Inula salicina L.: Insights into Its Polyphenolic Constituents and Biological Activity.

In this study, UHPLC-HRMS analysis of the defatted methanol extract obtained from Inula salicina L. led to the identification of 58 compounds-hydroxycinnamic and hydroxybenzoic acids and their glycosides, acylquinic and caffeoylhexaric acids, and flavonoids and their glycosides. In addition, a new natural compound, N-(8-methylnepetin)-3-hydroxypiperidin-2-one was isolated and its structure was elucidated by NMR spectroscopy. The presence of a flavoalkaloid in genus Inula is described now for the first time. Chlorogenic acid was the main compound followed by 3,5-, 1,5- and 4,5-dicaffeoylquinic acids. The methanol extract was studied for its antioxidant potential by DPPH, ABTS, and FRAP assays and sun protective properties. In addition, a study was conducted to assess the effectiveness of the tested extract in inhibiting biofilm formation by Gram-positive and Gram-negative strains. Results from crystal violet tests revealed a notable decrease in biofilm mass due to the extract. The anti-biofilm efficacy was confirmed through the observation of the biofilm viability by live/dead staining. The obtained results showed that this plant extract could be used in the development of cosmetic products with antibacterial and sun protection properties.

Pathogenic Potential of Opportunistic Gram-Negative Bacteria Isolated from the Cloacal Microbiota of Free-Living Reptile Hosts Originating from Bulgaria.

Reptiles are known to be asymptomatic carriers of various zoonotic pathogens. A number of Gram-negative opportunistic commensals are causative agents of bacterial infections in immunocompromised or stressed hosts and are disseminated by reptiles, whose epidemiological role should not be neglected. Since most studies have focused on exotic species, in captivity or as pet animals, the role of wild populations as a potential source of pathogens still remains understudied. In the present study, we isolated a variety of Gram-negative bacteria from the cloacal microbiota of free-living lizard and tortoise hosts (Reptilia: Sauria and Testudines) from the Bulgarian herpetofauna. We evaluated their pathogenic potential according to their antibiotic susceptibility patterns, biofilm-forming capacity, and extracellular production of some enzymes considered to play roles as virulence factors. To our knowledge, the phenotypic manifestation of virulence factors/enzymatic activity and biofilm formation in wild reptile microbiota has not yet been widely investigated. All isolates were found to be capable of forming biofilms to some extent and 29.6% of them could be categorized as strong producers. Two strains proved to be excellent producers. The majority of the isolated strains showed extracellular production of at least one exoenzyme. The most pronounced pathogenicity could be attributed to the newly isolated Pseudomonas aeruginosa strain due to its multiresistance, excellent biofilm formation, and expression of exoenzymes.

Anti-Biofilm and Anti-Quorum-Sensing Activity of Inula Extracts: A Strategy for Modulating Chromobacterium violaceum Virulence Factors.

The formation of microbial biofilm is a self-organizing process among bacterial cells, regulated by quorum-sensing (QS) mechanisms, contributing to development of infections. These processes, either separately or in combination, significantly contribute to bacterial resistance to antibiotics and disinfectants. A novel approach to addressing the challenge of treating infections due to antibacterial resistance involves the use of plant metabolites. In recent years, there has been increasing recognition of different phytochemicals as potential modulators. In our study, we evaluated the synergistic effect of chloroform and methanol extracts from Inula species against key virulence factors, including biofilm formation, violacein production, and swarming motility. Each of the 11 examined plant extracts demonstrated the ability to reduce biofilms and pigment synthesis in C. violaceum. Two of the extracts from I. britannica exhibited significant anti-biofilm and anti-quorum-sensing effects with over 80% inhibition. Their inhibitory effect on violacein synthesis indicates their potential as anti-QS agents, likely attributed to their high concentration of terpenoids (triterpenoids, sesquiterpene lactones, and diterpenoids). Scanning electron microscopy revealed a notable reduction in biofilm biomass, along with changes in biofilm architecture and cell morphology. Additionally, fluorescence microscopy revealed the presence of metabolically inactive cells, indicating the potent activity of the extracts during treatment. These new findings underscore the effectiveness of the plant extracts from the genus Inula as potential anti-virulent agents against C. violaceum. They also propose a promising strategy for preventing or treating its biofilm formation.

An Overview of Biofilm-Associated Infections and the Role of Phytochemicals and Nanomaterials in Their Control and Prevention.

Biofilm formation is considered one of the primary virulence mechanisms in Gram-positive and Gram-negative pathogenic species, particularly those responsible for chronic infections and promoting bacterial survival within the host. In recent years, there has been a growing interest in discovering new compounds capable of inhibiting biofilm formation. This is considered a promising antivirulence strategy that could potentially overcome antibiotic resistance issues. Effective antibiofilm agents should possess distinctive properties. They should be structurally unique, enable easy entry into cells, influence quorum sensing signaling, and synergize with other antibacterial agents. Many of these properties are found in both natural systems that are isolated from plants and in synthetic systems like nanoparticles and nanocomposites. In this review, we discuss the clinical nature of biofilm-associated infections and some of the mechanisms associated with their antibiotic tolerance. We focus on the advantages and efficacy of various natural and synthetic compounds as a new therapeutic approach to control bacterial biofilms and address multidrug resistance in bacteria.

From Traditional Dairy Product "Katak" to Beneficial Lactiplantibacillus plantarum Strains.

Traditional milk products, widely consumed in many countries for centuries, have been drawing renewed attention in recent years as sources of bacteria with possible bioprotective properties. One such product for which only limited information exists is the traditional Bulgarian "katak". This fermented yogurt-like product, renowned for its taste and long-lasting properties, possesses specific sensory characteristics. In this study, 18 lactic acid bacteria (LABs) were isolated from artisanal samples made in the Northwest part of Bulgaria. A polyphasic taxonomic approach combining classical phenotypic and molecular taxonomic methods, such as multiplex PCR, 16S rDNA sequencing, and MALDI-TOF MS, was applied, leading to the identification of 13 strains. The dominance of Lactiplantibacillus plantarum was confirmed. In vitro tests with the identified strains in model systems showed a promising broad strain-specific spectrum of activity against food-borne and human pathogens (Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli). Non-purified Lactobacillus postbiotics, produced during fermentation in skimmed and soya milks and in MRS broth, were estimated as limiting agents of virulence factors. The LAB's production of lactate, acetate, and butyrate is a promising probiotic feature. A further characterization of the active strains and analysis of the purified post-metabolites are needed and are still in progress.

Ultra-Short Laser-Assisted Micro-Structure Formations on Mg/Zn Double-Doped Calcium Phosphate Ceramics for Enhanced Antimicrobial Activity.

Bacterial infection is one of the most common and harmful medical issues following the implantation of materials and devices in the body leading to antibiotic resistance of diverse bacterial strains. In this work, a novel approach is presented combining adopted laser-based patterning method in addition to doping (Mg and Zn) metal ions to prepare calcium phosphate ceramic substrate, applicable in medicine, with enhanced surface antimicrobial characteristics. The preparation of tablets containing Mg (8.44 mol%) and Zn (2.63 mol%) ß-tricalcium phosphate involved biomimetic precipitation of amorphous calcium phosphate in media of simulated body fluid enriched with Mg2+ and Zn2+ ions as well as the presence of valine as an organic additive, followed by step-wise calcination up to 1000 °C. The results from laser processing showed formation of deeper patterns with increased surface roughness (from 4.9 µm to 9.4 µm) as laser power and velocity increase, keeping constant the hatch sizes of 50 µm. The textured surfaces consist of peaks and valleys arrangement that change the morphology of Escherichia coli cells and decrease of cell viability. Our study reveals the possibilities of the application of ultra-short laser radiation as a potential alternative therapy for controlling the antimicrobial effect of the ceramic surface.

Multiple cry Genes in Bacillus thuringiensis Strain BTG Suggest a Broad-Spectrum Insecticidal Activity.

The properties of Bacillus thuringiensis strains as a biopesticide with potent action against moths, beetles, and mosquitoes have been known for decades, with individual subspecies showing specific activity against a particular pest. The aim of the present work is to characterize strains that can be used for broad-spectrum pest control in agriculture. Twenty strains of B. thuringiensis were isolated from Bulgarian soil habitats. The strains were screened for genes encoding 12 different crystal (Cry) endotoxins by PCR with specific primer pairs. Seven of the isolates contained cry genes in their genomes. B. thuringiensis strains PL1, PL3, and PL20 contained at least three different cry genes, while B. thuringiensis serovar galleriae BTG contained at least four. Moreover, scanning electron microscopy (SEM) investigation revealed the production of bipyramidal (PL1, PL3, PL20), polygonal (PL1), cubic (BTG), and spherical crystals (BTG and PL20). Potentially containing the most cry genes, the BTG genome was sequenced and annotated. It comprises 6,275,416 base pairs, does not contain plasmids, has a GC content of 35.05%, and contained 7 genes encoding crystal toxins: cry1Ab35, cry1Db, cry1Fb, cry1Ib, cry2Ab, cry8Ea1, and cry9Ba. This unique combination would possibly enable the simultaneous pesticidal action against pest species from orders Lepidoptera, Coleoptera, Diptera, and Hemiptera, as well as class Gastropoda. Whole-genome sequencing provided accurate information about the presence, localization, and classification of Cry toxins in B. thuringiensis BTG, revealing the great potential of the strain for the development of new broad-spectrum bio-insecticides.

Ciprofloxacin-Loaded Mixed Polymeric Micelles as Antibiofilm Agents.

In this work, mixed polymeric micelles (MPMs) based on a cationic poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA29-b-PCL70-b-PDMAEMA29) and a non-ionic poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (PEO99-b-PPO67-b-PEO99) triblock copolymers, blended at different molar ratios, were developed. The key physicochemical parameters of MPMs, including size, size distribution, and critical micellar concentration (CMC), were evaluated. The resulting MPMs are nanoscopic with a hydrodynamic diameter of around 35 nm, and the ζ-potential and CMC values strongly depend on the MPM's composition. Ciprofloxacin (CF) was solubilized by the micelles via hydrophobic interaction with the micellar core and electrostatic interaction between the polycationic blocks, and the drug localized it, to some extent, in the micellar corona. The effect of a polymer-to-drug mass ratio on the drug-loading content (DLC) and encapsulation efficiency (EE) of MPMs was assessed. MPMs prepared at a polymer-to-drug mass ratio of 10:1 exhibited very high EE and a prolonged release profile. All micellar systems demonstrated their capability to detach pre-formed Gram-positive and Gram-negative bacterial biofilms and significantly reduced their biomass. The metabolic activity of the biofilm was strongly suppressed by the CF-loaded MPMs indicating the successful drug delivery and release. The cytotoxicity of empty and CF-loaded MPMs was evaluated. The test reveals composition-dependent cell viability without cell destruction or morphological signs of cell death.

Effects of cationic polymers on the viability of microbial biofilms.

INTRODUCTION: The number of published biofilm studies and novel ways for studying them has risen dramatically in recent years, ow-ing to the broad application of biofilms in medicine. Some bacteria develop biofilms that are highly resistant to antimicrobial agents, resulting in persistent infections. This necessitates the development of alternative methods for combating biofilms. In this regard, the application of cationic polymers is a good candidate for realization of this strategy. AIM: The aim of our study was to investigate the potential of a newly synthesized covalently attached star copolymer of N,N'-dimeth-ylaminoethyl methacrylate and hydroxyl-bearing oligo(ethylene glycol) methacrylate [P(DMAEMA-co-HOEGMA)] to silica surfaces and its quaternized version [P(QDMAEMA-co-HOEGMA)] for destruction of biofilms formed by Bacillus subtilis or Pseudomonas aeruginosa. MATERIALS AND METHODS: Model strains representing different genera and taxonomic groups were selected for the study. The anti-biofilm activities of two different newly synthesized cationic polymers were investigated by observation (live/dead staining) of the viability of bacterial cells within the biofilm. RESULTS: The results obtained by the live/dead labeling of bacterial biofilms show a substantial decrease in the viability of population in the presence of cationic polymers, better expressed at B. subtilis. CONCLUSIONS: The studied two immobilized on silica wafers newly synthesized star copolymers exhibited potential for anti-biofilm effects. The results demonstrated combined potential for reducing the viability of bacterial cells within the biofilms and probably for loosening the biofilm matrix. The effect was better expressed in B. subtilis.

Bioactive Azadirachta indica and Melia azedarach leaves extracts with anti-SARS-CoV-2 and antibacterial activities.

The leaves of Azadirachta indica L. and Melia azedarach L., belonging to Meliaceae family, have been shown to have medicinal benefits and are extensively employed in traditional folk medicine. Herein, HPLC analysis of the ethyl acetate fraction of the total methanolic extract emphasized the enrichment of both A. indica L., and M. azedarach L. leaves extracts with phenolic and flavonoids composites, respectively. Besides, 4 limonoids and 2 flavonoids were isolated using column chromatography. By assessing the in vitro antiviral activities of both total leaves extracts against Severe Acute Respiratory Syndrome Corona virus 2 (SARS-CoV-2), it was found that A. indica L. and M. azedarach L. have robust anti-SARS-CoV-2 activities at low half-maximal inhibitory concentrations (IC50) of 8.451 and 6.922 µg/mL, respectively. Due to the high safety of A. indica L. and M. azedarach L. extracts with half-maximal cytotoxic concentrations (CC50) of 446.2 and 351.4 µg/ml, respectively, both displayed extraordinary selectivity indices (SI>50). A. indica L. and M. azedarach L. leaves extracts could induce antibacterial activities against both Gram-negative and positive bacterial strains. The minimal inhibitory concentrations of A. indica L. and M. azedarach L. leaves extracts varied from 25 to 100 mg/mL within 30 min contact time towards the tested bacteria. Our findings confirm the broad-spectrum medicinal value of A. indica L. and M. azedarach L. leaves extracts. Finally, additional in vivo investigations are highly recommended to confirm the anti-COVID-19 and antimicrobial activities of both plant extracts.

A Thermostable Lipase Isolated from Brevibacillus thermoruber Strain 7 Degrades Ɛ-Polycaprolactone.

The tremendous problem with plastic waste accumulation has determined an interest in biodegradation by effective degraders and their enzymes, such as thermophilic enzymes, which are characterized by high catalytic rates, thermostability, and optimum temperatures close to the melting points of some plastics. In the present work, we report on the ability of a thermophilic lipase, by Brevibacillus thermoruber strain 7, to degrade Ɛ-polycaprolactone (PCL), as well as the enzyme purification, the characterization of its physicochemical properties, the product degradation, and its disruptive effect on the PCL surface. The pure enzyme showed the highest reported optimum temperature at 55 °C and a pH of 7.5, while its half-life at 60 °C was more than five hours. Its substrate specificity referred the enzyme to the subgroup of lipases in the esterase group. A strong inhibitory effect was observed by detergents, inhibitors, and Fe3+ while Ca2+ enhanced its activity. The monomer Ɛ-caprolactone was a main product of the enzyme degradation. Similar elution profiles of the products received after treatment with ultra-concentrate and pure enzyme were observed. The significant changes in PCL appearance comprising the formation of shallower or deeper in-folds were observed after a week of incubation. The valuable enzyme properties of the lipase from Brevibacillus thermoruber strain 7, which caused a comparatively quick degradation of PCL, suggests further possible exploration of the enzyme for effective and environment-friendly degradation of PCL wastes in the area of thermal basins, or in thermophilic remediation processes.

Polyphasic characterisation of Microcoleusautumnalis (Gomont, 1892) Strunecky, Komárek & J.R.Johansen, 2013 (Oscillatoriales, Cyanobacteria) using a metabolomic approach as a complementary tool.

As a result of the continuous revision of cyanobacterial taxonomy, Phormidiumautumnale (Agardh) Trevisan ex Gomont, 1892 has been transferred to the genus Microcoleus as Microcoleusautumnalis (Gomont, 1892) Strunecky, Komárek & J.R.Johansen, 2013. This transfer was based on a single strain and literature data. In the present study, we revise the taxonomic position of Microcoleusautumnalis by applying the classical approach of polyphasic taxonomy and additionally using metabolomics. Cyanobacterial strains identified as Phormidiumautumnale and Microcoleusvaginatus (type species of the genus Microcoleus) were used for comparative analyses. In addition, the taxonomic relationship between the species Phormidiumautumnale and Phormidiumuncinatum was determined on the basis of polyphasic characteristics. Monitoring of the morphological variability of Phormidiumautumnale and Microcoleusvaginatus strains showed a difference in the morphology concerning the ends of the trichomes, the shape of the apical cells, as well as the presence/absence of the calyptra and its shape. The performed TEM analysis of the thylakoid arrangement of the studied strains showed parietal arrangement of the thylakoids in the representatives of genus Phormidium and fascicular arrangement in genus Microcoleus. Molecular genetic analyses, based on 16S rDNA, revealed grouping of the investigated P.autumnale strains in a separate clade. This clade is far from the subtree, which is very clearly formed by the representatives of the type species of genus Microcoleus, namely M.vaginatus. The metabolomic analysis involving P.autumnale and M.vaginatus strains identified 39 compounds that could be used as potential biochemical markers to distinguish the two cyanobacterial species. Based on the data obtained, we suggest changing of the current status of Microcoleusautumnalis by restoring its previous appurtenance to the genus Phormidium under the name Phormidiumautumnale (Agardh) Trevisan ex Gomont, 1892 and distinguishing this species from genus Microcoleus.

Bulgarian Medicinal Extracts as Natural Inhibitors with Antiviral and Antibacterial Activity.

BACKGROUND: Bulgaria is a country with a wide range of medicinal plants, with uses in traditional medicine dating back for centuries. METHODS: Disc diffusion assay was used to evaluate the antimicrobial activity of the plant extracts. A cytopathic effect inhibition test was used for the assessment of the antiviral activity of the extracts. The virucidal activity of the extracts, their influence on the stage of viral adsorption, and their protective effect on uninfected cells were reported using the end-point dilution method, and Δlgs was determined as compared to the untreated controls. RESULTS: The results of the study reveal that the antibacterial potential of G. glabra and H. perforatum extracts in Gram-positive bacteria is more effective than in Gram-negative bacteria. When applied during the replication of HSV-1 and HCov-OC-43, only some of the extracts showed weak activity, with SI between 2 to 8.5. Almost all tested extracts inhibited the extracellular virions of the studied enveloped viruses (HSV-1 and HCov-OC-43) to a greater extent than of the non-enveloped viruses (PV-1 and HAdV-5). They inhibited the stage of viral adsorption (HSV-1) in the host cell (MDBK) to varying degrees and showed a protective effect on healthy cells (MDBK) before they were subjected to viral invasion (HSV-1). CONCLUSION: The antipathogenic potential of extracts of H. perforatum and G. glabra suggests their effectiveness as antimicrobial agents. All 13 extracts of the Bulgarian medicinal plants studied can be used to reduce viral yield in a wide range of viral infections.

Degradation of Poly(ε-caprolactone) by a Thermophilic Community and Brevibacillus thermoruber Strain 7 Isolated from Bulgarian Hot Spring.

The continual plastic accumulation in the environment and the hazardous consequences determine the interest in thermophiles as possible effective plastic degraders, due to their unique metabolic mechanisms and change of plastic properties at elevated temperatures. PCL is one of major biodegradable plastics with promising application to replace existing non-biodegradable polymers. Metagenomic analysis of the phylogenetic diversity in plastic contaminated area of Marikostinovo hot spring, Bulgaria revealed a higher number taxonomic groups (11) in the sample enriched without plastic (Marikostinovo community, control sample, MKC-C) than in that enriched in the presence of poly-ε-caprolactone (PCL) (MKC-P), (7). A strong domination of the phylum Proteobacteria was observed for MKC-C, while the dominant phyla in MKC-P were Deinococcus-Thermus and Firmicutes. Among the strains isolated from MKC-P, the highest esterase activity was registered for Brevibacillus thermoruber strain 7 at 55 °C. Its co-cultivation with another isolate resulted in ~10% increase in enzyme activity. During a 28-day biodegradation process, a decrease in PCL molecular weight and weight loss were established resulting in 100% degradation by MKC-P and 63.6% by strain 7. PCL degradation intermediate profiles for MKC-P and pure strain were similar. Broken plastic pieces from PCL surface and formation of a biofilm by MKC-P were observed by SEM, while the pure strain caused significant deformation of PCL probes without biofilm formation.

Plastic Degradation by Extremophilic Bacteria.

Intensive exploitation, poor recycling, low repeatable use, and unusual resistance of plastics to environmental and microbiological action result in accumulation of huge waste amounts in terrestrial and marine environments, causing enormous hazard for human and animal life. In the last decades, much scientific interest has been focused on plastic biodegradation. Due to the comparatively short evolutionary period of their appearance in nature, sufficiently effective enzymes for their biodegradation are not available. Plastics are designed for use in conditions typical for human activity, and their physicochemical properties roughly change at extreme environmental parameters like low temperatures, salt, or low or high pH that are typical for the life of extremophilic microorganisms and the activity of their enzymes. This review represents a first attempt to summarize the extraordinarily limited information on biodegradation of conventional synthetic plastics by thermophilic, alkaliphilic, halophilic, and psychrophilic bacteria in natural environments and laboratory conditions. Most of the available data was reported in the last several years and concerns moderate extremophiles. Two main questions are highlighted in it: which extremophilic bacteria and their enzymes are reported to be involved in the degradation of different synthetic plastics, and what could be the impact of extremophiles in future technologies for resolving of pollution problems.

Structural, Thermal, and Storage Stability of Rapana Thomasiana Hemocyanin in the Presence of Cholinium-Amino Acid-Based Ionic Liquids.

Novel biocompatible compounds that stabilize proteins in solution are in demand for biomedical and/or biotechnological applications. Here, we evaluated the effect of six ionic liquids, containing mono- or dicholinium [Chol]1or2 cation and anions of charged amino acids such as lysine [Lys], arginine [Arg], aspartic acid [Asp], or glutamic acid [Glu], on the structure, thermal, and storage stability of the Rapana thomasiana hemocyanin (RtH). RtH is a protein with huge biomedicinal potential due to its therapeutic, drug carrier, and adjuvant properties. Overall, the ionic liquids (ILs) induce changes in the secondary structure of RtH. However, the structure near the Cu-active site seems unaltered and the oxygen-binding capacity of the protein is preserved. The ILs showed weak antibacterial activity when tested against three Gram-negative and three Gram-positive bacterial strains. On the contrary, [Chol][Arg] and [Chol][Lys] exhibited high anti-biofilm activity against E. coli 25213 and S. aureus 29213 strains. In addition, the two ILs were able to protect RtH from chemical and microbiological degradation. Maintained or enhanced thermal stability of RtH was observed in the presence of all ILs tested, except for RtH-[Chol]2[Glu].

Enhanced cellular uptake of platinum by a tetracationic Pt(II) nanocapsule and its implications to cancer treatment.

Despite the known limitations of cisplatin chemotherapy, the treatment of cancer by platinum-based drugs remains the method of choice for many oncologists. The advancement in drug delivery formulations and protocols of combined treatments provided effective tools to ameliorate the side effects of platinum-based therapies. Another approach to improve the pharmacological profiles of anticancer platinum drugs is to properly modify their structure and composition, which has produced numerous platinum complexes with improved therapeutic effect. Recently, we have demonstrated the strong anticancer potency of supramolecular nanocapsules that form by self-assembly of four bis-anthracene ligands with two metal ions, either Pt(II) or Pd(II). Herein, we focus our study on the Pt(II) nanocapsule and its uptake by two types of cancer cells, suspension cultures of HL-60 cells and the adherent cancer cells HT-29. Comparison of the platinum uptake by cancer cells treated with the nanocapsule and with cisplatin evidenced superior uptake of platinum caused by the nanocapsule, which in HT-29 and HL-60 cells prevails by 21 and 31 times, respectively. Morphological changes in the HL-60 cells induced by the Pt(II) nanocapsule were studied by transmission electron microscopy (TEM) which provided plausible explanation of the uptake results. These data corroborate also with the known nanocapsule's very high cytotoxicity, better selectivity, and lack of cross-resistance with cisplatin. Additionally, our estimations of the drug-drug interactions in combined treatments established the propensity of the nanocapsule to exert supra-additive cytotoxicity in combination with cisplatin against the bladder cancer T-24 cells. All these findings define the scope for more detailed pharmacological characterization of the presented Pt(II) nanocapsule.

Destruction of Pseudomonas aeruginosa pre-formed biofilms by cationic polymer micelles bearing silver nanoparticles.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen often associated with biofilm infections. This study evaluated the capacity for biofilm destruction of a novel combination of cationic polymer micelles formed from poly(2-(dimethylamino)ethyl methacrylate)-b-poly(ε-caprolactone)-b-poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA-PCL-PDMAEMA) triblock copolymer either alone, or loaded with silver nanoparticles (M_AgNPs). Pre-formed P. aeruginosa biofilms were incubated with either blank micelles, AgNO3, or M_AgNPs. Biofilm biomass (crystal violet assay), metabolic activity (Alamar blue reduction), structure (SEM) and viability (CLSM after Live/Dead staining, or plating for CFU) were checked. The results showed that the micelles alone loosened the biofilm matrix, and caused some alterations in the bacterial surface. AgNO3 killed the bacteria in situ leaving dead biofilm bacteria on the surface. M_AgNPs combined the two types of activities causing significant biofilm reduction, and alteration and death of biofilm bacteria. Therefore, the applied PDMAEMA-based micelles appear to be a successful candidate for the treatment of P. aeruginosa biofilm infections.

Application of cationic polymer micelles for the dispersal of bacterial biofilms.

Contamination of surfaces in hospitals and food industry by bacterial biofilms is a serious health risk. Of concern is their resistance to routine antibacterials and disinfectants. This requires the development of novel approaches to biofilm detachment. The study evaluates the effectiveness of cationic polymer micelles (CPMs) against pre-formed biofilms. CPMs based on different polycations were used. The hydrodynamic radius of the particles ranged from 16 to 360 nm. Biofilms of Escherichia coli 420, Pseudomonas aeruginosa PAO1, Staphylococcus aureus 29213 and Bacillus subtilis 168 were cultivated for 24 h then the pre-formed biofilms were treated with the CPMs for 2, 4 or 6 h. Biofilm biomass was evaluated by the crystal violet assay, and live/dead fluorescence test was applied for bacterial viability. The ability of CPMs to interact with pre-formed biofilms of the model strains was evaluated. We observed that the most effective CPMs were those based on poly(2-(dimethylamino)ethyl methacrylate) copolymers which reduced the biofilm biomass three- to four-fold compared with the treatment of the biofilm with water. Significantly reduced vitality of the bacteria in the biofilms was registered by the live/dead stain. The results indicate the applicability of the CPMs for disinfection of biofilm-contaminated surfaces and the treatment of wounds.

Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains.

Pseudomonas aeruginosa causes chronic lung infections in people with cystic fibrosis (CF) and acute opportunistic infections in people without CF. Forty-two P. aeruginosa strains from a range of clinical and environmental sources were collated into a single reference strain panel to harmonise research on this diverse opportunistic pathogen. To facilitate further harmonized and comparable research on P. aeruginosa, we characterized the panel strains for growth rates, motility, virulence in the Galleria mellonella infection model, pyocyanin and alginate production, mucoid phenotype, LPS pattern, biofilm formation, urease activity, and antimicrobial and phage susceptibilities. Phenotypic diversity across the P. aeruginosa panel was apparent for all phenotypes examined, agreeing with the marked variability seen in this species. However, except for growth rate, the phenotypic diversity among strains from CF versus non-CF sources was comparable. CF strains were less virulent in the G. mellonella model than non-CF strains (P = 0.037). Transmissible CF strains generally lacked O-antigen, produced less pyocyanin and had low virulence in G. mellonella. Furthermore, in the three sets of sequential CF strains, virulence, O-antigen expression and pyocyanin production were higher in the earlier isolate compared to the isolate obtained later in infection. Overall, this full phenotypic characterization of the defined panel of P. aeruginosa strains increases our understanding of the virulence and pathogenesis of P. aeruginosa and may provide a valuable resource for the testing of novel therapies against this problematic pathogen.