissue concentrations and predict CC prognosis.