Ceragenin-mediated disruption of Pseudomonas aeruginosa biofilms.

BACKGROUND: Microbial biofilms, as a hallmark of cystic fibrosis (CF) lung disease and other chronic infections, remain a desirable target for antimicrobial therapy. These biopolymer-based viscoelastic structures protect pathogenic organisms from immune responses and antibiotics. Consequently, treatments directed at disrupting biofilms represent a promising strategy for combating biofilm-associated infections. In CF patients, the viscoelasticity of biofilms is determined mainly by their polymicrobial nature and species-specific traits, such as Pseudomonas aeruginosa filamentous (Pf) bacteriophages. Therefore, we examined the impact of microbicidal ceragenins (CSAs) supported by mucolytic agents-DNase I and poly-aspartic acid (pASP), on the viability and viscoelasticity of mono- and bispecies biofilms formed by Pf-positive and Pf-negative P. aeruginosa strains co-cultured with Staphylococcus aureus or Candida albicans. METHODS: The in vitro antimicrobial activity of ceragenins against P. aeruginosa in mono- and dual-species cultures was assessed by determining minimum inhibitory concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC). Inhibition of P. aeruginosa mono- and dual-species biofilms formation by ceragenins alone and in combination with DNase I or poly-aspartic acid (pASP) was estimated by the crystal violet assay. Additionally, the viability of the biofilms was measured by colony-forming unit (CFU) counting. Finally, the biofilms' viscoelastic properties characterized by shear storage (G') and loss moduli (G"), were analyzed with a rotational rheometer. RESULTS: Our results demonstrated that ceragenin CSA-13 inhibits biofilm formation and increases its fluidity regardless of the Pf-profile and species composition; however, the Pf-positive biofilms are characterized by elevated viscosity and elasticity parameters. CONCLUSION: Due to its microbicidal and viscoelasticity-modifying properties, CSA-13 displays therapeutic potential in biofilm-associated infections, especially when combined with mucolytic agents.

Degradation of Polylactide and Polycaprolactone as a Result of Biofilm Formation Assessed under Experimental Conditions Simulating the Oral Cavity Environment.

Polylactide (PLA) and polycaprolactone (PCL) are biodegradable and bioabsorbable thermoplastic polymers considered as promising materials for oral applications. However, any abiotic surface used, especially in areas naturally colonized by microorganisms, provides a favorable interface for microbial growth and biofilm development. In this study, we investigated the biofilm formation of C. krusei and S. mutans on the surface of PLA and PCL immersed in the artificial saliva. Using microscopic (AFM, CLSM) observations and spectrometric measurements, we assessed the mass and topography of biofilm that developed on PLA and PCL surfaces. Incubated up to 56 days in specially prepared saliva and microorganisms medium, solid polymer samples were examined for surface properties (wettability, roughness, elastic modulus of the surface layer), structure (molecular weight, crystallinity), and mechanical properties (hardness, tensile strength). It has been shown that biofilm, especially S. mutans, promotes polymer degradation. Our findings indicate the need for additional antimicrobial strategies for the effective oral applications of PLA and PCL.

Modulation of Biofilm Mechanics by DNA Structure and Cell Type.

Deoxyribonucleic acid (DNA) evolved as a tool for storing and transmitting genetic information within cells, but outside the cell, DNA can also serve as "construction material" present in microbial biofilms or various body fluids, such as cystic fibrosis, sputum, and pus. In the present work, we investigate the mechanics of biofilms formed from Pseudomonas aeruginosa Xen 5, Staphylococcus aureus Xen 30, and Candida albicans 1408 using oscillatory shear rheometry at different levels of compression and recreate these mechanics in systems of entangled DNA and cells. The results show that the compression-stiffening and shear-softening effects observed in biofilms can be reproduced in DNA networks with the addition of an appropriate number of microbial cells. Additionally, we observe that these effects are cell-type dependent. We also identify other mechanisms that may significantly impact the viscoelastic behavior of biofilms, such as the compression-stiffening effect of DNA cross-linking by bivalent cations (Mg2+, Ca2+, and Cu2+) and the stiffness-increasing interactions of P. aeruginosa Xen 5 biofilm with Pf1 bacteriophage produced by P. aeruginosa. This work extends the knowledge of biofilm mechanobiology and demonstrates the possibility of modifying biopolymers toward obtaining the desired biophysical properties.

N-Acetyl-Cysteine Increases Activity of Peanut-Shaped Gold Nanoparticles Against Biofilms Formed by Clinical Strains of Pseudomonas aeruginosa Isolated from Sputum of Cystic Fibrosis Patients.

Background: Extracellular polymeric substances (EPS) produced by bacteria, as they form a biofilm, determine the stability and viscoelastic properties of biofilms and prevent antibiotics from penetrating this multicellular structure. To date, studies demonstrated that an appropriate optimization of the chemistry and morphology of nanotherapeutics might provide a favorable approach to control their interaction with EPS and/or diffusion within the biofilm matrix. Targeting the biofilms' EPS, which in certain conditions can adopt liquid crystal structure, was demonstrated to improve the anti-biofilm activity of antibiotics and nanoparticles. A similar effect is achievable by interfering EPS' production by mucoactive agents, such as N-acetyl-cysteine (NAC). In our previous study, we demonstrated the nanogram efficiency of non-spherical gold nanoparticles, which due to their physicochemical features, particularly morphology, were noted to be superior in antimicrobial activity compared to their spherical-shaped counterparts. Methods: To explore the importance of EPS matrix modulation in achieving a suitable efficiency of peanut-shaped gold nanoparticles (AuP NPs) against biofilms produced by Pseudomonas aeruginosa strains isolated from cystic fibrosis patients, fluorescence microscopy, as well as resazurin staining were employed. Rheological parameters of AuP NPs-treated biofilms were investigated by rotational and creep-recovery tests using a rheometer in a plate-plate arrangement. Results: We demonstrated that tested nanoparticles significantly inhibit the growth of mono- and mixed-species biofilms, particularly when combined with NAC. Notably, gold nanopeanuts were shown to decrease the viscosity and increase the creep compliance of Pseudomonas biofilm, similarly to EPS-targeting NAC. Synergistic activity of AuP NPs with tobramycin was also observed, and the AuP NPs were able to eradicate bacteria within biofilms formed by tobramycin-resistant isolates. Conclusion: We propose that peanut-shaped gold nanoparticles should be considered as a potent therapeutic agent against Pseudomonas biofilms.

The Effect of Physiological Incubation on the Properties of Elastic Magnetic Composites for Soft Biomedical Sensors.

Magnetic micro- and nanoparticles (MPs)-based composite materials are widely used in various applications in electronics, biotechnology, and medicine. This group of silicone composites have advantageous magnetic and mechanical properties as well as sufficient flexibility and biocompatibility. These composites can be applied in medicine for biological sensing, drug delivery, tissue engineering, and as remote-controlled microrobots operating in vivo. In this work, the properties of polydimethylsiloxane (PDMS)-based composites with different percentages (30 wt.%, 50 wt.%, 70 wt.%) of NdFeB microparticles as a filler were characterized. The novelty of the work was to determine the influence of the percentage of MP content and physiological conditioning on the properties of the PDMS-MP composites after in vitro incubation. An important essence of the work was a comprehensive study of the properties of materials important from the point of view of medical applications. Materials were tested before and after conditioning in 0.9 wt.% NaCl solution at a temperature of 37 °C. Several studies were carried out, including thermal, physicochemical, and rheological tests. The results show that with an increase of the incubation time, most of the measured thermal and physicochemical parameters decreased. The presence of the magnetic filler, especially at a concentration of 70 wt.%, has a positive effect on thermal stability and physicochemical and rheological properties. The performed tests provided important results, which can lead to further research for a broader application of magnetic composites in the biomedical field.

Tissue Rheology as a Possible Complementary Procedure to Advance Histological Diagnosis of Colon Cancer.

In recent years, rheological measurements of cells and tissues at physiological and pathological stages have become an essential method to determine how forces and changes in mechanical properties contribute to disease development and progression, but there is no standardization of this procedure so far. In this study, we evaluate the potential of nanoscale atomic force microscopy (AFM) and macroscopic shear rheometry to assess the mechanical properties of healthy and cancerous human colon tissues. The direct comparison of tissue mechanical behavior under uniaxial and shear deformation shows that cancerous tissues not only are stiffer compared to healthy tissue but also respond differently when shear and compressive stresses are applied. These results suggest that rheological parameters can be useful measures of colon cancer mechanopathology. Additionally, we extend the list of biological materials exhibiting compressional stiffening and shear weakening effects to human colon tumors. These mechanical responses might be promising mechanomarkers and become part of the new procedures in colon cancer diagnosis. Enrichment of histopathological grading with rheological assessment of tissue mechanical properties will potentially allow more accurate colon cancer diagnosis and improve prognosis.

Biofilm Growth Causes Damage to Silicone Voice Prostheses in Patients after Surgical Treatment of Locally Advanced Laryngeal Cancer.

Voice prosthesis implantation with the creation of a tracheoesophageal fistula is the gold standard procedure for voice rehabilitation in patients after a total laryngectomy. All patients implanted with a voice prosthesis (VP) have biofilms of fungi and bacteria grow on their surface. Biofilm colonization is one of the main reasons for VP degradation that can lead to VP dysfunction, which increases the high risk of pneumonia. In a 20-month evaluation period, 129 cases of prostheses after replacement procedures were investigated. Microbiological examination of the biofilms revealed that there were four of the most common fungi species (Candida spp.) and a large variety of bacterial species present. We studied the relationship between the time of proper function of Provox VP, the microorganism composition of the biofilm present on it, and the degradation level of the silicone material. Evaluation of the surface of the removed VP using an atomic force microscope (AFM) has demonstrated that biofilm growth might drastically change the silicone's mechanical properties. Changes in silicone stiffness and thermal properties might contribute to the failure of VP function. Our data can serve in future studies for the development of methods to prevent or inhibit biofilm formation on the VP surface that would translate to an increase in their durability and safety.

Antimicrobial and Physicochemical Properties of Artificial Saliva Formulations Supplemented with Core-Shell Magnetic Nanoparticles.

Saliva plays a crucial role in oral cavity. In addition to its buffering and moisturizing properties, saliva fulfills many biofunctional requirements, including antibacterial activity that is essential to assure proper oral microbiota growth. Due to numerous extra- and intra-systemic factors, there are many disorders of its secretion, leading to oral dryness. Saliva substitutes used in such situations must meet many demands. This study was design to evaluate the effect of core-shell magnetic nanoparticles (MNPs) adding (gold-coated and aminosilane-coated nanoparticles NPs) on antimicrobial (microorganism adhesion, biofilm formation), rheological (viscosity, viscoelasticity) and physicochemical (pH, surface tension, conductivity) properties of three commercially available saliva formulations. Upon the addition of NPs (20 µg/mL), antibacterial activity of artificial saliva was found to increase against tested microorganisms by 20% to 50%. NPs, especially gold-coated ones, decrease the adhesion of Gram-positive and fungal cells by 65% and Gram-negative bacteria cells by 45%. Moreover, the addition of NPs strengthened the antimicrobial properties of tested artificial saliva, without influencing their rheological and physicochemical properties, which stay within the range characterizing the natural saliva collected from healthy subjects.

The Influence of Mucin-Based Artificial Saliva on Properties of Polycaprolactone and Polylactide.

Polycaprolactone (PCL) and polylactide (PLA) are the two most common biodegradable polymers with potential use in oral applications. Both polymers undergo mainly slow hydrolytic degradation in the human body. However, specific conditions of the oral cavity, like elevated temperature, low pH, and presence of saliva affect the rate of hydrolysis. The study examined the properties of solid samples of PCL and PLA subjected to degradation in phosphate buffered saline (PBS) and artificial saliva (AS) at temperatures of 37 or 42 °C, and pH values 2 or 7.4. A number of tests were performed, including measurement of the degree of swelling, weight loss, molecular weight, differential scanning calorimetry, and thermogravimetry of polymers, as well as hardness and tensile strength. Additionally, topography and stiffness of surfaces using atomic force microscopy are presented. It has been noticed that in the artificial saliva, the processes of polymer degradation occur slightly more slowly, and the effects of temperature and pH are less pronounced. We believe that a layer of porcine gastric mucin from artificial saliva that adsorbed on the surface of polymers may have a key role in the observed differences; this layer resembles protective mucin coating tissues in the oral cavity.

Artificial Saliva: Challenges and Future Perspectives for the Treatment of Xerostomia.

The chronic sensation of a dry mouth is a disease condition called xerostomia and affects a large part of the population. Xerostomia is associated with decreased secretion, or more often, qualitative changes in saliva proteins and immunoglobulin concentrations that develop as a result of salivary gland dysfunction. Several reasons causing dry mouth were described, and usually, they include taking medications, diseases or radiotherapy. In some situations, when it is difficult to use salivary stimulants or salivary gland damage is irreversible, the only option might seem to be saliva substitutes. The paper presents the most important aspects considering saliva preparations. The rheological and lubricating properties and the reconstruction of the complex saliva structure has been the main purpose of research. The biological properties of saliva preparations were also widely discussed. As part of the work, the antimicrobial effect of three commercial saliva preparations was tested. Finally, inadequate antimicrobial properties against the strains isolated from the oral cavity were demonstrated. The development of salivary substitutes, in particular, the improvement of antimicrobial properties, can be achieved using nanotechnology, including drug delivery systems containing nanocarriers.

The Role of Oral Cavity Biofilm on Metallic Biomaterial Surface Destruction-Corrosion and Friction Aspects.

Metallic biomaterials in the oral cavity are exposed to many factors such as saliva, bacterial microflora, food, temperature fluctuations, and mechanical forces. Extreme conditions present in the oral cavity affect biomaterial exploitation and significantly reduce its biofunctionality, limiting the time of exploitation stability. We mainly refer to friction, corrosion, and biocorrosion processes. Saliva plays an important role and is responsible for lubrication and biofilm formation as a transporter of nutrients for microorganisms. The presence of metallic elements in the oral cavity may lead to the formation of electro-galvanic cells and, as a result, may induce corrosion. Transitional microorganisms such as sulfate-reducing bacteria may also be present among the metabolic microflora in the oral cavity, which can induce biological corrosion. Microorganisms that form a biofilm locally change the conditions on the surface of biomaterials and contribute to the intensification of the biocorrosion processes. These processes may enhance allergy to metals, inflammation, or cancer development. On the other hand, the presence of saliva and biofilm may significantly reduce friction and wear on enamel as well as on biomaterials. This work summarizes data on the influence of saliva and oral biofilms on the destruction of metallic biomaterials.