The oral microbiota and periodontal health in orthodontic patients.

The oral microbiota develops within the first 2 years of childhood and becomes distinct from the parents by 4 years-of-age. The oral microbiota plays an important role in the overall health/symbiosis of the individual. Deviations from the state of symbiosis leads to dysbiosis and an increased risk of pathogenicity. Deviations can occur not only from daily life activities but also from orthodontic interventions. Orthodontic appliances are formed from a variety of biomaterials. Once inserted, they serve as a breeding ground for microbial attachment, not only from new surface areas and crevices but also from material physicochemical interactions different than in the symbiotic state. Individuals undergoing orthodontic treatment show, compared with untreated people, qualitative and quantitative differences in activity within the oral microbiota, induced by increased retention of supra- and subgingival microbial plaque throughout the treatment period. These changes are at the root of the main undesirable effects, such as gingivitis, white spot lesions (WSL), and more severe caries lesions. Notably, the oral microbiota profile in the first weeks of orthodontic intervention might be a valuable indicator to predict and identify higher-risk individuals with respect to periodontal health and caries risk within an otherwise healthy population. Antimicrobial coatings have been used to dissuade microbes from adhering to the biomaterial; however, they disrupt the host microbiota, and several bacterial strains have become resistant. Smart biomaterials that can reduce the antimicrobial load preventing microbial adhesion to orthodontic appliances have shown promising results, but their complexity has kept many solutions from reaching the clinic. 3D printing technology provides opportunities for complex chemical syntheses to be performed uniformly, reducing the cost of producing smart biomaterials giving hope that they may reach the clinic in the near future. The purpose of this review is to emphasize the importance of the oral microbiota during orthodontic therapy and to use innovative technologies to better maintain its healthy balance during surgical procedures.

Nano-architectonics of Pt single-atoms and differently-sized nanoparticles supported by manganese-oxide nanosheets and impact on catalytic and anti-biofilm activities.

Hybrid-nanozymes are promising in various applications, but comprehensive comparison of hybrid-nanozymes composed of single-atoms or nanoparticles on the same support has never been made. Here, manganese-oxide nanosheets were loaded with Pt-single-atoms or differently-sized nanoparticles and their oxidase- and-peroxidase activities compared. High-resolution Transmission-Electron-Microscopy and corresponding Fast Fourier Transform imaging showed that Pt-nanoparticles (1.5 nm diameter) had no clear (111) crystal-planes, while larger nanoparticles had clear (111) crystal-planes. X-ray Photo-electron Spectroscopy demonstrated that unloaded nanosheets were composed of MnO2 with a high number of oxygen vacancies (Vo/Mn 0.4). Loading with 7.0 nm Pt-nanoparticles induced a change to Mn2O3, while loading with 1.5 nm nanoparticles increased the number of vacancies (Vo/Mn 1.2). Nanosheets loaded with 3.0 nm Pt-nanoparticles possessed similarly high catalytic activities as Pt-single-atoms. However, loading with 1.5 nm or 7.0 nm Pt-nanoparticles yielded lower catalytic activities. A model is proposed explaining the low catalytic activity of under- and over-sized Pt-nanoparticles as compared with intermediately-sized (3.0 nm) Pt-nanoparticles and single-atoms. Herewith, catalytic activities of hybrid-nanozymes composed of single-atoms and intermediately-sized nanoparticles are put a par, as confirmed here with respect to bacterial biofilm eradication. This conclusion facilitates a balanced choice between using Pt-single-atoms or nanoparticles in further development and application of hybrid-nanozymes.

Left ventricular shape index and eccentricity index with ECG-gated Nitrogen-13 ammonia PET/CT in patients with myocardial infarction, ischemia, and normal perfusion.

BACKGROUND: LV geometry with shape index (SI) and eccentricity index (EI) measured by myocardial perfusion positron emission tomography/computed tomography (PET/CT) may allow the evaluation of left ventricular (LV) adverse remodeling. This first study aims to explore the relationship of SI and EI values acquired by Nitrogen-13 ammonia PET/CT in patients with normal perfusion, ischemia, and myocardial infarction. And evaluate the correlations between the variables of LV geometry, and with the variables of LV function. METHODS AND RESULTS: One hundred and forty patients who underwent an electrocardiogram (ECG)-gated PET/CT were selected and classified into 4 groups according to ischemia or infarction burden (normal perfusion, mild ischemia, moderate-severe ischemia, and infarction). The variables were automatically retrieved using dedicated software (QPS/QGS; Cedars-Sinai, Los Angeles, CA, USA). On multicomparison analysis (one-way ANOVA and Dunnett's Test), subjects in the infarction group had significant higher values of SI end-diastolic rest (P < 0.001), and stress (P = 0.003), SI end-systolic rest (P = 0.002) and stress (P < 0.001) as well as statistically significant lower values of EI rest (P < 0.001) and stress (P < 0.001) when compared with all other groups. Regarding Pearson correlation, in the infarcted group all the variables of SI and EI were significantly correlated (P < 0.001) with strong correlation coefficients (>0.60). SI end-systolic correlated significantly with the variables of LV function independently of the group of patients (P < 0.05). CONCLUSIONS: Shape and eccentricity indices differ in patients with myocardial infarction as compared to patients with ischemia or normal perfusion. This encourage further research in their potential for detecting LV adverse remodeling.

Water in bacterial biofilms: pores and channels, storage and transport functions.

Bacterial biofilms occur in many natural and industrial environments. Besides bacteria, biofilms comprise over 70 wt% water. Water in biofilms occurs as bound- or free-water. Bound-water is adsorbed to bacterial surfaces or biofilm (matrix) structures and possesses different Infra-red and Nuclear-Magnetic-Resonance signatures than free-water. Bound-water is different from intra-cellularly confined-water or water confined within biofilm structures and bacteria are actively involved in building water-filled structures by bacterial swimmers, dispersion or lytic self-sacrifice. Water-filled structures can be transient due to blocking, resulting from bacterial growth, compression or additional matrix formation and are generally referred to as "channels and pores." Channels and pores can be distinguished based on mechanism of formation, function and dimension. Channels allow transport of nutrients, waste-products, signalling molecules and antibiotics through a biofilm provided the cargo does not adsorb to channel walls and channels have a large length/width ratio. Pores serve a storage function for nutrients and dilute waste-products or antimicrobials and thus should have a length/width ratio close to unity. The understanding provided here on the role of water in biofilms, can be employed to artificially engineer by-pass channels or additional pores in industrial and environmental biofilms to increase production yields or enhance antimicrobial penetration in infectious biofilms.

On-demand pulling-off of magnetic nanoparticles from biomaterial surfaces through implant-associated infectious biofilms for enhanced antibiotic efficacy.

Biomaterial-associated infections can occur any time after surgical implantation of biomaterial implants and limit their success rates. On-demand, antimicrobial release coatings have been designed, but in vivo release triggers uniquely relating with infection do not exist, while inadvertent leakage of antimicrobials can cause exhaustion of a coating prior to need. Here, we attach magnetic-nanoparticles to a biomaterial surface, that can be pulled-off in a magnetic field through an adhering, infectious biofilm. Magnetic-nanoparticles remained stably attached to a surface upon exposure to PBS for at least 50 days, did not promote bacterial adhesion or negatively affect interaction with adhering tissue cells. Nanoparticles could be magnetically pulled-off from a surface through an adhering biofilm, creating artificial water channels in the biofilm. At a magnetic-nanoparticle coating concentration of 0.64 mg cm-2, these by-pass channels increased the penetrability of Staphylococcus aureus and Pseudomonas aeruginosa biofilms towards different antibiotics, yielding 10-fold more antibiotic killing of biofilm inhabitants than in absence of artificial channels. This innovative use of magnetic-nanoparticles for the eradication of biomaterial-associated infections requires no precise targeting of magnetic-nanoparticles and allows more effective use of existing antibiotics by breaking the penetration barrier of an infectious biofilm adhering to a biomaterial implant surface on-demand.

Encapsulation of Photothermal Nanoparticles in Stealth and pH-Responsive Micelles for Eradication of Infectious Biofilms In Vitro and In Vivo.

Photothermal nanoparticles can be used for non-antibiotic-based eradication of infectious biofilms, but this may cause collateral damage to tissue surrounding an infection site. In order to prevent collateral tissue damage, we encapsulated photothermal polydopamine-nanoparticles (PDA-NPs) in mixed shell polymeric micelles, composed of stealth polyethylene glycol (PEG) and pH-sensitive poly(ß-amino ester) (PAE). To achieve encapsulation, PDA-NPs were made hydrophobic by electrostatic binding of indocyanine green (ICG). Coupling of ICG enhanced the photothermal conversion efficacy of PDA-NPs from 33% to 47%. Photothermal conversion was not affected by micellar encapsulation. No cytotoxicity or hemolytic effects of PEG-PAE encapsulated PDA-ICG-NPs were observed. PEG-PAE encapsulated PDA-ICG-NPs showed good penetration and accumulation in a Staphylococcus aureus biofilm. Penetration and accumulation were absent when nanoparticles were encapsulated in PEG-micelles without a pH-responsive moiety. PDA-ICG-NPs encapsulated in PEG-PAE-micelles found their way through the blood circulation to a sub-cutaneous infection site after tail-vein injection in mice, yielding faster eradication of infections upon near-infrared (NIR) irradiation than could be achieved after encapsulation in PEG-micelles. Moreover, staphylococcal counts in surrounding tissue were reduced facilitating faster wound healing. Thus, the combined effect of targeting and localized NIR irradiation prevented collateral tissue damage while eradicating an infectious biofilm.

Escherichia coli Colonization of Intestinal Epithelial Layers In Vitro in the Presence of Encapsulated Bifidobacterium breve for Its Protection against Gastrointestinal Fluids and Antibiotics.

Encapsulation of probiotic bacteria can enhance their functionality when used in combination with antibiotics for treating intestinal tract infections. The interaction strength of encapsulating shells, however, varies among the encapsulation methods and impacts encapsulation. Here, we compared the protection offered by encapsulating shells with different interaction strengths toward probiotic Bifidobacterium breve against simulated gastric fluid and tetracycline, including protamine-assisted SiO2 nanoparticle yolk-shell packing (weak interaction across a void), alginate gelation (intermediate interaction due to hydrogen binding), and ZIF-8 mineralization (strong interaction due to coordinate covalent binding). The presence of encapsulating shells was demonstrated using X-ray-photoelectron spectroscopy, particulate microelectrophoresis, and dynamic light scattering. Strong interaction upon ZIF-8 encapsulation caused demonstrable cell wall damage to B. breve and slightly reduced bacterial viability, delaying the growth of encapsulated bacteria. Cell wall damage and reduced viability did not occur upon encapsulation with weakly interacting yolk-shells. Only alginate-hydrogel-based shells yielded protection against simulated gastric acid and tetracycline. Accordingly, only alginate-hydrogel-encapsulated B. breve operated synergistically with tetracycline in killing tetracycline-resistant Escherichia coli adhering to intestinal epithelial layers and maintained surface coverage of transwell membranes by epithelial cell layers and their barrier integrity. This synergy between alginate-hydrogel-encapsulated B. breve and an antibiotic warrants further studies for treating antibiotic-resistant E. coli infections in the gastrointestinal tract.

Antimicrobial loading of nanotubular titanium surfaces favoring surface coverage by mammalian cells over bacterial colonization.

Titanium is frequently used for dental implants, percutaneous pins and screws or orthopedic joint prostheses. Implant surfaces can become peri-operatively contaminated by surgically introduced bacteria during implantation causing lack of surface coverage by mammalian cells and subsequent implant failure. Especially implants that have to function in a bacteria-laden environment such as dental implants or percutaneous pins, cannot be surgically implanted while being kept sterile. Accordingly, contaminating bacteria adhering to implant surfaces hamper successful surface coverage by mammalian cells required for long-term functioning. Here, nanotubular titanium surfaces were prepared and loaded with Ag nanoparticles or gentamicin with the aim of killing contaminating bacteria in order to favor surface coverage by mammalian cells. In mono-cultures, unloaded nanotubules did not cause bacterial killing, but loading of Ag nanoparticles or gentamicin reduced the number of adhering Staphylococcus aureus or Pseudomonas aeruginosa CFUs. A gentamicin-resistant Staphylococcus epidermidis was only killed upon loading with Ag nanoparticles. However, unlike low-level gentamicin loading, loading with Ag nanoparticles also caused tissue-cell death. In bi-cultures, low-level gentamicin-loading of nanotubular titanium surfaces effectively eradicated contaminating bacteria favoring surface coverage by mammalian cells. Thus, care must be taken in loading nanotubular titanium surfaces with Ag nanoparticles, while low-level gentamicin-loaded nanotubular titanium surfaces can be used as a local antibiotic delivery system to negate failure of titanium implants due to peri-operatively introduced, contaminating bacteria without hampering surface coverage by mammalian cells.

Influence of interaction between surface-modified magnetic nanoparticles with infectious biofilm components in artificial channel digging and biofilm eradication by antibiotics in vitro and in vivo.

Magnetic targeting of antimicrobial-loaded magnetic nanoparticles to micrometer-sized infectious biofilms is challenging. Bacterial biofilms possess water channels that facilitate transport of nutrient and metabolic waste products, but are insufficient to allow deep penetration of antimicrobials and bacterial killing. Artificial channel digging in infectious biofilms involves magnetically propelling nanoparticles through a biofilm to dig additional channels to enhance antimicrobial penetration. This does not require precise targeting. However, it is not known whether interaction of magnetic nanoparticles with biofilm components impacts the efficacy of antibiotics after artificial channel digging. Here, we functionalized magnetic-iron-oxide-nanoparticles (MIONPs) with polydopamine (PDA) to modify their interaction with staphylococcal pathogens and extracellular-polymeric-substances (EPS) and relate the interaction with in vitro biofilm eradication by gentamicin after magnetic channel digging. PDA-modified MIONPs had less negative zeta potentials than unmodified MIONPs due to the presence of amino groups and accordingly more interaction with negatively charged staphylococcal cell surfaces than unmodified MIONPs. Neither unmodified nor PDA-modified MIONPs interacted with EPS. Concurrently, use of non-interacting unmodified MIONPs for artificial channel digging in in vitro grown staphylococcal biofilms enhanced the efficacy of gentamicin more than the use of interacting, PDA-modified MIONPs. In vivo experiments in mice using a sub-cutaneous infection model confirmed that non-interacting, unmodified MIONPs enhanced eradication by gentamicin of Staphylococcus aureus Xen36 biofilms about 10 fold. Combined with the high biocompatibility of magnetic nanoparticles, these results form an important step in understanding the mechanism of artificial channel digging in infectious biofilms for enhancing antibiotic efficacy in hard-to-treat infectious biofilms in patients.

Thermo-resistance of ESKAPE-panel pathogens, eradication and growth prevention of an infectious biofilm by photothermal, polydopamine-nanoparticles in vitro.

Nanotechnology offers many novel infection-control strategies that may help prevent and treat antimicrobial-resistant bacterial infections. Here, we synthesized polydopamine, photothermal-nanoparticles (PDA-NPs) without further surface-functionalization to evaluate their potential with respect to biofilm-control. Most ESKAPE-panel pathogens in suspension with photothermal-nanoparticles showed three- to four-log-unit reductions upon Near-Infra-Red (NIR)-irradiation, but for enterococci only less than two-log unit reduction was observed. Exposure of existing Staphylococcus aureus biofilms to photothermal-nanoparticles followed by NIR-irradiation did not significantly kill biofilm-inhabitants. This indicates that the biofilm mode of growth poses a barrier to penetration of photothermal-nanoparticles, yielding dissipation of heat to the biofilm-surrounding rather than in its interior. Staphylococcal biofilm-growth in the presence of photothermal-nanoparticles could be significantly prevented after NIR-irradiation because PDA-NPs were incorporated in the biofilm and heat dissipated inside it. Thus, unmodified photothermal nanoparticles have potential for prophylactic infection-control, but data also constitute a warning for possible development of thermo-resistance in infectious pathogens.

Two-Stage Interpretation of Changes in TEER of Intestinal Epithelial Layers Protected by Adhering Bifidobacteria During E. coli Challenges.

Mechanisms of gastrointestinal protection by probiotic bacteria against infection involve amongst others, modulation of intestinal epithelial barrier function. Trans-epithelial electrical resistance (TEER) is widely used to evaluate cellular barrier functions. Here, we developed a two-stage interpretative model of the time-dependence of the TEER of epithelial layers grown in a transwell during Escherichia coli challenges in the absence or presence of adhering bifidobacteria. E. coli adhesion in absence or presence of adhering bifidobacteria was enumerated using selective plating. After 4-8 h, E. coli challenges increased TEER to a maximum due to bacterial adhesion and increased expression of a tight-junction protein [zonula occludens-1 (ZO-1)], concurrent with a less dense layer structure, that is indicative of mild epithelial layer damage. Before the occurrence of a TEER-maximum, decreases in electrical conductance (i.e., the reciprocal TEER) did not relate with para-cellular dextran-permeability, but after occurrence of a TEER-maximum, dextran-permeability and conductance increased linearly, indicative of more severe epithelial layer damage. Within 24 h after the occurrence of a TEER maximum, TEER decreased to below the level of unchallenged epithelial layers demonstrating microscopically observable holes and apoptosis. Under probiotic protection by adhering bifidobacteria, TEER-maxima were delayed or decreased in magnitude due to later transition from mild to severe damage, but similar linear relations between conductance and dextran permeability were observed as in absence of adhering bifidobacteria. Based on the time-dependence of the TEER and the relation between conductance and dextran-permeability, it is proposed that bacterial adhesion to epithelial layers first causes mild damage, followed by more severe damage after the occurrence of a TEER-maximum. The mild damage caused by E. coli prior to the occurrence of TEER maxima was reversible upon antibiotic treatment, but the severe damage after occurrence of TEER maxima could not be reverted by antibiotic treatment. Thus, single-time TEER is interpretable in two ways, depending whether increasing to or decreasing from its maximum. Adhering bifidobacteria elongate the time-window available for antibiotic treatment to repair initial pathogen damage to intestinal epithelial layers.

Visualization of Bacterial Colonization and Cellular Layers in a Gut-on-a-Chip System Using Optical Coherence Tomography.

Imaging of cellular layers in a gut-on-a-chip system has been confined to two-dimensional (2D)-imaging through conventional light microscopy and confocal laser scanning microscopy (CLSM) yielding three-dimensional- and 2D-cross-sectional reconstructions. However, CLSM requires staining and is unsuitable for longitudinal visualization. Here, we compare merits of optical coherence tomography (OCT) with those of CLSM and light microscopy for visualization of intestinal epithelial layers during protection by a probiotic Bifidobacterium breve strain and a simultaneous pathogen challenge by an Escherichia coli strain. OCT cross-sectional images yielded film thicknesses that coincided with end-point thicknesses derived from cross-sectional CLSM images. Light microscopy on histological sections of epithelial layers at the end-point yielded smaller layer thicknesses than OCT and CLSM. Protective effects of B. breve adhering to an epithelial layer against an E. coli challenge included the preservation of layer thickness and membrane surface coverage by epithelial cells. OCT does not require staining or sectioning, making OCT suitable for longitudinal visualization of biological films, but as a drawback, OCT does not allow an epithelial layer to be distinguished from bacterial biofilms adhering to it. Thus, OCT is ideal to longitudinally evaluate epithelial layers under probiotic protection and pathogen challenges, but proper image interpretation requires the application of a second method at the end-point to distinguish bacterial and epithelial films.

Role of adhesion forces in mechanosensitive channel gating in Staphylococcus aureus adhering to surfaces.

Mechanosensitive channels in bacterial membranes open or close in response to environmental changes to allow transmembrane transport, including antibiotic uptake and solute efflux. In this paper, we hypothesize that gating of mechanosensitive channels is stimulated by forces through which bacteria adhere to surfaces. Hereto, channel gating is related with adhesion forces to different surfaces of a Staphylococcus aureus strain and its isogenic ΔmscL mutant, deficient in MscL (large) channel gating. Staphylococci becoming fluorescent due to uptake of calcein, increased with adhesion force and were higher in the parent strain (66% when adhering with an adhesion force above 4.0 nN) than in the ΔmscL mutant (40% above 1.2 nN). This suggests that MscL channels open at a higher critical adhesion force than at which physically different, MscS (small) channels open and contribute to transmembrane transport. Uptake of the antibiotic dihydrostreptomycin was monitored by staphylococcal killing. The parent strain exposed to dihydrostreptomycin yielded a CFU reduction of 2.3 log-units when adhering with an adhesion force above 3.5 nN, but CFU reduction remained low (1.0 log-unit) in the mutant, independent of adhesion force. This confirms that large channels open at a higher critical adhesion-force than small channels, as also concluded from calcein transmembrane transport. Collectively, these observations support our hypothesis that adhesion forces to surfaces play an important role, next to other established driving forces, in staphylococcal channel gating. This provides an interesting extension of our understanding of transmembrane antibiotic uptake and solute efflux in infectious staphylococcal biofilms in which bacteria experience adhesion forces from a wide variety of surfaces, like those of other bacteria, tissue cells, or implanted biomaterials.

Eradicating Infecting Bacteria while Maintaining Tissue Integration on Photothermal Nanoparticle-Coated Titanium Surfaces.

Photothermal nanoparticles locally release heat when irradiated by near-infrared (NIR). Clinical applications initially involved tumor treatment, but currently extend toward bacterial infection control. Applications toward much smaller, micrometer-sized bacterial infections, however, bear the risk of collateral damage by dissipating heat into tissues surrounding an infection site. This can become a complication when photothermal nanoparticle coatings are clinically applied on biomaterial surfaces requiring tissue integration, such as titanium-made, bone-anchored dental implants. Dental implants can fail due to infection in the pocket formed between the implant screw and the surrounding soft tissue ("peri-implantitis"). We address the hitherto neglected potential complication of collateral tissue damage by evaluating photothermal, polydopamine nanoparticle (PDA-NP) coatings on titanium surfaces in different coculture models. NIR irradiation of PDA-NP-coated (200 µg/cm2) titanium surfaces with adhering Staphylococcus aureus killed staphylococci within an irradiation time window of around 3 min. Alternatively, when covered with human gingival fibroblasts, this irradiation time window maintained surface coverage by fibroblasts. Contaminating staphylococci on PDA-NP-coated titanium surfaces, as can be per-operatively introduced, reduced surface coverage by fibroblasts, and this could be prevented by NIR irradiation for 5 min or longer prior to allowing fibroblasts to adhere and grow. Negative impacts of early postoperative staphylococcal challenges to an existing fibroblast layer covering a coated surface were maximally prevented by 3 min NIR irradiation. Longer irradiation times caused collateral fibroblast damage. Late postoperative staphylococcal challenges to a protective keratinocyte layer covering a fibroblast layer required 10 min NIR irradiation for adverting a staphylococcal challenge. This is longer than foreseen from monoculture studies because of additional heat uptake by the keratinocyte layer. Summarizing, photothermal treatment of biomaterial-associated infection requires precise timing of NIR irradiation to prevent collateral damage to tissues surrounding the infection site.

Homogeneous Distribution of Magnetic, Antimicrobial-Carrying Nanoparticles through an Infectious Biofilm Enhances Biofilm-Killing Efficacy.

Magnetic, antimicrobial-carrying nanoparticles provide a promising, new and direly needed antimicrobial strategy against infectious bacterial biofilms. Penetration and accumulation of antimicrobials over the thickness of a biofilm is a conditio sine qua non for effective killing of biofilm inhabitants. Simplified schematics on magnetic-targeting always picture homogeneous distribution of magnetic, antimicrobial-carrying nanoparticles over the thickness of biofilms, but this is not easy to achieve. Here, gentamicin-carrying magnetic nanoparticles (MNPs-G) were synthesized through gentamicin conjugation with iron-oxide nanoparticles and used to demonstrate the importance of their homogeneous distribution over the thickness of a biofilm. Diameters of MNPs-G were around 60 nm, well below the limit for reticuloendothelial rejection. MNPs-G killed most ESKAPE-panel pathogens, including Escherichia coli, equally as well as gentamicin in solution. MNPs-G distribution in a Staphylococcus aureus biofilm was dependent on magnetic-field exposure time and most homogeneous after 5 min magnetic-field exposure. Exposure of biofilms to MNPs-G with 5 min magnetic-field exposure yielded not only homogeneous distribution of MNPs-G, but concurrently better staphylococcal killing at all depths than that of MNPs, gentamicin in solution, and MNPs-G, or after other magnet-field exposure times. In summary, homogeneous distribution of gentamicin-carrying magnetic nanoparticles over the thickness of a staphylococcal biofilm was essential for killing biofilm inhabitants and required optimizing of the magnetic-field exposure time. This conclusion is important for further successful development of magnetic, antimicrobial-carrying nanoparticles toward clinical application.

Artificial Channels in an Infectious Biofilm Created by Magnetic Nanoparticles Enhanced Bacterial Killing by Antibiotics.

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.

Physico-chemistry from initial bacterial adhesion to surface-programmed biofilm growth.

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.

Surface enhanced fluorescence and nanoscopic cell wall deformation in adhering Staphylococcus aureus upon exposure to cell wall active and non-active antibiotics.

In infections, bacteria often adhere to surfaces and become deformed by the forces with which they adhere. Nanoscopic cell wall deformation defines bacterial responses to environmental conditions and is likely influenced by antibiotics. Here, staphylococcal cell wall deformation upon exposure to cell wall active and non-active antibiotics or their combinations is compared for two green-fluorescent (GFP) isogenic Staphylococcus aureus strains adhering to a gold surface, of which one lacks peptidoglycan cross-linking. Exposure to cell wall active antibiotics caused greater cell wall deformation than a buffer control in the GFP parent and in the Δpbp4GFP isogenic mutant, as measured by surface-enhanced-fluorescence. Cell wall non-active antibiotics only yielded greater deformation than a buffer control in the parent strain, while combinations of cell wall active and non-active antibiotics did not cause greater cell wall deformation. 3D-analysis of the impact of adhesion forces and Young's moduli of the cell wall, both measured using atomic force microscopy, led to the conclusion that increased deformation was mainly due to cell wall weakening and not due to the effects of antibiotics on adhesion forces. Interactions between bacteria and antibiotics are mostly studied using planktonic bacteria, while during infection, bacteria are in an adhering state that deforms their cell wall and therewith influences their adaptive responses. We anticipate that the demonstration of cell wall weakening in adhering bacteria under the influence of antibiotics and the role of peptidoglycan herein will aid in the development of new antibiotics. Surface-enhanced-fluorescence may accordingly develop into a new, highly-sensitive method for diagnosing antibiotic-resistant bacteria.