Osteomyelitis is a significant clinical challenge that leads to bone destruction and loss. Systemic antibiotic treatments do not reach infected sites at therapeutic levels, necessitating high dosages, which in turn can lead to toxicity and promote the emergence of antibiotic-resistant bacteria. To overcome these challenges, our objective was to synthesize a biocompatible drug-delivery system that has the potential to achieve localized antibiotic delivery to completely eradicate bacteria. We synthesized four formulations of microparticles using thiol-ene click chemistry by varying the thiol monomer-to-crosslinker (2-thiol:4-thiol) ratio and the ene monomer-to-surfactant (2-ene:Hitenol) ratio. The microparticles were loaded with doxycycline by soaking them in 20 or 50 mg/mL doxycycline (D20 or D50) over 24 h. The microparticles exhibited a payload-dependent release, with the D20 groups releasing approximately 11%-22% of their total doxycycline content, while the D50 groups released less than 10% of the total doxycycline content by 96 h. All doxycycline-loaded microparticle formulations showed antibacterial activity against Methicillin-resistant Staphylococcus aureus (MRSA) over a 7-day period, with the formulation with a 2-thiol:4-thiol ratio of 0.1:0.9 and a 2-ene:Hitenol ratio of 0.8:0.2 showing complete eradication of bacteria by Day 7. The MTT and Live/Dead assays showed that doses of 12 mg or less of this formulation were not cytotoxic to mouse pre-osteoblast cells. Taken together, we successfully synthesized a biocompatible microparticle-based drug delivery system that can be loaded with doxycycline and that exhibits drug release under physiological conditions.

Purpose:Streptococcus pneumoniae (S. pneumoniae) causes disease in multiple body systems, including at the ocular surface and in the lungs. S. pneumoniae IgA1 protease cleaves and inactivates human IgA1, which is present in high concentrations at mucosal surfaces. Mice lack IgA1, but studies have shown that the gene encoding IgA1 protease is required for virulence in pneumonia in mice. This study aimed to determine if the gene is required for virulence in the mouse keratitis model. Methods:S. pneumoniae D39 or the isogenic IgA1 protease mutant, D39Δiga, was applied to the scratched ocular surface of A/J mice at a dose of 108 colony-forming units (CFUs). Disease severity was scored 8 and 24 hours post-infection, and the infected eyes were removed and homogenized for quantification of bacterial loads and host proteins associated with inflammation. In addition, the number of cornea-infiltrating neutrophils was quantified by flow cytometry. Results:At 24 hours post-infection, D39Δiga-infected corneas had significantly higher remaining mean bacterial loads (5.59 log10 CFU) and clinical severity scores (2.5) compared with those infected with D39 (3.01 log10 CFU and 0.812, P = 0.002 and 0.0011, respectively). At 8 hours post-infection, these corneas contained significantly greater quantities of 16 cytokines and chemokines associated with inflammation than those infected with D39 (P < 0.05). D39Δiga infected corneas also contained significantly larger quantities of neutrophils 24 hours post-infection (P = 0.0013). Conclusions:Absence of S. pneumoniae IgA1 protease enhances bacterial survival at the mouse ocular surface and contributes to corneal inflammation.

Proteases serve important functions for eukaryotes and bacteria, such as processing other proteins to activate them or degrading other proteins to create smaller peptides or eliminate malformed proteins. Proteases in pathogenic bacteria can also act as virulence factors by virtue of degradation of host proteins, activating other proteins that then enhance virulence, or eliciting bystander inflammatory damage through stimulation of the host immune response, to name a few functions. Zymography of a bacterial protein preparation is a powerful tool for visualizing and identifying the protease profile of a given bacterial strain. A sodium dodecyl sulfate (SDS)-polyacrylamide gel is impregnated with a protease substrate such as gelatin. Following electrophoresis, removal of SDS, incubation in buffer favoring protease activity, and staining, the substrate within the gel will retain the stain while clear bands will emerge where the protease activity degrades the gelatin. This method can be modified with different substrates, buffers, and other variables as detailed in the notes.

Porosity and roughened surfaces of implant materials have been shown to lead to improved cellular attachment and enhanced osseointegration. These topography changes in the surface also aid in the mechanical interlocking of the material to the bone. Polyetheretherketone (PEEK) has emerged as a popular alternative to titanium-based implants due to its lack of stress-shielding effect, radiolucency, and high chemical resistance. However, PEEK is bioinert, thus requiring surface modifications to elicit appropriate cellular responses that lead to successful osteointegration of the material in vivo. Sulfonation is a process used to modify the surface of PEEK, which can be controlled by varying parameters such as soak time and soak temperature, thereby fabricating a porous surface on the material. This work aimed to ensure the repeatability of a previously optimized sulfonated and hydrothermally treated PEEK surface and subsequently observe the mechanical properties, bacterial attachment, and cellular response of pre-osteoblast MC3T3-E1 cells on the surface. This study found that while all PEEK surfaces had similar cell and Staphylococcus aureus attachment, the sulfonated and hydrothermally treated PEEK (peak mean load of 605 N, p ≤ 0.0001) and the sulfonated only PEEK (peak mean load of 495 N, p = 0.0240) had a higher level of performance in expulsion testing than smooth PEEK due to its mechanical interlocking ability. Imaging and contact angle analysis confirm that a surface with repeatable porosity can be achieved.

Titanium alloys are commonly used for implants, but the naturally forming oxides are bioinert and not ideal for bacterial resistance or osseointegration. Anodization processes are a modification technique that can crystallize the oxides, alter oxide surface topography, and introduce beneficial chemistries. Crystalline titanium oxides are known to exhibit photocatalytic activity (PCA) under UVA light. Anodization was used to create mixed-phase oxides on six titanium alloys including commercially pure titanium (CPTi), Ti-6Al-4V (TAV), Ti-6Al-7Nb (TAN), two forms of Ti-15Mo (TiMo-β and TiMo-αβ), and Ti-35Nb-7Zr-5Ta (TNZT). Combined EDS and XPS analyses showed uptake of the electrolyte and substrate alloying elements into the oxides. The relative oxide PCA was measured using methylene blue degradation assays. CPTi and TAN oxides exhibited increased PCA compared to other alloys. Combined XRD and EBSD oxide phase analyses revealed an unfavorable arrangement of anatase and rutile phases near the outermost surfaces, which may have reduced PCA for other oxides. The relative Staphylococcus aureus attachment to each oxide was also assessed. The CPTi and TiMo-αβ oxides showed significantly reduced S. aureus attachment after 1 h of UVA compared to un-anodized CPTi. Cell culture results verified that the UVA irradiation did not negatively influence the MC3T3-E1 attachment or proliferation on the mixed-phase oxides.

Titanium has a long history of clinical use, but the naturally forming oxide is not ideal for bacterial resistance. Anodization processes can modify the crystallinity, surface topography, and surface chemistry of titanium oxides. Anatase, rutile, and mixed phase oxides are known to exhibit photocatalytic activity (PCA)-driven bacterial resistance under UVA irradiation. Silver additions are reported to enhance PCA and reduce bacterial attachment. This study investigated the effects of silver-doping additions to three established anodization processes. Silver doping showed no significant influence on oxide crystallinity, surface topography, or surface wettability. Oxides from a sulfuric acid anodization process exhibited significantly enhanced PCA after silver doping, but silver-doped oxides produced from phosphoric-acid-containing electrolytes did not. Staphylococcus aureus attachment was also assessed under dark and UVA-irradiated conditions on each oxide. Each oxide exhibited a photocatalytic antimicrobial effect as indicated by significantly decreased bacterial attachment under UVA irradiation compared to dark conditions. However, only the phosphorus-doped mixed anatase and rutile phase oxide exhibited an additional significant reduction in bacteria attachment under UVA irradiation as a result of silver doping. The antimicrobial success of this oxide was attributed to the combination of the mixed phase oxide and higher silver-doping uptake levels.

Bacteria-related infections remain a leading cause of dental implant failures. Despite the successful history of titanium implants, naturally forming oxides lack antibacterial properties. Crystalline oxides, modified through anodization processes, have shown photocatalytic-induced antibacterial properties when exposed to sufficient energy sources such as UVA light. Chemically doping these oxides with some metallic and non-metallic elements has been shown to enhance their photocatalytic activity (PCA). The present study’s objectives were to assess the relative UVA and violet-light-irradiated PCA levels, bacterial attachment levels, and pre-osteoblast early cell viability levels of phosphorus-doped and phosphorus-and-silver-doped anatase-phase oxides. Each oxide revealed similar surface topographies and surface porosity levels. However, the phosphorus-and-silver-doped oxides exhibited significantly higher PCA levels compared to the phosphorus-doped oxide counterpart after irradiation with 365 nm UVA (p < 0.0001) or 410 nm violet (p = 0.007 and 0.03) light. The phosphorus-doped oxides and phosphorus-and-silver-doped oxides revealed similar Staphylococcus aureus attachment levels after 60 min of UVA irradiation. The phosphorus-and-silver-doped oxides exhibited significantly increased 7-day cell viability compared to their phosphorus-doped oxide counterparts. Thus, it was concluded that the silver doping additions to the oxides show much promise for biomaterials applications and warrant further exploration.

Titanium anodization has been shown to produce crystalline oxides exhibiting photocatalytic reactions that form reactive oxygen species (ROS) when exposed to UV light. The ROS subsequently attack bacteria cells, and thus reduce bacteria attachment on titanium implant surfaces. Polyaniline (PANI) is a conductive polymer that has shown antibacterial properties when electropolymerized onto titanium. Our research group hypothesized the addition of PANI to crystalline titanium oxide surfaces would increase the available free electrons and thus increase photocatalytic activity (PCA). This research led to the development of a novel single-step anodization approach for PANI doping crystalline titanium oxide layers. The objective of the present study was to determine the proper aniline electrolyte concentration needed to maximize the PCA and reduce bacterial attachment on the formed oxides. Aniline concentrations up to 1 M were added into a 1 M sulfuric acid electrolyte. The formed oxides exhibited increased PANI surface coverage but decreased anatase and rutile crystalline titanium oxide phase formation with increasing aniline electrolyte concentrations. Despite exhibiting the lowest levels of anatase and rutile formation, the 0.75 M and 1 M aniline oxides with the greatest PANI surface coverage also exhibited the highest PCA levels. 1 M aniline oxides showed significantly higher PCA under UVA irradiation compared to oxides formed from aniline concentrations up to 0.5 M (p < 0.001). 0.75 M aniline oxides exhibited significant reductions in Staphylococcus aureus attachment with or without UVA irradiation compared to control oxides without PANI. MTT and live/dead assays confirmed cytocompatibility and nearly 100% cell viability for the PANI doped oxides.

Bacterial keratitis is a vision-threatening infection of the cornea that is typically treated with antibiotics. However, antibiotics sometimes fail to eradicate the infection and do not prevent or repair the damage caused directly by the bacteria or the host immune response to the infection. Our group previously demonstrated that treatment of Pseudomonas aeruginosa keratitis in rabbits with innovative cold atmospheric plasma (iCAP) resulted in reduced edema, ulcer formation, and bacterial load. In this study, we investigated the efficacy of iCAP treatment in methicillin-resistant Staphylococcus aureus (MRSA). New Zealand white rabbits were infected intrastromally with MRSA then treated with iCAP, moxifloxacin, vancomycin, or combination of iCAP with each antibiotic to assess the safety and efficacy of iCAP treatment compared to untreated controls and antibiotics. iCAP treatment significantly reduced bacterial loads and inflammation, improved anterior chamber clarity, and prevented corneal ulceration compared to untreated controls and antibiotic treatment. Safety assessments of grimace test scores and tear production showed that iCAP was not significantly different from either antibiotic treatment in terms of distress or tear production. Combination iCAP/antibiotic treatment did not appear to provide significant added benefit over iCAP alone. Our findings suggest that the addition of iCAP may be a viable tool in reducing damage to the cornea and anterior chamber of the eye following S. aureus keratitis.

Crystalline titanium oxides have shown photocatalytic activity (PCA) and the formation of antibacterial reactive oxygen species (ROS) when stimulated with UV light. Polyaniline (PANI) is a conductive polymer that has shown antibacterial effects. Previously, titanium oxides have been PANI-doped using a multi-step approach. In the present study, we compared PANI-doped specimens produced with a two-step method (ACV), to PANI-doped specimens produced by a novel single-step direct anodization (AAn) method, and a control group of anodized un-doped specimens. The surface morphology, oxide crystallinity, surface elemental composition, surface roughness, surface wettability, oxide adhesion, corrosion resistance, PCA, and ROS generation of each oxide group were evaluated. All groups exhibited mixed anatase and rutile phase oxides. The AAn group revealed less anatase and rutile, but more PANI-surface coverage. The AAn group exhibited significantly increased PCA after 60 min of direct UVA illumination compared to the ACV group, despite containing lower amounts of anatase and rutile. The ACV and AAn groups showed significant increases in ROS production after 4 h UVA illumination while the control group showed similar ROS production. These findings suggested that PANI doping using the novel direct anodization technique significantly improved PCA even for oxides containing less crystallinity. The S. aureus attachment response to each oxide group was also compared under UVA pre-illumination, UVA direct illumination, and no illumination (dark) lighting conditions. Although no significant differences were shown in the bacterial response, both PANI-doped groups exhibited less average bacterial attachment compared to the control group. The response of MC3T3-E1 pre-osteoblast cells to each oxide group was evaluated using MTT and live/dead assays, and no evidence of cytotoxicity was found. Since many, if not most, titanium implant devices are routinely anodized as a part of the manufacturing processes, these study findings are applicable to a wide variety of implant applications.

Three unique 5,6-seco-hexahydrodibenzopyrans (seco-HHDBP) machaeridiols A–C, reported previously from Machaerium Pers., have displayed potent activities against methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecium, and E. faecalis (VRE). In order to enrich the pipeline of natural product-derived antimicrobial compounds, a series of novel machaeridiol-based analogs (1–17) were prepared by coupling stemofuran, pinosylvin, and resveratrol legends with monoterpene units R-(−)-α-phellandrene, (−)-p-mentha-2,8-diene-1-ol, and geraniol, and their inhibitory activities were profiled against MRSA ATCC 1708, VRE ATCC 700221, and cancer signaling pathways. Compounds 5 and 11 showed strong in vitro activities with MIC values of 2.5 μg/mL and 1.25 μg/mL against MRSA, respectively, and 2.50 μg/mL against VRE, while geranyl analog 14 was found to be moderately active (MIC 5 μg/mL). The reduction of the double bonds of the monoterpene unit of compound 5 resulted in 17, which had the same antibacterial potency (MIC 1.25 μg/mL and 2.50 μg/mL) as its parent, 5. Furthermore, a combination study between seco-HHDBP 17 and HHDBP machaeriol C displayed a synergistic effect with a fractional inhibitory concentrations (FIC) value of 0.5 against MRSA, showing a four-fold decrease in the MIC values of both 17 and machaeriol C, while no such effect was observed between vancomycin and 17. Compounds 11 and 17 were further tested in vivo against nosocomial MRSA at a single intranasal dose of 30 mg/kg in a murine model, and both compounds were not efficacious under these conditions. Finally, compounds 1–17 were profiled against a panel of luciferase genes that assessed the activity of complex cancer-related signaling pathways (i.e., transcription factors) using T98G glioblastoma multiforme cells. Among the compounds tested, the geranyl-substituted analog 14 exhibited strong inhibition against several signaling pathways, notably Smad, Myc, and Notch, with IC50 values of 2.17 μM, 1.86 μM, and 2.15 μM, respectively. In contrast, the anti-MRSA actives 5 and 17 were found to be inactive (IC50 > 20 μM) across the panel of these cancer-signaling pathways.

The role of the pneumococcal polysaccharide capsule is largely unclear for Streptococcus pneumoniae keratitis, an ocular inflammatory disease that develops as a result of bacterial infection of the cornea. In this study, capsule-deficient strains were compared to isogenic parent strains in their ability to adhere to human corneal epithelial cells. One isogenic pair was further used in topical ocular infection of mice to assess the contribution of the capsule to keratitis. The results showed that non-encapsulated pneumococci were significantly more adherent to cells, persisted in significantly higher numbers on mouse corneas in vivo, and caused significant increases in murine ocular IL9, IL10, IL12-p70, MIG, and MIP-1-gamma compared to encapsulated S. pneumoniae. These findings indicate that the bacterial capsule impedes virulence and the absence of capsule impacts inflammation following corneal infection.

The purpose of this study was to evaluate the effect of polyaniline doping of mixed anatase and rutile phase titanium oxide layers on the resulting photocatalytic activity (PCA) and antibacterial behavior. Commercially pure titanium grade 4 (CPTi) samples were divided into three groups: (1) anodized in 0.5 M sulfuric acid (control); (2) electropolymerized using cyclic voltammetry in a mixture of 0.5 M sulfuric acid and 0.25 M aniline after anodization in 0.5 M sulfuric acid (ACV); and (3) anodized in 0.5 M sulfuric acid and 0.25 M aniline (AAn). SEM, XRD, and water contact angles were used to evaluate the surface morphology, oxide layer crystallinity, and hydrophilicity of the oxide layers (n= 3). The PCA was determined through methylene blue (MB) degradation assay using 365 nm UVA illumination (n= 7). The antibacterial behavior was evaluated by assessing the reduction in percentage of S.aureus attachment under three UVA lighting conditions: (1) a pre-illumination group in which samples were irradiated under UVA light for one hour prior to bacteria exposure; (2) direct illumination group in which samples were irradiated for one hour after bacteria exposure; and (3) a no illumination group in which the samples were kept in the dark for one hour after bacteria exposure (n= 5). SEM confirmed the presence of polyaniline on AAN and ACV group surfaces. XRD confirmed the presence of anatase and rutile peaks for all groups with lower peak intensities for the AAn group. AAn, with an average contact angle of 29.7°, showed significantly higher hydrophilicity (p= 0.041) compared to the ACV and control groups with average contact angles of 46.4° and 47.9°, respectively. AAn showed significantly higher MB degradation at 60 minutes when compared to ACV (p= 0.034). Bacterial testing showed no significance differences between the three groups. However, an increased trend of bacterial attachment reduction was shown for the AAn and ACV groups, averaging 90.7% and 87.7%, compared to the control group, averaging 72.7%, under direct UVA illumination. Doping of polyaniline into the titanium oxide layer by direct anodization in sulfuric acid and aniline improves the hydrophilicity and photocatalytic activity of the titanium surfaces. Although polyaniline doped titanium oxide showed promising trends, no significant differences were shown in the antibacterial behaviors of the surfaces that were tested in this study. Additional studies using different concentration of aniline in the anodizing electrolyte are needed to attempt to improve photocatalytic activity and reduce bacterial attachment.Download : Download high-res image (187KB)Download : Download full-size image

The ongoing COVID-19 pandemic has increased the use of single-use medical fabrics such as surgical masks, respirators, and other personal protective equipment (PPE), which have faced worldwide supply chain shortages. Reusable PPE is desirable in light of such shortages; however, the use of reusable PPE is largely restricted by the difficulty of rapid sterilization. In this work, we demonstrate successful bacterial and viral inactivation through remote and rapid radio frequency (RF) heating of conductive textiles. The RF heating behavior of conductive polymer-coated fabrics was measured for several different fabrics and coating compositions. Next, to determine the robustness and repeatability of this heating response, we investigated the textile's RF heating response after multiple detergent washes. Finally, we show a rapid reduction of bacteria and virus by RF heating our conductive fabric. 99.9% of methicillin-resistant Staphylococcus aureus (MRSA) was removed from our conductive fabrics after only 10 min of RF heating; human cytomegalovirus (HCMV) was completely sterilized after 5 min of RF heating. These results demonstrate that RF heating conductive polymer-coated fabrics offer new opportunities for applications of conductive textiles in the medical and/or electronic fields.

The viridans streptococci are a group of bacteria that are commensals of the oral cavity and pharynx. These species tend to cause severe cases of bacterial endophthalmitis with poor prognoses but remain largely uncharacterized in this context. Here, we report the whole-genome sequences of 21 strains of viridans streptococci isolated from endophthalmitis in humans.

Bacterial proteases and peptidases are integral to cell physiology and stability, and their necessity in Streptococcus pneumoniae is no exception. Protein cleavage and processing mechanisms within the bacterial cell serve to ensure that the cell lives and functions in its commensal habitat and can respond to new environments presenting stressful conditions. For S. pneumoniae, the human nasopharynx is its natural habitat. In the context of virulence, movement of S. pneumoniae to the lungs, blood, or other sites can instigate responses by the bacteria that result in their proteases serving dual roles of self-protein processors and virulence factors of host protein targets.

ABSTRACT Purpose S. epidermidis is an ocular pathogen and a leading cause of keratitis. It produces hemolysins and at least 3 proteases. The purpose of the present study is to compare the secretion of hemolysins and proteases between 28 ocular isolates and one non-ocular strain and to determine their relationship to ocular virulence in selected strains using a rabbit model of infection. Materials and Methods Culture supernatants were compared for protease production and hemolysis. Selected strains were injected into rabbit corneas and their virulence and pathology recorded. The major protease activity in a virulent strain was identified and the gene was cloned and expressed as a recombinant protein. The corneal toxicity of this protease was determined. Antibodies to the native protease were generated and tested for neutralizing activity in vivo and in vitro. The corneal pathology of the S. epidermidis protease was compared to the pathology of S. aureus V8 protease. Results Strains that exhibited the least protease activity in vitro caused significantly less ocular pathology in vivo (p ≤ 0.003). Strains that were hemolytic and secreted a major protease had numerically higher SLE scores. This protease was identified as the serine protease Esp. The recombinant Esp protease caused extensive pathology when injected into the corneal stroma (7.62 ± 0.33). Antibody generated against native Esp did not neutralize the activity of the protease in vivo or in vitro. The antibody reacted with Esp proteases secreted by other S. epidermidis strains. S. epidermidis Esp protease and its homologue in S. aureus caused similar ocular pathology when injected in the rabbit corneal stroma. Conclusion Hemolysins and proteases seem to be important in corneal pathology caused by S. epidermidis infections. The Esp protease mediates significant corneal damage. S. epidermidis Esp and S. aureus V8 protease caused similar and extensive edema in rabbit corneas.