Silver nanoparticles impede the biofilm formation by Pseudomonas aeruginosa and Staphylococcus epidermidis

Biofilms are ensued due to bacteria that attach to surfaces and aggregate in a hydrated polymeric matrix. Formation of these sessile communities and their inherent resistance to anti-microbial agents are the source of many relentless and chronic bacterial infections. Such biofilms are responsible play a major role in development of ocular related infectious diseases in human namely microbial keratitis. Different approaches have been used for preventing biofilm related infections in health care settings. Many of these methods have their own demerits that include chemical based complications; emergent antibiotic resistant strains, etc. silver nanoparticles are renowned for their influential anti-microbial activity. Hence the present study over the biologically synthesized silver nanoparticles, exhibited a potential anti-biofilm activity that was tested in vitro on biofilms formed by Pseudomonas aeruginosa and Staphylococcus epidermidis during 24-h treatment. Treating these organisms with silver nanoparticles resulted in more than 95% inhibition in biofilm formation. The inhibition was known to be invariable of the species tested. As a result this study demonstrates the futuristic application of silver nanoparticles in treating microbial keratitis based on its potential anti-biofilm activity.     

Squalenyl Hydrogen Sulfate Nanoparticles for Simultaneous Delivery of Tobramycin and an Alkylquinolone Quorum Sensing Inhibitor Enable the Eradication of P. aeruginosa Biofilm Infections

Elimination of pulmonary Pseudomonas aeruginosa (PA) infections is challenging to accomplish with antibiotic therapies, mainly due to resistance mechanisms. Quorum sensing inhibitors (QSIs) interfering with biofilm formation can thus complement antibiotics. For simultaneous and improved delivery of both active agents to the infection sites, self‐assembling nanoparticles of a newly synthesized squalenyl hydrogen sulfate (SqNPs) were prepared. These nanocarriers allowed for remarkably high loading capacities of hydrophilic antibiotic tobramycin (Tob) and a novel lipophilic QSI at 30 % and circa 10 %, respectively. The drug‐loaded SqNPs showed improved biofilm penetration and enhanced efficacy in relevant biological barriers (mucin/human tracheal mucus, biofilm), leading to complete eradication of PA biofilms at circa 16‐fold lower Tob concentration than Tob alone. This study offers a viable therapy optimization and invigorates the research and development of QSIs for clinical use.     

Squalenyl Hydrogen Sulfate Nanoparticles for Simultaneous Delivery of Tobramycin and an Alkylquinolone Quorum Sensing Inhibitor Enable the Eradication of P. aeruginosa Biofilm Infections

Elimination of pulmonary Pseudomonas aeruginosa (PA) infections is challenging to accomplish with antibiotic therapies, mainly due to resistance mechanisms. Quorum sensing inhibitors (QSIs) interfering with biofilm formation can thus complement antibiotics. For simultaneous and improved delivery of both active agents to the infection sites, self-assembling nanoparticles of a newly synthesized squalenyl hydrogen sulfate (SqNPs) were prepared. These nanocarriers allowed for remarkably high loading capacities of hydrophilic antibiotic tobramycin (Tob) and a novel lipophilic QSI at 30 % and circa 10 %, respectively. The drug-loaded SqNPs showed improved biofilm penetration and enhanced efficacy in relevant biological barriers (mucin/human tracheal mucus, biofilm), leading to complete eradication of PA biofilms at circa 16-fold lower Tob concentration than Tob alone. This study offers a viable therapy optimization and invigorates the research and development of QSIs for clinical use.     

Synthetically Tuning the 2‐Position of Halogenated Quinolines: Optimizing Antibacterial and Biofilm Eradication Activities via Alkylation and Reductive Amination Pathways

Agents capable of eradicating bacterial biofilms are of great importance to human health as biofilm‐associated infections are tolerant to our current antibiotic therapies. We have recently discovered that halogenated quinoline (HQ) small molecules are: 1) capable of eradicating methicillin‐resistant Staphylococcus aureus (MRSA), methicillin‐resistant Staphylococcus epidermidis (MRSE) and vancomycin‐resistant Enterococcus faecium (VRE) biofilms, and 2) synthetic tuning of the 2‐position of the HQ scaffold has a significant impact on antibacterial and antibiofilm activities. Here, we report the chemical synthesis and biological evaluation of 39 HQ analogues that have a high degree of structural diversity at the 2‐position. We identified diverse analogues that are alkylated and aminated at the 2‐position of the HQ scaffold and demonstrate potent antibacterial (MIC≤0.39 μm ) and biofilm eradication (MBEC 1.0–93.8 μm ) activities against drug‐resistant Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecium strains while demonstrating <5 % haemolysis activity against human red blood cells (RBCs) at 200 μm . In addition, these HQs demonstrated low cytotoxicity against HeLa cells. Halogenated quinolines are a promising class of antibiofilm agents against Gram‐positive pathogens that could lead to useful treatments against persistent bacterial infections.     

Bioprospecting the Antibiofilm and Antimicrobial Activity of Soil and Insect Gut Bacteria

Antimicrobial resistance is a growing concern in public health and current research shows an important role for bacterial biofilms in recurrent or chronic infections. New strategies, therefore, are necessary to overcome antimicrobial resistance, through the development of new therapies that could alter or inhibit biofilm formation. In this sense, antibiofilm natural products are very promising. In this work, a bioprospection of antimicrobial and antibiofilm extracts from Uruguayan soil bacteria and insect gut bacteria was carried out. Extracts from extracellular broths were tested for their ability to inhibit planktonic cell growth and biofilm formation. Genomic analysis of Bacillus cereus ILBB55 was carried out. All extracts were able to inhibit the growth of, at least, one microorganism and several extracts showed MICs lower than 500 µg mL⁻¹ against microorganisms of clinical relevance (Staphylococcus aureus, Pseudomonas aeruginosa, and Enterobacter cloacae). Among the extracts evaluated for biofilm inhibition only ILBB55, from B. cereus, was able to inhibit, S. aureus (99%) and P. aeruginosa (62%) biofilms. Genomic analysis of this strain showed gene clusters similar to other clusters that code for known antimicrobial compounds. Our study revealed that extracts from soil bacteria and insect gut bacteria, especially from B. cereus ILBB55, could be potential candidates for drug discovery to treat infectious diseases and inhibit S. aureus and P. aeruginosa biofilms.     

Approaches for Mitigating Microbial Biofilm-Related Drug Resistance: A Focus on Micro- and Nanotechnologies

Biofilms play an essential role in chronic and healthcare-associated infections and are more resistant to antimicrobials compared to their planktonic counterparts due to their (1) physiological state, (2) cell density, (3) quorum sensing abilities, (4) presence of extracellular matrix, (5) upregulation of drug efflux pumps, (6) point mutation and overexpression of resistance genes, and (7) presence of persister cells. The genes involved and their implications in antimicrobial resistance are well defined for bacterial biofilms but are understudied in fungal biofilms. Potential therapeutics for biofilm mitigation that have been reported include (1) antimicrobial photodynamic therapy, (2) antimicrobial lock therapy, (3) antimicrobial peptides, (4) electrical methods, and (5) antimicrobial coatings. These approaches exhibit promising characteristics for addressing the impending crisis of antimicrobial resistance (AMR). Recently, advances in the micro- and nanotechnology field have propelled the development of novel biomaterials and approaches to combat biofilms either independently, in combination or as antimicrobial delivery systems. In this review, we will summarize the general principles of clinically important microbial biofilm formation with a focus on fungal biofilms. We will delve into the details of some novel micro- and nanotechnology approaches that have been developed to combat biofilms and the possibility of utilizing them in a clinical setting.     

Identification of New Potential Inhibitors of Quorum Sensing through a Specialized Multi-Level Computational Approach

Biofilms are aggregates of microorganisms anchored to a surface and embedded in a self-produced matrix of extracellular polymeric substances and have been associated with 80% of all bacterial infections in humans. Because bacteria in biofilms are less amenable to antibiotic treatment, biofilms have been associated with developing antibiotic resistance, a problem that urges developing new therapeutic options and approaches. Interfering with quorum-sensing (QS), an important process of cell-to-cell communication by bacteria in biofilms is a promising strategy to inhibit biofilm formation and development. Here we describe and apply an in silico computational protocol for identifying novel potential inhibitors of quorum-sensing, using CviR—the quorum-sensing receptor from Chromobacterium violaceum—as a model target. This in silico approach combines protein-ligand docking (with 7 different docking programs/scoring functions), receptor-based virtual screening, molecular dynamic simulations, and free energy calculations. Particular emphasis was dedicated to optimizing the discrimination ability between active/inactive molecules in virtual screening tests using a target-specific training set. Overall, the optimized protocol was used to evaluate 66,461 molecules, including those on the ZINC/FDA-Approved database and to the Mu.Ta.Lig Virtual Chemotheca. Multiple promising compounds were identified, yielding good prospects for future experimental validation and for drug repurposing towards QS inhibition.     

Covalently Immobilized Battacin Lipopeptide Gels with Activity against Bacterial Biofilms

Novel antibiotic treatments are in increasing demand to tackle life-threatening infections from bacterial pathogens. In this study, we report the use of a potent battacin lipopeptide as an antimicrobial gel to inhibit planktonic and mature biofilms of S. aureus and P. aeruginosa. The antimicrobial gels were made by covalently linking the N-terminal cysteine containing lipopeptide (GZ3.163) onto the polyethylene glycol polymer matrix and initiating gelation using thiol-ene click chemistry. The gels were prepared both in methanol and in water and were characterised using rheology, Fourier transform infrared (FT-IR) spectroscopy and scanning electron microscopy (SEM). Antibacterial and antibiofilm analyses revealed that the gels prepared in methanol have better antibacterial and antibiofilm activity. Additionally, a minimum peptide content of 0.5 wt% (relative to polymer content) is required to successfully inhibit the planktonic bacterial growth and disperse mature biofilms of P. aeruginosa and S. aureus. The antibacterial activity of these lipopeptide gels is mediated by a contact kill mechanism of action. The gels are non-haemolytic against mouse red blood cells and are non-cytotoxic against human dermal fibroblasts. Findings from this study show that battacin lipopeptide gels have the potential to be developed as novel topical antibacterial agents to combat skin infections, particularly caused by S. aureus.     

Sonochemical Coatings of ZnO and CuO Nanoparticles Inhibit Streptococcus mutans Biofilm Formation on Teeth Model

Antibiotic resistance has prompted the search for new agents that can inhibit bacterial growth. We recently reported on the antibiofilm activities of nanosized ZnO and CuO nanoparticles (NPs) synthesized by using sonochemical irradiation. In this study, we examined the antibacterial activity of ZnO and CuO NPs in a powder form and also examined the antibiofilm behavior of teeth surfaces that were coated with ZnO and CuO NPs using sonochemistry. Free ZnO and CuO NPs inhibited biofilm formation of Streptococcus mutans . Furthermore, by using the sonochemical procedure, we were able to coat teeth surfaces that inhibited bacterial colonization.     

Surface-enhanced Raman scattering (SERS) revealing chemical variation during biofilm formation: from initial attachment to mature biofilm

Surface-enhanced Raman scattering (SERS) has recently been proved to be a promising technique for characterizing the chemical composition of the biofilm matrix. In the present study, to fully understand the chemical variations during biofilm formation, SERS based on silver colloidal nanoparticles was applied to evaluate the chemical components in the matrix of biofilm at different growth phases, including initial attached bacteria, colonies, and mature biofilm. Meanwhile, atomic force microscopy was also applied to study the changes of biofilm morphology. Three model bacteria, including Escherichia coli, Pseudomonas putida, and Bacillus subtilis, were used to cultivate biofilms. The results showed that the content of carbohydrates, proteins, and nucleic acids in the biofilm matrix increased significantly along with the biofilm growth of the three bacteria judging from the intensities and appearance probabilities of related marker peaks in the SERS spectra. The content of lipids, however, only increased in the Gram-negative biofilms (E. coli and P. putida) rather than the Gram-positive biofilm (B. subtilis). Our findings strongly suggest the SERS has significant potential for studying chemical variations during biofilm formation.     

Antibiofilm approaches: prevention of catheter colonization

Bacteria frequently attach to medical devices such as intravascular catheters by forming sessile multicellular communities known as biofilms, which can be the source of persistent infections that are recalcitrant to systemic antibiotic therapy. As a result of this persistence, a number of technologies have been developed to prevent catheter-associated biofilm formation. Whereas the most straightforward approaches focus on impregnating catheter material with classical antimicrobial agents, these approaches are not universally effective, thereby underscoring the need for more potent and more sophisticated approaches to the prevention of catheter-related biofilm infections.     

Development of Chitosan-Based Surfaces to Prevent Single- and Dual-Species Biofilms of Staphylococcus aureus and Pseudomonas aeruginosa

Implantable medical devices (IMDs) are susceptible to microbial adhesion and biofilm formation, which lead to several clinical complications, including the occurrence of implant-associated infections. Polylactic acid (PLA) and its composites are currently used for the construction of IMDs. In addition, chitosan (CS) is a natural polymer that has been widely used in the medical field due to its antimicrobial and antibiofilm properties, which can be dependent on molecular weight (Mw). The present study aims to evaluate the performance of CS-based surfaces of different Mw to inhibit bacterial biofilm formation. For this purpose, CS-based surfaces were produced by dip-coating and the presence of CS and its derivatives onto PLA films, as well surface homogeneity were confirmed by contact angle measurements, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The antimicrobial activity of the functionalized surfaces was evaluated against single- and dual-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa. Chitosan-based surfaces were able to inhibit the development of single- and dual-species biofilms by reducing the number of total, viable, culturable, and viable but nonculturable cells up to 79%, 90%, 81%, and 96%, respectively, being their activity dependent on chitosan Mw. The effect of CS-based surfaces on the inhibition of biofilm formation was corroborated by biofilm structure analysis using confocal laser scanning microscopy (CLSM), which revealed a decrease in the biovolume and thickness of the biofilm formed on CS-based surfaces compared to PLA. Overall, these results support the potential of low Mw CS for coating polymeric devices such as IMDs where the two bacteria tested are common colonizers and reduce their biofilm formation.     

Polycationic chitosan-conjugated photosensitizer for antibacterial photodynamic therapy

The complex nature of bacterial cell membrane and structure of biofilm has challenged the efficacy of antimicrobial photodynamic therapy. This study was aimed to synthesize a polycationic chitosan-conjugated rose bengal (CSRB) photosensitizer and test its antibiofilm efficacy on Enterococcus faecalis (gram positive) and Pseudomonas aeruginosa (gram negative) using photodynamic therapy. During experiments, CSRB was tested along with an anionic photosensitizer rose bengal (RB) and a cationic photosensitizer methylene blue (MB) for uptake and killing efficacy on 7-day-old E. faecalis and P. aeruginosa biofilms. Microbiological culture based analysis was used to analyze the cell viability, while laser scanning confocal microscopy (LSCM) was used to examine the structure of biofilm. The synthesized CSRB showed absorbance spectrum similar to the RB. The concentration of CSRB uptaken by both the bacterial biofilms was significantly higher than that of RB and MB (P < 0.05). Photoactivation resulted in significantly higher elimination of both bacterial biofilms sensitized with CSRB than RB and MB. The structure of biofilm under LSCM was found to be disrupted following CSRB treatment. The present study highlighted the importance of inherent cell membrane permeabilizing effect of chitosan and increased cell/biofilm uptake of conjugated photosensitizer to produce significant antibiofilm efficacy during photodynamic therapy.     

Halogenated Phenazines that Potently Eradicate Biofilms,MRSA Persister Cells in Non‐Biofilm Cultures,and Mycobacterium tuberculosis

Conventional antibiotics are ineffective against non‐replicating bacteria (for example, bacteria within biofilms). We report a series of halogenated phenazines (HP), inspired by marine antibiotic 1 , that targets persistent bacteria. HP 14 demonstrated the most potent biofilm eradication activities to date against MRSA, MRSE, and VRE biofilms (MBEC=0.2–12.5 μM), as well as the effective killing of MRSA persister cells in non‐biofilm cultures. Frontline MRSA treatments, vancomycin and daptomycin, were unable to eradicate MRSA biofilms or non‐biofilm persisters alongside 14 . HP 13 displayed potent antibacterial activity against slow‐growing M. tuberculosis (MIC=3.13 μM), the leading cause of death by bacterial infection around the world. HP analogues effectively target persistent bacteria through a mechanism that is non‐toxic to mammalian cells and could have a significant impact on treatments for chronic bacterial infections.     

Halogenated Phenazines that Potently Eradicate Biofilms,MRSA Persister Cells in Non‐Biofilm Cultures,and Mycobacterium tuberculosis

Conventional antibiotics are ineffective against non‐replicating bacteria (for example, bacteria within biofilms). We report a series of halogenated phenazines (HP), inspired by marine antibiotic 1 , that targets persistent bacteria. HP 14 demonstrated the most potent biofilm eradication activities to date against MRSA, MRSE, and VRE biofilms (MBEC=0.2–12.5 μM), as well as the effective killing of MRSA persister cells in non‐biofilm cultures. Frontline MRSA treatments, vancomycin and daptomycin, were unable to eradicate MRSA biofilms or non‐biofilm persisters alongside 14 . HP 13 displayed potent antibacterial activity against slow‐growing M. tuberculosis (MIC=3.13 μM), the leading cause of death by bacterial infection around the world. HP analogues effectively target persistent bacteria through a mechanism that is non‐toxic to mammalian cells and could have a significant impact on treatments for chronic bacterial infections.     

Hydrophobic Chitosan Nanoparticles Loaded with Carvacrol against Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa infections have become more challenging to treat and eradicate due to their ability to form biofilms. This study aimed to produce hydrophobic nanoparticles by grafting 11-carbon and three-carbon alkyl chains to a chitosan polymer as a platform to carry and deliver carvacrol for improving its antibacterial and antibiofilm properties. Carvacrol–chitosan nanoparticles showed ζ potential values of 10.5–14.4 mV, a size of 140.3–166.6 nm, and an encapsulation efficiency of 25.1–68.8%. Hydrophobic nanoparticles reduced 46–53% of the biomass and viable cells (7–25%) within P. aeruginosa biofilms. Diffusion of nanoparticles through the bacterial biofilm showed a higher penetration of nanoparticles created with 11-carbon chain chitosan than those formulated with unmodified chitosan. The interaction of nanoparticles with a 50:50 w/w phospholipid mixture at the air–water interface was studied, and values suggested that viscoelasticity and fluidity properties were modified. The modified nanoparticles significantly reduced viable P. aeruginosa in biofilms (0.078–2.0 log CFU·cm⁻²) and swarming motility (40–60%). Furthermore, the formulated nanoparticles reduced the quorum sensing in Chromobacterium violaceum. This study revealed that modifying the chitosan polarity to synthesize more hydrophobic nanoparticles could be an effective treatment against P. aeruginosa biofilms to decrease its virulence and pathogenicity, mainly by increasing their ability to interact with the membrane phospholipids and penetrate preformed biofilms.     

Covalent Lectin Inhibition and Application in Bacterial Biofilm Imaging

Biofilm formation by pathogenic bacteria is a hallmark of chronic infections. In many cases, lectins play key roles in establishing biofilms. The pathogen Pseudomonas aeruginosa often exhibiting various drug resistances employs its lectins LecA and LecB as virulence factors and biofilm building blocks. Therefore, inhibition of the function of these proteins is thought to have potential in developing “pathoblockers” preventing biofilm formation and virulence. A covalent lectin inhibitor specific to a carbohydrate binding site is described for the first time. Its application in the LecA‐specific in vitro imaging of biofilms formed by P. aeruginosa is also reported.     

Polymeric Nanoparticles Active against Dual-Species Bacterial Biofilms

Biofilm infections are a global public health threat, necessitating new treatment strategies. Biofilm formation also contributes to the development and spread of multidrug-resistant (MDR) bacterial strains. Biofilm-associated chronic infections typically involve colonization by more than one bacterial species. The co-existence of multiple species of bacteria in biofilms exacerbates therapeutic challenges and can render traditional antibiotics ineffective. Polymeric nanoparticles offer alternative antimicrobial approaches to antibiotics, owing to their tunable physico-chemical properties. Here, we report the efficacy of poly(oxanorborneneimide) (PONI)-based antimicrobial polymeric nanoparticles (PNPs) against multi-species bacterial biofilms. PNPs showed good dual-species biofilm penetration profiles as confirmed by confocal laser scanning microscopy. Broad-spectrum antimicrobial activity was observed, with reduction in both bacterial viability and overall biofilm mass. Further, PNPs displayed minimal fibroblast toxicity and high antimicrobial activity in an in vitro co-culture model comprising fibroblast cells and dual-species biofilms of Escherichia coli and Pseudomonas aeruginosa. This study highlights a potential clinical application of the presented polymeric platform.     

Thiazole Analogues of the Marine Alkaloid Nortopsentin as Inhibitors of Bacterial Biofilm Formation

Anti-virulence strategy is currently considered a promising approach to overcome the global threat of the antibiotic resistance. Among different bacterial virulence factors, the biofilm formation is recognized as one of the most relevant. Considering the high and growing percentage of multi-drug resistant infections that are biofilm-mediated, new therapeutic agents capable of counteracting the formation of biofilms are urgently required. In this scenario, a new series of 18 thiazole derivatives was efficiently synthesized and evaluated for its ability to inhibit biofilm formation against the Gram-positive bacterial reference strains Staphylococcus aureus ATCC 25923 and S. aureus ATCC 6538 and the Gram-negative strain Pseudomonas aeruginosa ATCC 15442. Most of the new compounds showed a marked selectivity against the Gram-positive strains. Remarkably, five compounds exhibited BIC₅₀ values against S. aureus ATCC 25923 ranging from 1.0 to 9.1 µM. The new compounds, affecting the biofilm formation without any interference on microbial growth, can be considered promising lead compounds for the development of a new class of anti-virulence agents.     

Recent Advances and Future Prospects on Adaptive Biomaterials for Antimicrobial Applications

Bacterial infection is becoming the biggest threat to human health. The scenario is partly due to the ineffectiveness of the conventional antibiotic treatments against the emergence of multidrug‐resistant bacteria and partly due to the bacteria living in biofilms or cells. Adaptive biomaterials can change their physicochemical properties in the microenvironment of bacterial infection, thereby facilitating either their interactions with bacteria or drug release. The trends in treating bacterial infections using adaptive biomaterials‐based systems are flourishing and generate innumerous possibility to design novel antimicrobial therapeutics. This feature article aims to summarize the recent developments in the formulations, mechanisms, and advances of adaptive materials in bacterial infection diagnosis, contact killing of bacteria, and antimicrobial drug delivery. Also, the challenges and limitations of current antimicrobial treatments based on adaptive materials and their clinical and industrial future prospects are discussed.