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
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.
Biofilms cause persistent bacterial infections and are extremely recalcitrant to antimicrobials, due in part to reduced penetration of antimicrobials into biofilms that allows bacteria residing in ...the depth of a biofilm to survive antimicrobial treatment. Here, we describe the preparation of surface-adaptive, Triclosan-loaded micellar nanocarriers showing (1) enhanced biofilm penetration and accumulation, (2) electrostatic targeting at acidic pH toward negatively charged bacterial cell surfaces in a biofilm, and (3) antimicrobial release due to degradation of the micelle core by bacterial lipases. First, it was established that mixed-shell-polymeric-micelles (MSPM) consisting of a hydrophilic poly(ethylene glycol) (PEG)-shell and pH-responsive poly(β-amino ester) become positively charged at pH 5.0, while being negatively charged at physiological pH. This is opposite to single-shell-polymeric-micelles (SSPM) possessing only a PEG-shell and remaining negatively charged at pH 5.0. The stealth properties of the PEG-shell combined with its surface-adaptive charge allow MSPMs to penetrate and accumulate in staphylococcal biofilms, as demonstrated for fluorescent Nile red loaded micelles using confocal-laser-scanning-microscopy. SSPMs, not adapting a positive charge at pH 5.0, could not be demonstrated to penetrate and accumulate in a biofilm. Once micellar nanocarriers are bound to a staphylococcal cell surface, bacterial enzymes degrade the MSPM core to release its antimicrobial content and kill bacteria over the depth of a biofilm. This constitutes a highly effective pathway to control blood-accessible staphylococcal biofilms using antimicrobials, bypassing biofilm recalcitrance to antimicrobial penetration.
The existence of stress de-activation was further supported by the observation that an external mechanical stress on adhering bacteria enhances the antimicrobial efficacy of quaternary ammonium ...compounds in solution 17. Since the great majority of bacterial strains and species possess a negative surface charge 18, strong adhesion forces can be found on positively charged surfaces, such as quaternary ammonium-coated surfaces that are known to kill bacteria upon contact 19 in this "lethal" regime of strong adhesion forces (see Figure 1).\n EPS was stained with calcofluor white, rendering blue fluorescence. Upon first approach of a bacterium to a surface, it becomes attached to a layer of highly viscous water adjacent to the surface that is subsequently slowly penetrated to allow stronger contact with the surface, after which protein structures on the cell surface re-orient themselves to allow optimal binding. Since it is unlikely that metabolic processes and phenotypic changes occur within minutes, we envisage that adhesion forces after physico-chemical strengthening represent the transition forces between the three adhesion force regimes depicted in Figure 1.
Bacterial infections are mostly due to bacteria in their biofilm mode‐of‐growth, making them recalcitrant to antibiotic penetration. In addition, the number of bacterial strains intrinsically ...resistant to available antibiotics is alarmingly growing. This study reports that micellar nanocarriers with a poly(ethylene glycol) shell fully penetrate staphylococcal biofilms due to their biological invisibility. However, when the shell is complemented with poly(β‐amino ester), these mixed‐shell micelles become positively charged in the low pH environment of a biofilm, allowing not only their penetration but also their accumulation in biofilms without being washed out, as do single‐shell micelles lacking the pH‐adaptive feature. Accordingly, bacterial killing of multidrug resistant staphylococcal biofilms exposed to protoporphyrin IX‐loaded mixed‐shell micelles and after light‐activation is superior compared with single‐shell micelles. Subcutaneous infections in mice, induced with vancomycin‐resistant, bioluminescent staphylococci can be eradicated by daily injection of photoactivatable protoporphyrin IX‐loaded, mixed‐shell micelles in the bloodstream and light‐activation at the infected site. Micelles, which are not degraded by bacterial enzymes in the biofilm, are degraded in the liver and spleen and cleared from the body through the kidneys. Thus, adaptive micellar nanocarriers loaded with light‐activatable antimicrobials constitute a much‐needed alternative to current antibiotic therapies.
Photodynamic treatment with protoporphyrin IX‐loaded micelles is successful in eradicating a subcutaneous, multidrug‐resistant infection in mice, while unused micelles are demonstrated to be cleared from the blood circuation.
Biomaterial-associated infections occur on both permanent implants and temporary devices for restoration or support of human functions. Despite increasing use of biomaterials in an aging society, ...comparatively few biomaterials have been designed that effectively reduce the incidence of biomaterial-associated infections. This review provides design guidelines for infection-reducing strategies based on the concept that the fate of biomaterial implants or devices is a competition between host tissue cell integration and bacterial colonization at their surfaces.
Many nanotechnology-based antimicrobials and antimicrobial-delivery-systems have been developed over the past decades with the aim to provide alternatives to antibiotic treatment of ...infectious-biofilms across the human body. Antimicrobials can be loaded into nanocarriers to protect them against de-activation, and to reduce their toxicity and potential, harmful side-effects. Moreover, antimicrobial nanocarriers such as micelles, can be equipped with stealth and pH-responsive features that allow self-targeting and accumulation in infectious-biofilms at high concentrations. Micellar and liposomal nanocarriers differ in hydrophilicity of their outer-surface and inner-core. Micelles are self-assembled, spherical core-shell structures composed of single layers of surfactants, with hydrophilic head-groups and hydrophobic tail-groups pointing to the micellar core. Liposomes are composed of lipids, self-assembled into bilayers. The hydrophilic head of the lipids determines the surface properties of liposomes, while the hydrophobic tail, internal to the bilayer, determines the fluidity of liposomal-membranes. Therefore, whereas micelles can only be loaded with hydrophobic antimicrobials, hydrophilic antimicrobials can be encapsulated in the hydrophilic, aqueous core of liposomes and hydrophobic or amphiphilic antimicrobials can be inserted in the phospholipid bilayer. Nanotechnology-derived liposomes can be prepared with diameters <100-200 nm, required to prevent reticulo-endothelial rejection and allow penetration into infectious-biofilms. However, surface-functionalization of liposomes is considerably more difficult than of micelles, which explains while self-targeting, pH-responsive liposomes that find their way through the blood circulation toward infectious-biofilms are still challenging to prepare. Equally, development of liposomes that penetrate over the entire thickness of biofilms to provide deep killing of biofilm inhabitants still provides a challenge. The liposomal phospholipid bilayer easily fuses with bacterial cell membranes to release high antimicrobial-doses directly inside bacteria. Arguably, protection against de-activation of antibiotics in liposomal nanocarriers and their fusogenicity constitute the biggest advantage of liposomal antimicrobial carriers over antimicrobials free in solution. Many Gram-negative and Gram-positive bacterial strains, resistant to specific antibiotics, have been demonstrated to be susceptible to these antibiotics when encapsulated in liposomal nanocarriers. Recently, also progress has been made concerning large-scale production and long-term storage of liposomes. Therewith, the remaining challenges to develop self-targeting liposomes that penetrate, accumulate and kill deeply in infectious-biofilms remain worthwhile to pursue.
In a healthy host, a balance exists between members of the microbiota, such that potential pathogenic and non-pathogenic organisms can be found in apparent harmony. During infection, this balance can ...become disturbed, leading to often dramatic changes in the composition of the microbiota. For most bacterial infections, nonspecific antibiotics are used, killing the non-pathogenic members of the microbiota as well as the pathogens and leading to a substantial delay in the restoration of a healthy microbiota. However, in some cases, infections can self-resolve without the intervention of antibiotics. In this Review, we explore the mechanisms underlying microbiota restoration following insult (antibiotic or otherwise) to the skin, oral cavity, and gastrointestinal and urogenital tracts, highlighting recovery by natural processes and after probiotic administration.
Quaternary‐ammonium‐compounds are potent cationic antimicrobials used in everyday consumer products. Surface‐immobilized, quaternary‐ammonium‐compounds create an antimicrobial contact‐killing ...coating. We describe the preparation of a shape‐adaptive, contact‐killing coating by tethering quaternary‐ammonium‐compounds onto hyperbranched polyurea coatings, able to kill adhering bacteria by partially enveloping them. Even after extensive washing, coatings caused high contact‐killing of Staphylococcus epidermidis, both in culture‐based assays and through confocal‐laser‐scanning‐microscopic examination of the membrane‐damage of adhering bacteria. In culture‐based assays, at a challenge of 1600 CFU/cm2, contact‐killing was >99.99%. The working‐mechanism of dissolved quaternary‐ammonium‐compounds is based on their interdigitation in bacterial membranes, but it is difficult to envisage how immobilized quaternary‐ammonium‐molecules can exert such a mechanism of action. Staphylococcal adhesion forces to hyperbranched quaternary‐ammonium coatings were extremely high, indicating that quaternary‐ammonium‐molecules on hyperbranched polyurea partially envelope adhering bacteria upon contact. These lethally strong adhesion forces upon adhering bacteria then cause removal of membrane lipids and eventually lead to bacterial death.
Shape‐adaptive, hyperbranched polyurea with quaternary ammonium compounds. The preparation of AB2 monomers and the covalently attached hyperbranched polyurea coatings with polyethyleneimine covalently coupled to hyperbranched polyurea coatings. The coatings demonstrate high contact‐killing efficacies toward adhering staphylococci, without any demonstrable leaching of antibacterial compounds.
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