Rapid Bacterial Detection and Gram-Identification Using Bacterially Activated, Macrophage-Membrane-Coated Nanowired-Si Surfaces in a Microfluidic Device.

Bacterially induced sepsis requires rapid bacterial detection and identification. Hours count for critically ill septic patients, while current culture-based detection requires at least 10 h up to several days. Here, we apply a microfluidic device equipped with a bacterially activated, macrophage-membrane-coating on nanowired-Si adsorbent surfaces for rapid, bacterial detection and Gram-identification in bacterially contaminated blood. Perfusion of suspensions of Gram-negative or Gram-positive bacteria through a microfluidic device equipped with membrane-coated adsorbent surfaces detected low (<10 CFU/mL) bacterial levels. Subsequent, in situ fluorescence-staining yielded Gram-identification for guiding antibiotic selection. In mixed Escherichia coli and Staphylococcus aureus suspensions, Gram-negative and Gram-positive bacteria were detected in the same ratios as those fixed in suspension. Results were validated with a 100% correct score by blinded evaluation (two observers) of 15 human blood samples, spiked with widely different bacterial strains or combinations of strains, demonstrating the potential of the platform for rapid (1.5 h in total) diagnosis of bacterial sepsis.

Identification of Potential Antimicrobial Targets of Pseudomonas aeruginosa Biofilms through a Novel Screening Approach.

Pseudomonas aeruginosa is an opportunistic pathogen of considerable medical importance, owing to its pronounced antibiotic tolerance and association with cystic fibrosis and other life-threatening diseases. The aim of this study was to highlight the genes responsible for P. aeruginosa biofilm tolerance to antibiotics and thereby identify potential new targets for the development of drugs against biofilm-related infections. By developing a novel screening approach and utilizing a public P. aeruginosa transposon insertion library, several biofilm-relevant genes were identified. The Pf phage gene (PA0720) and flagellin gene (fliC) conferred biofilm-specific tolerance to gentamicin. Compared with the reference biofilms, the biofilms formed by PA0720 and fliC mutants were completely eliminated with a 4-fold-lower gentamicin concentration. Furthermore, the mreC, pprB, coxC, and PA3785 genes were demonstrated to play major roles in enhancing biofilm tolerance to gentamicin. The analysis of biofilm-relevant genes performed in this study provides important novel insights into the understanding of P. aeruginosa antibiotic tolerance, which will facilitate the detection of antibiotic resistance and the development of antibiofilm strategies against P. aeruginosa. IMPORTANCE Pseudomonas aeruginosa is an opportunistic pathogen of high medical importance and is one of the main pathogens responsible for the mortality of patients with cystic fibrosis. In addition to inherited antibiotic resistance, P. aeruginosa can form biofilms, defined as communities of microorganisms embedded in a self-produced matrix of extracellular polymeric substances adhering to each other and/or to a surface. Biofilms protect bacteria from antibiotic treatments and represent a major reason for antibiotic failure in the treatment of chronic infections caused by cystic fibrosis. Therefore, it is crucial to develop new therapeutic strategies aimed at specifically eradicating biofilms. The aim of this study was to generalize a novel screening method for biofilm research and to identify the possible genes involved in P. aeruginosa biofilm tolerance to antibiotics, both of which could improve the understanding of biofilm-related infections and allow for the identification of relevant therapeutic targets for drug development.

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms.

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

Nonviral Expression of LL-37 in a Human Skin Equivalent to Prevent Infection in Skin Wounds.

Inefficient autologous tissue recovery in skin wounds increases the susceptibility of patients to infections caused by multidrug resistant microorganisms, resulting in a high mortality rate. Genetic modification of skin cells has become an important field of study because it could lead to the construction of more functional skin grafts, through the overexpression of antimicrobial peptides that would prevent early contamination and infection with bacteria. In this study, we produce and evaluate human skin equivalents (HSEs) containing transfected human primary fibroblasts and keratinocytes by polyplexes to express the antimicrobial peptide LL-37. The effect of LL-37 on the metabolic activity of normal HSEs was evaluated before the construction of the transfected HSEs, and the antimicrobial efficacy against Pseudomonas aeruginosa and Staphylococcus aureus was evaluated. Subsequently, the levels of LL-37 in the culture supernatants of transfected HSEs, as well as the local expression, were determined. It was found that LL-37 treatment significantly promoted the cellular proliferation of HSEs. Furthermore, HSEs that express elevated levels of LL-37 were shown to possess histological characteristics close to the normal skin and display enhanced antimicrobial activity against S. aureus in vitro. These findings demonstrate that HSEs expressing LL-37 through nonviral modification of skin cells are a promising approach for the prevention of bacterial colonization in wounds.

Liposomes with Water as a pH-Responsive Functionality for Targeting of Acidic Tumor and Infection Sites.

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-H2 O). DCPA-H2 O liposomes became protonated relatively fast already at pH<6.8, due to the high HOMO binding energy of DCPA-H2 O. In murine models, DCPA-H2 O liposomes had longer blood circulation times than natural DPPC or cationic DCPM liposomes, while after tail-vein injection DCPA-H2 O 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-H2 O liposomes exceeded the ones of clinically applied ciprofloxacin as well as of ciprofloxacin-loaded DPPC or DCPM liposomes.

Candida Biofilm Formation Assay on Essential Oil Coated Silicone Rubber.

Development of biofilm associated candidemia for patients with implanted biomaterials causes an urgency to develop antimicrobial and biofilm inhibitive coatings in the management of recalcitrant Candida infections. Recently, there is an increase in the number of patients with biofilm formation and resistance to antifungal therapy. Therefore, there is a growing interest to use essential oils as coating agents in order to prevent biomaterial-associated Candida infections. Often high costs, complicated and laborious technologies are used for both applying the coating and determination of the antibiofilm effects hampering a rapid screening of essential oils. In order to determine biofilm formation of Candida on essential oil coated surfaces easier, cheaper and faster, we developed an essential oil (lemongrass oil) coated surface (silicone-rubber) by using a hypromellose ointment/essential oil mixture. Furthermore, we modified the "crystal violet binding assay" to quantify the biofilm mass of Candida biofilm formed on the lemongrass oil coated silicone rubber surface. The essential oil coating and the biomass determination of biofilms on silicone rubber can be easily applied with simple and accessible equipment, and will therefore provide rapid information about whether or not a particular essential oil is antiseptic, also when it is used as a coating agent.

Lubricating properties of chewing stimulated whole saliva from patients suffering from xerostomia.

OBJECTIVES: The study aimed to quantify the lubricating properties of chewing stimulated whole saliva from healthy controls (n = 22), from patients suffering from primary Sjögren's syndrome (n = 37) and from patients undergoing head-and-neck radiotherapy (n = 34). MATERIALS AND METHODS: All participants had to complete the Xerostomia Inventory questionnaire to score dry mouth sensation. Lubrication was measured using an ex vivo tongue-enamel friction system in terms of Relief and Relief period. MUC5b and total protein concentrations of the saliva samples were measured by an enzyme-linked immunosorbent assay and a bicinchoninic acid assay, respectively. RESULTS: Relief of Sjögren's patients' saliva and post-irradiation patients' saliva was similar compared with healthy controls, but saliva from post-irradiation patients lubricated significantly better than saliva from Sjögren's patients. The Relief period was similar between the three groups. The Relief and Relief period were higher for saliva samples post-irradiation compared to pre-irradiation. MUC5b and total protein concentrations were comparable in all groups. MUC5b and total protein output were significantly lower in patients subjected to radiotherapy compared to saliva from healthy controls and pre-irradiation patients. MUC5b concentrations positively correlated with lubricating properties of post-irradiation patient saliva. CONCLUSIONS: The lubricating properties of patient saliva were not any worse than healthy controls. Lower flow rate leads to lower availability of saliva in the oral cavity and decreases the overall output of protein and MUC5b, which might result in an insufficient replenishing of the mucosal salivary film. CLINICAL RELEVANCE: An insufficient replenishing might underlie the sensation of a dry mouth and loss of oral function.

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.

Antifungal and biofilm inhibitory effect of Cymbopogon citratus (lemongrass) essential oil on biofilm forming by Candida tropicalis isolates; an in vitro study.

ETHNOPHARMACOLOGICAL RELEVANCE: Cymbopogon citratus (lemongrass) essential oil has been widely used as a traditional medicine and is well known for antimicrobial properties. Therefore, it might be a potent anti-infective and biofilm inhibitive against Candida tropicalis infections. Until now, no ideal coating or cleaning method based on an essential oil has been described to prevent biofilm formation of Candida strains on silicone rubber maxillofacial prostheses, voice prostheses and medical devices susceptible to C. tropicalis infections. AIM OF THE STUDY: To investigate the antifungal and biofilm inhibitory effects of Cymbopogon citratus oil. Clinical isolates of C. tropicalis biofilms on different biomaterials were used to study the inhibitory effect. MATERIALS AND METHODS: The efficacy of Cymbopogon citratus, Cuminum cyminum, Citrus limon and Cinnamomum verum essential oils were compared on biofilm formation of three C. tropicalis isolates on 24 well polystyrene plates. C. citratus oil coated silicone rubber surfaces were prepared using hypromellose ointment as a vehicle. The antifungal tests to determine minimum inhibitory and minimum fungicidal concentrations were assessed by a microbroth dilution method and biofilm formation was determined by a crystal violet binding assay. RESULTS: C. tropicalis strains formed more biofilm on hydrophobic materials than on hydrophilic glass. C. citratus oil showed a high antifungal effect against all C. tropicalis strains. For comparison, C. limon oil and C. cyminum oil showed minor to no killing effect against the C. tropicalis strains. C. citratus oil had the lowest minimal inhibitory concentration of all essential oils tested and inhibited biofilm formation of all C. tropicalis strains. C. citratus oil coating on silicone rubber resulted in a 45-76% reduction in biofilm formation of all C. tropicalis strains. CONCLUSION: Cymbopogon citratus oil has good potential to be used as an antifungal and antibiofilm agent on silicone rubber prostheses and medical devices on which C. tropicalis biofilms pose a serious risk for skin infections and may cause a shorter lifespan of the prosthesis.

Polarization of Macrophages, Cellular Adhesion, and Spreading on Bacterially Contaminated Gold Nanoparticle-Coatings in Vitro.

Biomaterial-associated infections often arise from contaminating bacteria adhering to an implant surface that are introduced during surgical implantation and not effectively eradicated by antibiotic treatment. Whether or not infection develops from contaminating bacteria depends on an interplay between bacteria contaminating the biomaterial surface and tissue cells trying to integrate the surface with the aid of immune cells. The biomaterial surface plays a crucial role in defining the outcome of this race for the surface. Tissue integration is considered the best protection of a biomaterial implant against infectious bacteria. This paper aims to determine whether and how macrophages aid osteoblasts and human mesenchymal stem cells to adhere and spread over gold nanoparticle (GNP)-coatings with different hydrophilicity and roughness in the absence or presence of contaminating, adhering bacteria. All GNP-coatings had identical chemical surface composition, and water contact angles decreased with increasing roughness. Upon increasing the roughness of the GNP-coatings, the presence of contaminating Staphylococcus epidermidis in biculture with cells gradually decreased surface coverage by adhering and spreading cells, as in the absence of staphylococci. More virulent Staphylococcus aureus fully impeded cellular adhesion and spreading on smooth gold- or GNP-coatings, while Escherichia coli allowed minor cellular interaction. Murine macrophages in monoculture tended toward their pro-inflammatory "fighting" M1-phenotype on all coatings to combat the biomaterial, but in bicultures with contaminating, adhering bacteria, macrophages demonstrated Ym1 expression, indicative of polarization toward their anti-inflammatory "fix-and-repair" M2-phenotype. Damage repair of cells by macrophages improved cellular interactions on intermediately hydrophilic/rough (water contact angle 30 deg/surface roughness 118 nm) GNP-coatings in the presence of contaminating, adhering Gram-positive staphylococci but provided little aid in the presence of Gram-negative E. coli. Thus, the merits on GNP-coatings to influence the race for the surface and prevent biomaterial-associated infection critically depend on their hydrophilicity/roughness and the bacterial strain involved in contaminating the biomaterial surface.

Role of Viscoelasticity in Bacterial Killing by Antimicrobials in Differently Grown Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa colonizes the sputum of most adult cystic fibrosis patients, forming difficult-to-eradicate biofilms in which bacteria are protected in their self-produced extracellular polymeric substance (EPS) matrices. EPS provide biofilms with viscoelastic properties, causing time-dependent relaxation after stress-induced deformation, according to multiple characteristic time constants. These time constants reflect different biofilm (matrix) components. Since the viscoelasticity of biofilms has been related to antimicrobial penetration but not yet bacterial killing, this study aims to relate killing of P. aeruginosa, in its biofilm mode of growth, by three antimicrobials to biofilm viscoelasticity. P. aeruginosa biofilms were grown for 18 h in a constant-depth film fermenter, with mucin-containing artificial sputum medium (ASM+), artificial sputum medium without mucin (ASM-), or Luria-Bertani (LB) broth; this yielded 100-µm-thick biofilms that differed in their amounts of matrix environmental DNA (eDNA) and polysaccharides. Low-load compression testing, followed by three-element Maxwell analyses, showed that the fastest relaxation component, associated with unbound water, was most important in LB-medium-grown biofilms. Slower components due to water with dissolved polysaccharides, insoluble polysaccharides, and eDNA were most important in the relaxation of ASM+-grown biofilms. ASM--grown biofilms showed intermediate stress relaxation. P. aeruginosa in LB-medium-grown biofilms was killed most by exposure to tobramycin, colistin, or an antimicrobial peptide, while ASM+ provided the most protective matrix, with less water and most insoluble polysaccharides and eDNA. In conclusion, stress relaxation of P. aeruginosa biofilms grown in different media revealed differences in matrix composition that, within the constraints of the antimicrobials and growth media applied, correlated with the matrix protection offered against different antimicrobials.

Applications and Perspectives of Cascade Reactions in Bacterial Infection Control.

Cascade reactions integrate two or more reactions, of which each subsequent reaction can only start when the previous reaction step is completed. Employing natural substrates in the human body such as glucose and oxygen, cascade reactions can generate reactive oxygen species (ROS) to kill tumor cells, but cascade reactions may also have potential as a direly needed, novel bacterial infection-control strategy. ROS can disintegrate the EPS matrix of infectious biofilm, disrupt bacterial cell membranes, and damage intra-cellular DNA. Application of cascade reactions producing ROS as a new infection-control strategy is still in its infancy. The main advantages for infection-control cascade reactions include the fact that they are non-antibiotic based and induction of ROS resistance is unlikely. However, the amount of ROS generated is generally low and antimicrobial efficacies reported are still far <3-4 log units necessary for clinical efficacy. Increasing the amounts of ROS generated by adding more substrate bears the risk of collateral damage to tissue surrounding an infection site. Collateral tissue damage upon increasing substrate concentrations may be prevented by locally increasing substrate concentrations, for instance, using smart nanocarriers. Smart, pH-responsive nanocarriers can self-target and accumulate in infectious biofilms from the blood circulation to confine ROS production inside the biofilm to yield long-term presence of ROS, despite the short lifetime (nanoseconds) of individual ROS molecules. Increasing bacterial killing efficacies using cascade reaction components containing nanocarriers constitutes a first, major challenge in the development of infection-control cascade reactions. Nevertheless, their use in combination with clinical antibiotic treatment may already yield synergistic effects, but this remains to be established for cascade reactions. Furthermore, specific patient groups possessing elevated levels of endogenous substrate (for instance, diabetic or cancer patients) may benefit from the use of cascade reaction components containing nanocarriers.

Structural changes in S. epidermidis biofilms after transmission between stainless steel surfaces.

Transmission is a main route for bacterial contamination, involving bacterial detachment from a donor and adhesion to receiver surfaces. This work aimed to compare transmission of an extracellular polymeric substance (EPS) producing and a non-EPS producing Staphylococcus epidermidis strain from biofilms on stainless steel. After transmission, donor surfaces remained fully covered with biofilm, indicating transmission through cohesive failure in the biofilm. Counter to the numbers of biofilm bacteria, the donor and receiver biofilm thicknesses did not add up to the pre-transmission donor biofilm thickness, suggesting more compact biofilms after transmission, especially for non-EPS producing staphylococci. Accordingly, staphylococcal density per unit biofilm volume had increased from 0.20 to 0.52 µm-3 for transmission of the non-EPS producing strain under high contact pressure. The EPS producing strain had similar densities before and after transmission (0.17 µm-3). This suggests three phases in biofilm transmission: (1) compression, (2) separation and (3) relaxation of biofilm structure to its pre-transmission density in EPS-rich biofilms.

Influence of biofilm lubricity on shear-induced transmission of staphylococcal biofilms from stainless steel to silicone rubber.

In real-life situations, bacteria are often transmitted from biofilms growing on donor surfaces to receiver ones. Bacterial transmission is more complex than adhesion, involving bacterial detachment from donor and subsequent adhesion to receiver surfaces. Here, we describe a new device to study shear-induced bacterial transmission from a (stainless steel) pipe to a (silicone rubber) tube and compare transmission of EPS-producing and non-EPS-producing staphylococci. Transmission of an entire biofilm from the donor to the receiver tube did not occur, indicative of cohesive failure in the biofilm rather than of adhesive failure at the donor-biofilm interface. Biofilm was gradually transmitted over an increasing length of receiver tube, occurring mostly to the first 50 cm of the receiver tube. Under high-shearing velocity, transmission of non-EPS-producing bacteria to the second half decreased non-linearly, likely due to rapid thinning of the lowly lubricious biofilm. Oppositely, transmission of EPS-producing strains to the second tube half was not affected by higher shearing velocity due to the high lubricity and stress relaxation of the EPS-rich biofilms, ensuring continued contact with the receiver. The non-linear decrease of ongoing bacterial transmission under high-shearing velocity is new and of relevance in for instance, high-speed food slicers and food packaging.

Nanoengineered Superhydrophobic Surfaces of Aluminum with Extremely Low Bacterial Adhesivity.

Bacterial adhesion and biofilm formation on surfaces are troublesome in many industrial processes. Here, nanoporous and nanopillared aluminum surfaces were engineered by anodizing and postetching processes and made hydrophilic (using the inherent oxide layer) or hydrophobic (applying a Teflon coating) with the aim of discouraging bacterial adhesion. Adhesion of Staphylococcus aureus ATCC 12600 (Gram-positive, spherically shaped) and Escherichia coli K-12 (Gram-negative, rod-shaped) was evaluated to the nanoengineered surfaces under both static and flow conditions (fluid shear rate of 37 s-1). Compared to a nonstructured electropolished flat surface, the nanostructured surfaces significantly reduced the number of adhering colony forming units (CFUs) for both species, as measured using agar plating. For the hydrophilic surfaces, this was attributed to a decreased contact area, reducing bacterial adhesion forces on nanoporous and nanopillared surfaces to 4 and 2 nN, respectively, from 8 nN on flat surfaces. Reductions in the numbers of adhering CFUs were more marked on hydrophobic surfaces under flow, amounting to more than 99.9% and 99.4% for S. aureus and E. coli on nanopillared surfaces, respectively. Scanning electron microscopy revealed a few bacteria found on the hydrophobic nanopillared surfaces adhered predominantly to defective or damaged areas, whereas the intact area preserving the original nanopillared morphology was virtually devoid of adhering bacteria. The greater decrease in bacterial adhesion to hydrophobic nanopillared surfaces than to hydrophilic or nanoporous ones is attributed to effective air entrapment in the three-dimensional pillar morphology, rendering them superhydrophobic and slippery, in addition to providing a minimized contact area for bacteria to adhere to.

Lactobacilli require physical contact to reduce staphylococcal TSST-1 secretion and vaginal epithelial inflammatory response.

ITALIC! Staphylococcus aureusbiofilms can be found on vaginal epithelia, secreting toxins and causing inflammation. The co-vaginal species ITALIC! Lactobacilluscan alter staphylococcal-induced epithelial secretion of inflammatory cytokines and quench staphylococcal toxic shock syndrome toxin-1 secretion. It is hypothesized that these effects of lactobacilli require direct physical contact between lactobacilli, staphylococci and the epithelium. Indeed, lactobacilli only reduced ITALIC! S. aureus-induced inflammatory cytokine expression when allowed physical contact with vaginal epithelial cells. Furthermore, a reduction in toxic shock syndrome toxin-1 secretion only occurred when a probiotic ITALIC! Lactobacillusstrain was allowed contact, but not when being physically separated from ITALIC! S. aureus Bacterial-probe atomic force microscopy demonstrated that lactobacilli and staphylococci strongly adhere to epithelial cells, while lactobacilli adhere stronger to staphylococci than staphylococci to each other, giving lactobacilli opportunity to penetrate and reside in staphylococcal biofilms, as visualized using confocal laser scanning microscopy with fluorescence ITALIC! in situhybridization probes. These results identify that physical contact and biochemical signaling by lactobacilli are intrinsically linked mechanisms that reduce virulence of ITALIC! S. aureusbiofilm.

Vaginal epithelial cells regulate membrane adhesiveness to co-ordinate bacterial adhesion.

Vaginal epithelium is colonized by different bacterial strains and species. The bacterial composition of vaginal biofilms controls the balance between health and disease. Little is known about the relative contribution of the epithelial and bacterial cell surfaces to bacterial adhesion and whether and how adhesion is regulated over cell membrane regions. Here, we show that bacterial adhesion forces with cell membrane regions not located above the nucleus are stronger than with regions above the nucleus both for vaginal pathogens and different commensal and probiotic lactobacillus strains involved in health. Importantly, adhesion force ratios over membrane regions away from and above the nucleus coincided with the ratios between numbers of adhering bacteria over both regions. Bacterial adhesion forces were dramatically decreased by depleting the epithelial cell membrane of cholesterol or sub-membrane cortical actin. Thus, epithelial cells can regulate membrane regions to which bacterial adhesion is discouraged, possibly to protect the nucleus.

Osteoblast integration of dental implant materials after challenge by sub-gingival pathogens: a co-culture study in vitro.

Sub-gingival anaerobic pathogens can colonize an implant surface to compromise osseointegration of dental implants once the soft tissue seal around the neck of an implant is broken. In vitro evaluations of implant materials are usually done in monoculture studies involving either tissue integration or bacterial colonization. Co-culture models, in which tissue cells and bacteria battle simultaneously for estate on an implant surface, have been demonstrated to provide a better in vitro mimic of the clinical situation. Here we aim to compare the surface coverage by U2OS osteoblasts cells prior to and after challenge by two anaerobic sub-gingival pathogens in a co-culture model on differently modified titanium (Ti), titanium-zirconium (TiZr) alloys and zirconia surfaces. Monoculture studies with either U2OS osteoblasts or bacteria were also carried out and indicated significant differences in biofilm formation between the implant materials, but interactions with U2OS osteoblasts were favourable on all materials. Adhering U2OS osteoblasts cells, however, were significantly more displaced from differently modified Ti surfaces by challenging sub-gingival pathogens than from TiZr alloys and zirconia variants. Combined with previous work employing a co-culture model consisting of human gingival fibroblasts and supra-gingival oral bacteria, results point to a different material selection to stimulate the formation of a soft tissue seal as compared to preservation of osseointegration under the unsterile conditions of the oral cavity.

Impact of 3D Hierarchical Nanostructures on the Antibacterial Efficacy of a Bacteria-Triggered Self-Defensive Antibiotic Coating.

Titanium is often applied in implant surgery, but frequently implicated in infections associated with bacterial adhesion and growth on the implant surface. Here, we show that hierarchical nanostructuring of titanium and the subsequent coating of resulting topographical features with a self-defensive, antibacterial layer-by-layer (LbL) film enables a synergistic action of hierarchical nanotopography and localized, bacteria-triggered antibiotic release to dramatically enhance the antibacterial efficiency of surfaces. Although sole nanostructuring of titanium substrates did not significantly affect adhesion and growth of Staphylococcus aureus, the coating of 3D-nanopillared substrates with an ultrathin tannic acid/gentamicin (TA/G) LbL film resulted in a 10-fold reduction of the number of surface-attached bacteria. This effect is attributed to the enlarged surface area of the nanostructured coating available for localized bacteria-triggered release of antibiotics, as well as to the lower bacterial adhesion forces resulting in subsided activation of bacterial antibiotic-defense mechanisms when bacteria land on nanopillar tips. The result shows that a combination of 3D nanostructuring with a bacteria-triggered antibiotic-releasing coating presents a unique way to dramatically enhance antibacterial efficacy of biomaterial implants.

Mechanism of cell integration on biomaterial implant surfaces in the presence of bacterial contamination.

Bacterial contamination during biomaterial implantation is often unavoidable, yielding a combat between cells and bacteria. Here we aim to determine the modulatory function of bacterial components on stem-cell, fibroblast, and osteoblast adhesion to a titanium alloy, including the role of toll-like-receptors (TLRs). Presence of heat-sacrificed Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, or Pseudomonas aeruginosa induced dose and cell-type dependent responses. Stem-cells were most sensitive to bacterial presence, demonstrating decreased adhesion number yet increased adhesion effort with a relatively large focal adhesion contact area. Blocking TLRs had no effect on stem-cell adhesion in presence of S. aureus, but blocking both TLR2 and TLR4 induced an increased adhesion effort in presence of E. coli. Neither lipopolysaccharide, lipoteichoic acid, nor bacterial DNA provoked the same cell response as did whole bacteria. Herewith we suggest a new mechanism as to how biomaterials are integrated by cells despite the unavoidable presence of bacterial contamination. Stimulation of host cell integration of implant surfaces may open a new window to design new biomaterials with enhanced healing, thereby reducing the risk of biomaterial-associated infection of both "hardware-based" implants as well as of tissue-engineered constructs, known to suffer from similarly high infection risks as currently prevailing in "hardware-based" implants.