Bacterial-infections are mostly due to bacteria in an adhering, biofilm-mode of growth and not due to planktonically growing, suspended-bacteria. Biofilm-bacteria are much more recalcitrant to ...conventional antimicrobials than planktonic-bacteria due to (1) emergence of new properties of biofilm-bacteria that cannot be predicted on the basis of planktonic properties, (2) low penetration and accumulation of antimicrobials in a biofilm, (3) disabling of antimicrobials due to acidic and anaerobic conditions prevailing in a biofilm, and (4) enzymatic modification or inactivation of antimicrobials by biofilm inhabitants. In recent years, new nanotechnology-based antimicrobials have been designed to kill planktonic, antibiotic-resistant bacteria, but additional requirements rather than the mere killing of suspended bacteria must be met to combat biofilm-infections. The requirements and merits of nanotechnology-based antimicrobials for the control of biofilm-infection form the focus of this Tutorial Review.
Biofilm formation is initiated by adhesion of individual bacteria to a surface. However, surface adhesion alone is not sufficient to form the complex community architecture of a biofilm. ...Surface-sensing creates bacterial awareness of their adhering state on the surface and is essential to initiate the phenotypic and genotypic changes that characterize the transition from initial bacterial adhesion to a biofilm. Physico-chemistry has been frequently applied to explain initial bacterial adhesion phenomena, including bacterial mass transport, role of substratum surface properties in initial adhesion and the transition from reversible to irreversible adhesion. However, also emergent biofilm properties, such as production of extracellular-polymeric-substances (EPS), can be surface-programmed. This review presents a four-step, comprehensive description of the role of physico-chemistry from initial bacterial adhesion to surface-programmed biofilm growth: (1) bacterial mass transport towards a surface, (2) reversible bacterial adhesion and (3) transition to irreversible adhesion and (4) cell wall deformation and associated emergent properties. Bacterial transport mostly occurs from sedimentation or convective-diffusion, while initial bacterial adhesion can be described by surface thermodynamic and Derjaguin−Landau−Verwey−Overbeek (DLVO)-analyses, considering bacteria as smooth, inert colloidal particles. DLVO-analyses however, require precise indication of the bacterial cell surface, which is impossible due to the presence of bacterial surface tethers, creating a multi-scale roughness that impedes proper definition of the interaction distance in DLVO-analyses. Application of surface thermodynamics is also difficult, because initial bacterial adhesion is only an equilibrium phenomenon for a short period of time, when bacteria are attached to a substratum surface through few surface tethers. Physico-chemical bond-strengthening occurs in several minutes leading to irreversible adhesion due to progressive removal of interfacial water, conformational changes in cell surface proteins, re-orientation of bacteria on a surface and the progressive involvement of more tethers in adhesion. After initial bond-strengthening, adhesion forces arising from a substratum surface cause nanoscopic deformation of the bacterial cell wall against the elasticity of the rigid peptidoglycan layer positioned in the cell wall and the intracellular pressure of the cytoplasm. Cell wall deformation not only increases the contact area with a substratum surface, presenting another physico-chemical bond-strengthening mechanism, but is also accompanied by membrane surface tension changes. Membrane-located sensor molecules subsequently react to control emergent phenotypic and genotypic properties in biofilms, most notably adhesion-associated ones like EPS production. Moreover, also bacterial efflux pump systems may be activated or mechano-sensitive channels may be opened upon adhesion-induced cell wall deformation. The physico-chemical properties of the substratum surface thus control the response of initially adhering bacteria and through excretion of autoinducer molecules extend the awareness of their adhering state to other biofilm inhabitants who subsequently respond with similar emergent properties. Herewith, physico-chemistry is not only involved in initial bacterial adhesion to surfaces but also in what we here propose to call “surface-programmed” biofilm growth. This conclusion is pivotal for the development of new strategies to control biofilm formation on substratum surfaces, that have hitherto been largely confined to the initial bacterial adhesion phenomena.
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•Physico-chemistry dictates initial bacterial adhesion to substratum surfaces•Bond-strengthening leads to irreversible adhesion of bacteria (and inert colloids)•Through quorum-sensing, bacteria share information on substratum surface properties•Substratum surface properties program emergent biofilm properties•Regarding bacteria as inert colloids, does not yield a generalized biofilm model
Internalization of Staphylococcus aureus by macrophages can inactivate bacterial killing mechanisms, allowing intracellular residence and dissemination of infection. Concurrently, these staphylococci ...can evade antibiotics that are frequently unable to pass mammalian cell membranes. A binary, amphiphilic conjugate composed of triclosan and ciprofloxacin is synthesized that self‐assemble through micelle formation into antimicrobial nanoparticles (ANPs). These novel ANPs are stabilized through encapsulation in macrophage membranes, providing membrane‐encapsulated, antimicrobial‐conjugated NPs (Me‐ANPs) with similar protein activity, Toll‐like receptor expression and negative surface charge as their precursor murine macrophage/human monocyte cell lines. The combination of Toll‐like receptors and negative surface charge allows uptake of Me‐ANPs by infected macrophages/monocytes through positively charged, lysozyme‐rich membrane scars created during staphylococcal engulfment. Me‐ANPs are not engulfed by more negatively charged sterile cells possessing less lysozyme at their surface. The Me‐ANPs kill staphylococci internalized in macrophages in vitro. Me‐ANPs likewise kill staphylococci more effectively than ANPs without membrane‐encapsulation or clinically used ciprofloxacin in a mouse peritoneal infection model. Similarly, organ infections in mice created by dissemination of infected macrophages through circulation in the blood are better eradicated by Me‐ANPs than by ciprofloxacin. These unique antimicrobial properties of macrophage‐monocyte Me‐ANPs provide a promising direction for human clinical application to combat persistent infections.
A novel amphiphilic conjugate composed of triclosan and ciprofloxacin self‐assembles into antimicrobial nanoparticles (ANPs) and is stabilized through encapsulation in a macrophage–monocyte membrane. These membrane‐encapsulated ANPs exclusively enter bacterially infected macrophages. Accordingly, membrane‐encapsulated ANPs kill staphylococci internalized in macrophages in vitro. Staphylococcal killing in mouse peritoneal and organ infection models by membrane‐encapsulated ANPs is more effective than clinically applied ciprofloxacin.
Fungal biofilms cause a major clinical problem with a shrinking armamentarium for treatment. Here, the design and synthesis of voriconazole‐inbuilt zinc 2‐methylimidazolates frameworks (V‐ZIF) is ...reported. Voriconazole is built in through coordination‐binding between zinc and voriconazole. These metal–organic‐frameworks with inbuilt voriconazole, reduce inadvertent voriconazole‐leakage, yield a zero‐order release kinetics of voriconazole, aid antifungal penetration in Candida albicans biofilms, and prevent Candida aggregation yielding better dispersal. Once accumulated in an acidic C. albicans biofilm, voriconazole dissociates from the metal–organic framework to cause membrane‐damage and killing of inhabiting fungi. Moreover, in a murine model, the V‐ZIFs eradicate open‐wound infections caused by C. albicans better than voriconazole in solution, with negligible side effects to the healthy tissues of major organs. Thus, V‐ZIFs may provide a welcome addition to the antifungal armamentarium currently available for the treatment of fungal biofilms.
Voriconazole‐inbuilt zinc 2‐methylimidazolates frameworks are fabricated through coordination‐binding between zinc and voriconazole. These metal–organic frameworks (MOFs) with inbuilt voriconazole yield zero‐order release kinetics of voriconazole and aid antifungal penetration in C. albicans biofilms. Once accumulated in an acidic C. albicans biofilm, voriconazole dissociates from the MOF to cause membrane‐damage and killing of inhabiting fungi.
Diabetic foot ulcers infected with antibiotic‐resistant bacteria form a severe complication of diabetes. Antimicrobial‐loaded hydrogels are used as a dressing for infected wounds, but the ongoing ...rise in the number of antimicrobial‐resistant infections necessitates new, nonantibiotic based designs. Here, a guanosine‐quadruplex (G4)‐hydrogel composed of guanosine, 2‐formylphenylboronic acid, and putrescine is designed and used as a cascade‐reaction container. The G4‐hydrogel is loaded with glucose‐oxidase and hemin. The first cascade‐reaction, initiated by glucose‐oxidase, transforms glucose and O2 into gluconic acid and H2O2. In vitro, this reaction is most influential on killing Staphylococcus aureus or Pseudomonas aeruginosa in suspension, but showed limited killing of bacteria in biofilm‐modes of growth. The second cascade‐reaction, however, transforming H2O2 into reactive‐oxygen‐species (ROS), also enhances killing of biofilm bacteria due to hemin penetration into biofilms and interaction with eDNA G‐quadruplexes in the biofilm matrix. Therewith, the second cascade‐reaction generates ROS close to the target bacteria, facilitating killing despite the short life‐time of ROS. Healing of infected wounds in diabetic mice proceeds faster upon coverage by these G4‐hydrogels than by clinically common ciprofloxacin irrigation. Moreover, local glucose concentrations around infected wounds decrease. Concluding, a G4‐hydrogel loaded with glucose‐oxidase and hemin is a good candidate for infected wound dressings, particularly in diabetic patients.
A supramolecular guanosine‐quadruplex (G4)‐hydrogel containing glucose oxidase and hemin provides a cascade‐reaction container suitable as an antibacterial dressing for infected wounds. The cascade‐reaction in the G4‐hydrogel generates reactive‐oxygen‐species inside an infectious biofilm from endogenous glucose, killing multidrug resistant bacteria on a nonantibiotic basis, yielding faster healing of infected wounds, and reducing local glucose levels in diabetic mice.
Optical-coherence-tomography (OCT) is a non-destructive tool for biofilm imaging, not requiring staining, and used to measure biofilm thickness and putative comparison of biofilm structure based on ...signal intensity distributions in OCT-images. Quantitative comparison of biofilm signal intensities in OCT-images, is difficult due to the auto-scaling applied in OCT-instruments to ensure optimal quality of individual images. Here, we developed a method to eliminate the influence of auto-scaling in order to allow quantitative comparison of biofilm densities in different images. Auto- and re-scaled signal intensities could be qualitatively interpreted in line with biofilm characteristics for single and multi-species biofilms of different strains and species (cocci and rod-shaped organisms), demonstrating qualitative validity of auto- and re-scaling analyses. However, specific features of pseudomonas and oral multi-species biofilms were more prominently expressed after re-scaling. Quantitative validation was obtained by relating average auto- and re-scaled signal intensities across biofilm images with volumetric-bacterial-densities in biofilms, independently obtained using enumeration of bacterial numbers per unit biofilm volume. The signal intensities in auto-scaled biofilm images did not significantly relate with volumetric-bacterial-densities, whereas re-scaled intensities in images of biofilms of widely different strains and species increased linearly with independently determined volumetric-bacterial-densities in the biofilms. Herewith, the proposed re-scaling of signal intensity distributions in OCT-images significantly enhances the possibilities of biofilm imaging using OCT.
A lipid named DCPA was synthesized under microwave‐assisted heating. DCPA possesses a pyridine betaine, hydrophilic group that can be complexed with water through hydrogen bonding (DCPA‐H2O). ...DCPA‐H2O liposomes became protonated relatively fast already at pH<6.8, due to the high HOMO binding energy of DCPA‐H2O. In murine models, DCPA‐H2O liposomes had longer blood circulation times than natural DPPC or cationic DCPM liposomes, while after tail‐vein injection DCPA‐H2O liposomes targeted faster to solid tumors and intra‐abdominal infectious biofilms. Therapeutic efficacy in a murine, infected wound‐healing model of tail‐vein injected ciprofloxacin‐loaded DCPA‐H2O liposomes exceeded the ones of clinically applied ciprofloxacin as well as of ciprofloxacin‐loaded DPPC or DCPM liposomes.
A new lipid, DCPA, was prepared, possessing a hydrophilic, pyridine betaine group that can be hydrogen‐bonded with water yielding DCPA‐H2O. Self‐assembled DCPA‐H2O liposomes become rapidly protonated at relatively high pH (<7.0) due to the high HOMO binding energy of DCPA‐H2O. As a result, DCPA‐H2O liposomes target faster to solid tumors and infectious biofilms than natural DPPC or cationic DCPM liposomes.
Nanoparticles possess unique features due to their small size and can be composed of different surface chemistries. Carbon quantum dots possess several unique physico-chemical and antibacterial ...activities. This review provides an overview of different methods to prepare carbon quantum dots from different carbon sources in order to provide guidelines for choosing methods and carbon sources that yield carbon quantum dots with optimal antibacterial efficacy. Antibacterial activities of carbon quantum dots predominantly involve cell wall damage and disruption of the matrix of infectious biofilms through reactive oxygen species (ROS) generation to cause dispersal of infecting pathogens that enhance their susceptibility to antibiotics. Quaternized carbon quantum dots from organic carbon sources have been found to be equally efficacious for controlling wound infection and pneumonia in rodents as antibiotics. Carbon quantum dots derived through heating of natural carbon sources can inherit properties that resemble those of the carbon sources they are derived from. This makes antibiotics, medicinal herbs and plants or probiotic bacteria ideal sources for the synthesis of antibacterial carbon quantum dots. Importantly, carbon quantum dots have been suggested to yield a lower chance of inducing bacterial resistance than antibiotics, making carbon quantum dots attractive for large scale clinical use.
The poor penetrability of many biofilms contributes to the recalcitrance of infectious biofilms to antimicrobial treatment. Here, a new application for the use of magnetic nanoparticles in ...nanomedicine to create artificial channels in infectious biofilms to enhance antimicrobial penetration and bacterial killing is proposed. Staphylococcus aureus biofilms are exposed to magnetic‐iron‐oxide nanoparticles (MIONPs), while magnetically forcing MIONP movement through the biofilm. Confocal laser scanning microscopy demonstrates artificial channel digging perpendicular to the substratum surface. Artificial channel digging significantly (4–6‐fold) enhances biofilm penetration and bacterial killing efficacy by gentamicin in two S. aureus strains with and without the ability to produce extracellular polymeric substances. Herewith, this work provides a simple, new, and easy way to enhance the eradication of infectious biofilms using MIONPs combined with clinically applied antibiotic therapies.
A novel application of magnetic nanoparticles is described in which magnetic nanoparticles are used to create artificial channels in infectious biofilms. Artificially dug channels relieve the barrier posed by the biofilm matrix toward antibiotic penetration to enhance eradication of the biofilm. Since magnetic nanoparticles are clinically used, this new application will be relatively easy to translate to clinical practice.
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