Activity of N-Chlorotaurine against Periodontal Pathogens.

Dental plaque bacteria play an important role in the pathogenicity of periodontitis and peri-implantitis. Therefore, antimicrobial agents are one means of treatment. N-chlorotaurine (NCT) as an endogenous well-tolerated topical antiseptic could be of advantage for this purpose. Accordingly, its microbicidal activity against some dental plaque bacteria was investigated at therapeutic concentrations in vitro. In quantitative killing assays, the activity of NCT against planktonic bacteria and against biofilms grown for 48 h on implantation screws was tested. Electron microscopy was used to demonstrate the formation of biofilm and its morphological changes. The killing of planktonic bacteria of all tested species, namely Streptococcus sanguinis, Streptococcus salivarius, Streptococcus oralis, Streptococcus cristatus, Rothia aeria, and Capnocytophaga ochracea, was shown within 10-20 min by 1% NCT in 0.01 M phosphate-buffered saline at 37 °C. Bacteria grown on screws for 24 h were inactivated by 1% NCT after 15-20 min as well, but the formation of biofilm on the screws was visible in electron microscopy not before 48 h. The killing of biofilms by 1% NCT was demonstrated after 30 min (streptococci) and 40 min (R. aeria). As expected, NCT has broad activity against dental plaque bacteria as well and should be further investigated on its clinical efficacy in periodontitis and peri-implantitis.

Surface Topography, Microbial Adhesion, and Immune Responses in Silicone Mammary Implant-Associated Capsular Fibrosis.

Breast cancer is the most common cancer in women globally, often necessitating mastectomy and subsequent breast reconstruction. Silicone mammary implants (SMIs) play a pivotal role in breast reconstruction, yet their interaction with the host immune system and microbiome remains poorly understood. This study investigates the impact of SMI surface topography on host antimicrobial responses, wound proteome dynamics, and microbial colonization. Biological samples were collected from ten human patients undergoing breast reconstruction with SMIs. Mass spectrometry profiles were analyzed for acute and chronic wound proteomes, revealing a nuanced interplay between topography and antimicrobial response proteins. 16S rRNA sequencing assessed microbiome dynamics, unveiling topography-specific variations in microbial composition. Surface topography alterations influenced wound proteome composition. Microbiome analysis revealed heightened diversity around rougher SMIs, emphasizing topography-dependent microbial invasion. In vitro experiments confirmed staphylococcal adhesion, growth, and biofilm formation on SMI surfaces, with increased texture correlating positively with bacterial colonization. This comprehensive investigation highlights the intricate interplay between SMI topography, wound proteome dynamics, and microbial transmission. The findings contribute to understanding host-microbe interactions on SMI surfaces, essential for optimizing clinical applications and minimizing complications in breast reconstruction.

Lyophilized allogeneic bone tissue as an antibiotic carrier.

The rising number of primary joint replacements worldwide causes an increase of revision surgery of endoprostheses due bacterial infection. Revision surgery using non-cemented implants seems beneficial for the long-term outcome and the use of antibiotic-impregnated bone grafts might control the infection and give a good support for the implant. In this study we evaluated the release of antibiotics from fresh-frozen and lyophilized allogeneic bone grafts. Lyophilized bone chips and fresh frozen bone chips were mixed with gentamicin sulphate, gentamicin palmitate, vancomycin, calcium carbonate/calcium sulphate impregnated with gentamicin sulphate, and calcium carbonate/calcium sulphate bone substitute material impregnated with vancomycin. The efficacy of each preparation was measured by drug release tests and bacterial susceptibility using B. subtilis, S. aureus and methicillin-resistant Staphylococcus aureus. The release of gentamicin from lyophilized bone was similar to the release rate from fresh frozen bone during all the experimental time. That fact might be related to the similar porosity and microstructure of the bone chips. The release of gentamicin from lyophilized and fresh frozen bone was high in the first and second day, decreasing and keeping a low rate until the end of the second week. Depending on the surgical strategy either polymethylmethacrylate or allogeneic bone are able to deliver sufficient concentrations of gentamicin to achieve bacterial inhibition within two weeks after surgery. In case of uncemented revision of joint replacements allogeneic bone is able to deliver therapeutic doses of gentamicin and peak levels immediately after implantation during a fortnight. The use of lyophilized and fresh frozen bone allografts as antibiotic carriers is recommended for prophylaxis of bone infection.

Antibiotic-loaded calcium carbonate/calcium sulfate granules as co-adjuvant for bone grafting.

In this study HERAFILL(®) granules containing gentamicin was evaluated as a bone void filling material once mixed with allograft bone grafts. The efficacy of the bone grafts mixed with HERAFILL(®) was measured by drug release tests and bacterial susceptibility using Bacillus subtilis, Staphylococcus epidermidis and Staphylococcus aureus. The effect of storage at -80 °C on the delivery and efficacy of gentamicin from bone grafts mixed with HERAFILL(®) was also investigated. Higher elution of gentamicin was detected in all stored groups (1 and 6 months) in comparison with non-stored samples. The gentamicin elution released from all groups was efficient on reducing S. aureus and S. epidermidis CFU. The susceptibility tests using S. aureus showed less resistance of the strain after 1 month of the elution storage. That resistance was not observed after 6 months of storage. The capacity of bone grafts to act as gentamicin carriers has been confirmed in this study. The different granules sizes did not interfere in the delivery rate of the antibiotics or in the activity against the bacteria. Storage at -80 °C does not interfere on the antibiotic activity.

Bactericidal activity of N-chlorotaurine against biofilm-forming bacteria grown on metal disks.

Many orthopedic surgeons consider surgical irrigation and debridement with prosthesis retention as a treatment option for postoperative infections. Usually, saline solution with no added antimicrobial agent is used for irrigation. We investigated the activity of N-chlorotaurine (NCT) against various biofilm-forming bacteria in vitro and thereby gained significant information on its usability as a soluble and well-tolerated active chlorine compound in orthopedic surgery. Biofilms of Staphylococcus aureus were grown on metal alloy disks and in polystyrene dishes for 48 h. Subsequently, they were incubated for 15 min to 7 h in buffered solutions containing therapeutically applicable concentrations of NCT (1%, 0.5%, and 0.1%; 5.5 to 55 mM) at 37°C. NCT inactivated the biofilm in a time- and dose-dependent manner. Scanning electron microscopy revealed disturbance of the biofilm architecture by rupture of the extracellular matrix. Assays with reduction of carboxanilide (XTT) showed inhibition of the metabolism of the bacteria in biofilms. Quantitative cultures confirmed killing of S. aureus, Staphylococcus epidermidis, and Pseudomonas aeruginosa biofilms on metal alloy disks by NCT. Clinical isolates were slightly more resistant than ATCC type strains, but counts of CFU were reduced at least 10-fold by 1% NCT within 15 min in all cases. NCT showed microbicidal activity against various bacterial strains in biofilms. Whether this can be transferred to the clinical situation should be the aim of future studies.

Influence of poly-N-acetylglucosamine in the extracellular matrix on N-chlorotaurine mediated killing of Staphylococcus epidermidis.

N-chlorotaurine (NCT) has recently been shown to have bactericidal activity against bacterial biofilm on metal discs (Coraca-Huber et al., 2014). In a biofilm, Staphylococcus epidermidis polymerizes poly-N-acetylglucosamine (PNAG) to form an extracellular matrix (ECM). Pseudomonas aeruginosa does not express this PNAG and has been shown to be highly susceptible to NCT. We compared the action of NCT on S. epidermidis 1457, a PNAG positive strain (SE1457) and S. epidermidis 1457- M10 an isogenic PNAG negative mutant (SE1457 M10). NCT-mediated killing was more effective and quicker on the PNAG negative strain SE1457 M10. Bacteria hidden in biofilms for prolonged periods of time were generally more susceptible than freshly formed biofilms. The differences in NCT-mediated killing might not be direct effects since NCT did not react with the monomeric N-acetylglucosamine, but might be explained by denser growth in the PNAG-containing biofilm produced by the wild type strain, which results in delayed penetration of NCT. The higher susceptibility of older biofilms to NCTmediated killing could be explained by more pronounced 3D architecture and subsequent larger surface area for interactions with NCT.

Effect of storage temperature and antibiotic impregnation on the quantity of bone morphogenetic protein seven in human bone grafts.

PURPOSE: The aim of this study was to quantify the amount of bone morphogenic protein 7 (BMP-7) in bone samples in different storage and treatment conditions used in bone banks and thereby evaluate the benefit of this test as a routine measure before bone grafting. METHODS: Fresh as well as frozen bone chips, each with and without antibiotic impregnation, were screened for their BMP-7 content. Human bone chips were produced from femoral heads of two female donors who had undergone total hip replacement surgery. The amount of BMP-7 was detected using a commercially available enzyme-linked immunosorbent assay (ELISA) test. RESULTS: There were no significant differences between groups in samples obtained from the first femoral head. Bone-chip samples derived from the second femoral head showed significant differences between groups. The actual amount of these differences was small and most likely biologically irrelevant. It is important to note that there was a significant difference between groups when comparing both femoral heads, reflecting donor-to-donor variability. CONCLUSION: ELISA testing for BMP-7 as a qualitative measurement of bone grafts should be considered a routine quality-control test for bone banks.

Gentamicin palmitate as a new antibiotic formulation for mixing with bone tissue and local release.

During surgery with bone grafting, the impaction of bone tissue creates an avascular area where local circulation is disrupted. If infections arise, they may prevent systemically administered antibiotics from reaching the infected bone. In this study we evaluated gentamicin palmitate (GP) mixed with gentamicin sulfate (GS) as a coating for bone chips (BCh). The efficacy of the coated BCh was measured by gentamicin base release tests using B. subtilis, S. epidermidis and S. aureus. Gentamicin base release was evaluated in phosphate-buffered saline for up to 7 days using B. subtilis bioassay. Antimicrobial efficacy was tested with S. aureus and S. epidermidis. A significant difference on the release of gentamicin base between GS and GS + GP was observed. S. epidermidis are significantly more susceptible to GS + GP and GS than S. aureus. BCh can act as gentamicin carriers and showed efficacy against S. aureus and S. epidermidis.

Antibiotic-Polyphosphate Nanocomplexes: A Promising System for Effective Biofilm Eradication.

Purpose: The eradication of bacterial biofilms poses an enormous challenge owing to the inherently low antibiotic susceptibility of the resident microbiota. The complexation of antibiotics with polyphosphate can substantially improve antimicrobial performance. Methods: Nanoparticular complexes of the model drug colistin and polyphosphate (CP-NPs) were developed and characterized in terms of their particle size and morphology, polydispersity index (PDI), zeta potential, and cytotoxicity. Enzyme-triggered monophosphate and colistin release from the CP-NPs was evaluated in the presence of alkaline phosphatase (AP). Subsequently, antimicrobial efficacy was assessed by inhibition experiments on planktonic cultures, as well as time-kill assays on biofilms formed by the model organism Micrococcus luteus. Results: The CP-NPs exhibited a spherical morphology with particle sizes <200 nm, PDI <0.25, and negative zeta potential. They showed reduced cytotoxicity toward two human cell lines and significantly decreased hemotoxicity compared with native colistin. Release experiments with AP verified the enzymatic cleavage of polyphosphate and subsequent release of monophosphate and colistin from CP-NPs. Although CP-NPs were ineffective against planktonic M. luteus cultures, they showed major activity against bacterial biofilms, outperforming native colistin treatment. Strongly elevated AP levels in the biofilm state were identified as a potential key factor for the observed findings. Conclusion: Accordingly, polyphosphate-based nanocomplexes represent a promising tool to tackle bacterial biofilm.

Enzyme-responsive nanoparticles: enhancing the ability of endolysins to eradicate Staphylococcus aureus biofilm.

Stimuli-responsive nanomaterials show promise in eradicating Staphylococcus aureus biofilm from implants. Peptidoglycan hydrolases (PGHs) are cationic antimicrobials that can be bioengineered to improve the targeting of persisters and drug-resistant bacteria. However, these molecules can be degraded before reaching the target and/or present limited efficacy against biofilm. Therefore, there is an urgent need to improve their potency. Herein, PGH-polyphosphate nanoparticles (PGH-PP NPs) are formed by ionotropic gelation between cationic PGHs and anionic polyphosphate, with the aim of protecting PHGs and delivering them at the target site triggered by alkaline phosphatase (AP) from S. aureus biofilm. Optimized conditions for obtaining M23-PP NPs and GH15-PP NPs are presented. Size, zeta potential, and transmission electron microscopy imaging confirm the nanoscale size. The system demonstrates outstanding performance, as evidenced by a dramatic reduction in PGHs' minimum inhibitory concentration and minimum bactericidal concentration, together with protection against proteolytic effects, storage stability, and cytotoxicity towards the Caco-2 and HeLa cell lines. Time-kill experiments show the great potential of these negatively charged delivery systems in overcoming the staphylococcal biofilm barrier. Efficacy under conditions inhibiting AP proves the enzyme-triggered delivery of PGHs. The enzyme-responsive PGH-PP NPs significantly enhance the effectiveness of PGHs against bacteria residing in biofilm, offering a promising strategy for eradicating S. aureus biofilm.

The 2023 Orthopaedic Research Society's International Consensus Meeting on musculoskeletal infection: Summary from the host immunity section.

Musculoskeletal infections (MSKI), which are a major problem in orthopedics, occur when the pathogen eludes or overwhelms the host immune system. While effective vaccines and immunotherapies to prevent and treat MSKI should be possible, fundamental knowledge gaps in our understanding of protective, nonprotective, and pathogenic host immunity are prohibitive. We also lack critical knowledge of how host immunity is affected by the microbiome, implants, prior infection, nutrition, antibiotics, and concomitant therapies, autoimmunity, and other comorbidities. To define our current knowledge of these critical topics, a Host Immunity Section of the 2023 Orthopaedic Research Society MSKI International Consensus Meeting (ICM) proposed 78 questions. Systematic reviews were performed on 15 of these questions, upon which recommendations with level of evidence were voted on by the 72 ICM delegates, and another 12 questions were voted on with a recommendation of "Unknown" without systematic reviews. Two questions were transferred to another ICM Section, and the other 45 were tabled for future consideration due to limitations of available human resources. Here we report the results of the voting with internet access to the questions, recommendations, and rationale from the systematic reviews. Eighteen questions received a consensus vote of ≥90%, while nine recommendations failed to achieve this threshold. Commentary on why consensus was not achieved on these questions and potential ways forward are provided to stimulate specific funding mechanisms and research on these critical MSKI host defense questions.

The 2023 Orthopedic Research Society's international consensus meeting on musculoskeletal infection: Summary from the in vitro section.

Antimicrobial strategies for musculoskeletal infections are typically first developed with in vitro models. The In Vitro Section of the 2023 Orthopedic Research Society Musculoskeletal Infection international consensus meeting (ICM) probed our state of knowledge of in vitro systems with respect to bacteria and biofilm phenotype, standards, in vitro activity, and the ability to predict in vivo efficacy. A subset of ICM delegates performed systematic reviews on 15 questions and made recommendations and assessment of the level of evidence that were then voted on by 72 ICM delegates. Here, we report recommendations and rationale from the reviews and the results of the internet vote. Only two questions received a ≥90% consensus vote, emphasizing the disparate approaches and lack of established consensus for in vitro modeling and interpretation of results. Comments on knowledge gaps and the need for further research on these critical MSKI questions are included.

Sterilization Procedures for Titanium Alloy Surfaces Leads to Higher Expression of Biofilm-Related Staphylococcus aureus Genes.

Background: Around 1-2% of all implantation surgeries lead to implant-related infections, incurring costs of $40,000-$160,000 per total hip PJI. The 5-year mortality rate of prosthetic joint infections is up to 21%. To prevent infections during surgery, sterile surgery rooms and procedures have been developed and certified standards have been established. To guarantee the sterility, implants can be acquired already sterile from manufacturers. Some titanium implants can be delivered unsterilized with a manual for sterilization procedure in compliance with ISO 17664. The aim of this study is to evaluate if the most used sterilization methods (steam sterilization in an autoclave and UV light sterilization) of titanium alloys, can influence the biofilm forming capacity of Staphylococcus aureus. In this study, we examined the influence of sterilization methods on the gene expression of biofilm-associated genes and regulators. Methods: We compared gene expression of icaADBC, SarA, SigB, and SodA on titanium CP4 and Ti6Al4V alloys sterilized by UV-light and pressurized saturated steam sterilization. We performed RT-qPCR after RNA extraction of Staphylococcus aureus ATCC 29213. In addition, bacterial cell growth on the sterilized titanium surfaces was examined by colony forming unit counting on agar plates after 24 h of incubation. Results: Colony forming units of S. aureus on titanium CP4 samples showed a higher tendency in colony counts when sterilized with UV light than with pressurized saturated steam (autoclaved). Similarly, colony forming unit counts on Ti6Al4V samples showed tendencies of higher numbers on UV light sterilized samples than on autoclaved samples. Gene expression of icaADBC, SarA and SodA between steamed samples and UV light sterilized samples showed no difference on titanium CP4 samples, whereas SigB showed higher gene expression on titanium CP4 samples when sterilized with UV light than in an autoclave. On autoclaved Ti6Al4V samples, all examined genes showed 4 to 9 times higher fold changes in gene expression than on UV light sterilized samples. Conclusions: This study indicates that steam sterilization of Ti6Al4V can increase biofilm formation of S. aureus on its surface. The significantly increased gene expression of biofilm responsible genes may indicate a modification of titanium surfaces on alloy components. This may promote biofilm formation that can lead to implant-infections in vivo.

Antibiofilm Activity of Omega-3 Fatty Acids and Its Influence on the Expression of Biofilm Formation Genes on Staphylococcus aureus.

Background: Currently, 1-2% of all prosthetic joint surgeries are followed by an infection. These infections cause approximately 4% of deaths in the first year after surgery, while the 5-year mortality rate is up to 21%. Prosthetic joint infections are mainly caused by Staphylococcus aureus or Staphylococcus epidermis strains. Both species share the capability of biofilm formation and methicillin resistance. The formation of biofilm helps bacterial cells to withstand critical environmental conditions. Due to their tolerance against antibacterial substances, biofilms are a significant problem in modern medicine. Alternatives for the use of methicillin as a therapeutic are not yet widespread. The use of omega-3 fatty acids, such as docosahexaenoic acid, may help against prosthetic joint infections and lower mortality rates. The aim of this study is to evaluate if docosahexaenoic acid offers a safe anti-biofilm activity against Staphylococcus aureus and MRSA without enhancing icaADBC-dependent biofilm formation or additional stress responses, therefore enhancing antibiotic tolerance and resistance. Methods: In this study, we examined the gene expression of biofilm-associated genes and regulators. We performed RT-qPCR after RNA extraction of Staphylococcus aureus ATCC 29213 and one clinical MRSA strain. We compared gene expression of icaADBC, SarA, SigB, and agrAC under the influence of 1.25 mg /L and 0.625 mg/L of docosahexaenoic acid to their controls. Results: We found a higher expression of regulatory genes such as SarA, SigB, agrA, and agrC at 1.25 mg/L of docosahexaenoic acid in ATCC 29213 and a lower increase in gene expression levels in clinical MRSA isolates. icaADBC was not affected in both strains at both concentration levels by docosahexaenoic acid. Conclusions: Docosahexaenoic acid does not enhance icaADBC-dependent biofilm formation while still reducing bacterial CFU in biofilms. Docosahexaenoic acid can be considered an option as a therapeutic substance against biofilm formation and may be a good alternative in reducing the risk of MRSA formation.

An intraoperative assessment of bacterial contamination on surgical helmets and gloves during arthroplasty surgeries.

INTRODUCTION: Infection is a devasting complication after primary and revision arthroplasty. Therefore, identifying potential sources of infection can help to reduce infection rates. The aim of this study was to identify the impact and potential risk of contamination for glows and surgical helmets during arthroplasty procedures. METHODS: Surveillance cultures were used to detect contamination of the glow interface during the surgery and the surgical helmets immediately at the end of the surgery. The cultures were taken from 49 arthroplasty procedures from the surgeon as well as the assisting surgeon. RESULTS: In total, 196 cultures were taken. 31 (15.8%) of them showed a contamination. 12 (13.5%) of 98 cultures taken from the surgical helmets were positive, while 18 (18.3%) of 96 cultures taken from the gloves showed a contamination. DISCUSSION: The study showed that during arthroplasty procedures, surgical helmets and gloves were frequently contaminated with bacteria. In 20 of 49 (40.8%) arthroplasty surgeries, either the surgical helmet or the gloves showed a contamination. Surgeons should be aware that they might be a source for infection during arthroplasty surgeries.

3D bioprinted, vascularized neuroblastoma tumor environment in fluidic chip devices for precision medicine drug testing.

Neuroblastoma is an extracranial solid tumor which develops in early childhood and still has a poor prognosis. One strategy to increase cure rates is the identification of patient-specific drug responses in tissue models that mimic the interaction between patient cancer cells and tumor environment. We therefore developed a perfused and micro-vascularized tumor-environment model that is directly bioprinted into custom-manufactured fluidic chips. A gelatin-methacrylate/fibrin-based matrix containing multiple cell types mimics the tumor-microenvironment that promotes spontaneous micro-vessel formation by embedded endothelial cells. We demonstrate that both, adipocyte- and iPSC-derived mesenchymal stem cells can guide this process. Bioprinted channels are coated with endothelial cells post printing to form a dense vessel-tissue barrier. The tissue model thereby mimics structure and function of human soft tissue with endothelial cell-coated larger vessels for perfusion and micro-vessel networks within the hydrogel-matrix. Patient-derived neuroblastoma spheroids are added to the matrix during the printing process and grown for more than two weeks. We demonstrate that micro-vessels are attracted by and grow into tumor spheroids and that neuroblastoma cells invade the tumor-environment as soon as the spheroids disrupt. In summary, we describe the first bioprinted, micro-vascularized neuroblastoma-tumor-environment model directly printed into fluidic chips and a novel medium-throughput biofabrication platform suitable for studying tumor angiogenesis and metastasis in precision medicine approaches in future.

Lyophilized Human Bone Allograft as an Antibiotic Carrier: An In Vitro and In Vivo Study.

Background: Antibiotics delivered from implanted bone substitute materials (BSM) can potentially be used to prevent acute infections and biofilm formation, providing high concentrations of antibiotics at the surgical site without systemic toxicity. In addition, BSM should allow osteoconductivity supporting bone healing without further surgery. Promising results have been achieved using lyophilized bone allografts mixed with antibiotics. Methods: In this study specially prepared human bone allografts were evaluated as an antibiotic carrier in vitro and in vivo. The efficacy of different antibiotic-impregnated bone allografts was measured by drug release tests in vitro and in vivo and bacterial susceptibility tests using four bacterial species usually responsible for implant-associated infections. Results: The loading procedures of allograft bone substitutes with antibiotics were successful. Some of the antibiotic concentrations exceeded the MIC90 for up to 7 days in vitro and for up to 72 h in vivo. The susceptibility tests showed that S. epidermidis ATCC 12228 was the most susceptible bacterial species in comparison to the other strains tested for all antibiotic substances. Vancomycin and rifampicin showed the best results against standard and patient-isolated strains in vitro. In vivo, new bone formation was comparable in all study groups including the control group without antibiotic loading. Conclusions: Human bone allografts showed the capacity to act as customized loaded antibiotic carriers to prevent acute infections and should be considered in the management of bone infections in combination with systemic antimicrobial therapy.

Antibacterial and Anti-Biofilm Activity of Omega-3 Polyunsaturated Fatty Acids against Periprosthetic Joint Infections-Isolated Multi-Drug Resistant Strains.

Background: Implantable medical devices, such as prosthetics, catheters, and several other devices, have revolutionized medicine, but they increase the infection risk. In previous decades, commercially available antibiotics lost their activity against coagulase-negative Staphylococci (CoNS) and several other microorganisms. Docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are the two major omega-3 polyunsaturated fatty acids (ω-3 PUFAs) with antimicrobial properties. Materials and Methods: In this study, we tested the EPA and the DHA for its antibacterial and anti-biofilm activity in vitro against Staphylococcus epidermidis, Staphylococcus aureus, and different CoNS as reference strains and isolated from patients undergoing orthopedic treatment for implant infections. The tests were carried out with the strains in planktonic and biofilm form. Cytotoxicity assay was carried out with EPA and DHA using human gingival fibroblasts HGF-1. Results: The highest concentration of EPA and DHA promoted the complete killing of S. epidermidis 1457 and S. aureus ATCC 25923 in planktonic form. The fatty acids showed low activity against P. aeruginosa. EPA and DHA completely killed or significantly reduced the count of planktonic bacteria of the patient isolated strains. When incubated with media enriched with EPA and DHA, the biofilm formation was significantly reduced on S. epidermidis 1457 and not present on S. aureus ATCC 25923. The reduction or complete killing were also observed with the clinical isolates. The pre-formed biofilms showed reduction of the cell counting after treatment with EPA and DHA. Conclusion: In this study, the ω-3 PUFAs EPA and DHA showed antimicrobial and anti-biofilm activity in vitro against S. aureus, S. epidermidis, and P. aeruginosa, as well as against multi-drug resistant S. aureus and CoNS strains isolated from patients undergoing periprosthetic joint infections (PJI) treatment. Higher concentrations of the fatty acids showed killing activity on planktonic cells and inhibitory activity of biofilm formation. Although both substances showed antimicrobial activity, EPA showed better results in comparison with DHA. In addition, when applied on human gingival fibroblasts in vitro, EPA and DHA showed a possible protective effect on cells cultured in medium enriched with ethanol. Further studies are required to confirm the antimicrobial activity of EPA and DHA against multi-drug resistant strains and pan-drug resistant strains.

Activity of N-Chlorotaurine against Long-Term Biofilms of Bacteria and Yeasts.

Background: N-chlorotaurine (NCT), an antiseptic that originates from the human defense system, has broad-spectrum microbicidal activity and is well tolerated by human tissue and applicable to sensitive body regions. Bacteria in short-term biofilms, too, have been shown to be killed by NCT. It was the aim of the present study to demonstrate the activity of NCT against bacteria and yeasts in longer-lasting biofilms, including their co-culture. Materials and methods: Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella variicola biofilms were grown for 14 weeks in MBECTM inoculator with 96 well base. Some pegs were pinched off weekly and incubated in 1% NCT in PBS (PBS only for controls) at pH 7.1 and 37 °C, for 30 and 60 min. Subsequently, bacteria were resuspended by ultrasonication and subjected to quantitative cultures. Similar tests were conducted with C. albicans biofilms grown on metal (A2-steel) discs for 4 weeks. Mixed co-cultures of C. albicans plus each of the three bacterial strains on metal discs were grown for 5-7 weeks and weekly evaluated, as mentioned above. Results: Single biofilms of S. aureus, P. aeruginosa, and K. variicola grew to approximately 1 × 106 colony forming units (CFU)/mL and C. albicans to 1 × 105 CFU/mL. In combined biofilms, the CFU count was about 1 log10 lower. Viable counts of biofilms of single bacteria were reduced by 2.8 to 5.6 log10 in 1% NCT after 60 min (0.9 to 4.7 log10 after 30 min) with Gram-negative bacteria being more susceptible than S. aureus. Significant reduction of C. albicans by 2.0 to 2.9 log10 occurred after 4 h incubation. In combined biofilms, viable counts of C. albicans were reduced by 1.1 to 2.4 log10 after 4 h, while they reached the detection limit after 1 to 2 h with bacteria (2.0 to > 3.5 log10 reduction). Remarkably, older biofilms demonstrated no increase in resistance but constant susceptibility to NCT. This was valid for all tested pathogens. In electron microscopy, morphological differences between NCT-treated and non-treated biofilms could be found. Conclusions: NCT is active against long-term biofilms of up to several months irrespective of their age. Combined biofilm cultures of yeasts and bacteria show a similar susceptibility pattern to NCT as single ones. These results contribute to the explanation of the clinical efficacy of NCT, for instance, in infected chronic wounds and purulently coated crural ulcerations.

Bacterial toxins in musculoskeletal infections.

Musculoskeletal infections (MSKIs) remain a major health burden in orthopaedics. Bacterial toxins are foundational to pathogenesis in MSKI, but poorly understood by the community of providers that care for patients with MSKI, inducing an international group of microbiologists, infectious diseases specialists, orthopaedic surgeons and biofilm scientists to review the literature in this field to identify key topics and compile the current knowledge on the role of toxins in MSKI, with the goal of illuminating potential impact on biofilm formation and dispersal as well as therapeutic strategies. The group concluded that further research is needed to maximize our understanding of the effect of toxins on MSKIs, including: (i) further research to identify the roles of bacterial toxins in MSKIs, (ii) establish the understanding of the importance of environmental and host factors and in vivo expression of toxins throughout the course of an infection, (iii) establish the principles of drug-ability of antitoxins as antimicrobial agents in MSKIs, (iv) have well-defined metrics of success for antitoxins as antiinfective drugs, (v) design a cocktail of antitoxins against specific pathogens to (a) inhibit biofilm formation and (b) inhibit toxin release. The applicability of antitoxins as potential antimicrobials in the era of rising antibiotic resistance could meet the needs of day-to-day clinicians.