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.

Nanostructured Ti-13Nb-13Zr alloy for implant application-material scientific, technological, and biological aspects.

In dentistry, the most commonly used implant materials are CP-Titanium Grade 4 and Ti-6Al-4V ELI, possessing comparably high Young's modulus (>100 GPa). In the present study, the second-generation titanium alloy Ti-13Nb-13Zr is investigated with respect to the production of advanced dental implant systems. This should be achieved by the fabrication of long semi-finished bars with high strength and sufficient ductility to allow the automated production of small implants at low Young's modulus (<80 GPa) to minimize stress shielding, bone resorption, and gap formation between the bone and implant. In addition, bacterial colonization is to be reduced, and bone adhesion is to be enhanced by adjusting the microstructure. To do so, a dedicated thermo-mechanical treatment for Ti-13Nb-13Zr has been developed. This includes the adaption of equal channel angular swaging, a modern process of severe plastic deformation to continuously manufacture nanostructured materials, to Ti-13Nb-13Zr and short-time recrystallization and ageing treatments. In particular, two-pass equal channel angular swaging at a deformation temperature of 150°C and a counterpressure of 8 MPa has successfully been used to avoid shear band formation during deformation and to produce long Ti-13Nb-13Zr bars of 8 mm diameter. During recrystallization treatment at 700°C for 10 min followed by water quenching, a sub-micron-size primary α-phase in a matrix of α″-phase was developed. Subsequent ageing at 500°C for 1 h leads to martensite decomposition and, thus, to a homogeneously nanostructured microstructure of α- and ß-phase with substructures smaller than 200 nm. The resulting mechanical properties, especially the ultimate tensile strength of more than 990 MPa, fulfill the requirements of ASTM F1713 at Young's modulus of 73 GPa. Biological investigations show promising results in reducing bacterial biofilm formation and increased cell proliferation of osteoblasts compared to CP-Titanium Grade 4 and Ti-6Al-4V ELI, especially, if etched surfaces are applied.

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.

Ganglioside lipidomics of CNS myelination using direct infusion shotgun mass spectrometry.

Gangliosides are present and concentrated in axons and implicated in axon-myelin interactions, but how ganglioside composition changes during myelin formation is not known. Here, we present a direct infusion (shotgun) lipidomics method to analyze gangliosides in small amounts of tissue reproducibly and with high sensitivity. We resolve the mouse ganglioside lipidome during development and adulthood and determine the ganglioside content of mice lacking the St3gal5 and B4galnt1 genes that synthesize most ganglioside species. Our results reveal substantial changes in the ganglioside lipidome during the formation of myelinated nerve fibers. In sum, we provide insights into the CNS ganglioside lipidome with a quantitative and sensitive mass spectrometry method. Since this method is compatible with global lipidomic profiling, it will provide insights into ganglioside function in physiology and pathology.

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.

Influence of Nutrient Media Compared to Human Synovial Fluid on the Antibiotic Susceptibility and Biofilm Gene Expression of Coagulase-Negative Staphylococci In Vitro.

Bacterial antibiotic resistance and biofilm formation are mechanisms usually involved in the pathogeny of implant-related infections. Worldwide, antibiotic susceptibility tests are usually carried out using nutrient-rich media. Clinical routine laboratories and even research centers use for example EUCAST or CLSI for guidelines. In this study, we investigated the effect of different nutrient media on the antibiotic susceptibility and icaADBC gene expression of bacteria in biofilm. As media, Müller-Hinton Bouillon (MHB), Tryptic Soy Broth (TSB) and human synovial fluid (SF) diluted 1:4 in phosphate buffered saline (PBS), each also supplemented with 1% glucose, were used. The influence of different nutrient media on the antibiotic susceptibility of coagulase-negative staphylococci (CoNS) was evaluated by counting of colony-forming units (CFU) and by checking the metabolic activity of the bacteria. We used reverse transcriptase and real-time qPCR to investigate the influence of nutrient media on the biofilm gene expression. We used two-way analysis of variance (ANOVA). p < 0.05 was considered to be statistically significant. Significant differences in growth and antibiotic susceptibility were detected in all strains tested among the different media used. The nutrient media showed influence on the cell viability of all bacteria after antibiotic treatment. IcaADBC gene expression was significantly influenced by glucose and all nutrient media. The results highlight the influence of glucose on the antibiotic susceptibility, growth and gene expression of all strains tested. For all strains, a significant difference in bacterial recovery, viability and gene expression were found when compared to biofilm grown in SF.

Engineering of a target site-specific recombinase by a combined evolution- and structure-guided approach.

Site-specific recombinases (SSRs) can perform DNA rearrangements, including deletions, inversions and translocations when their naive target sequences are placed strategically into the genome of an organism. Hence, in order to employ SSRs in heterologous hosts, their target sites have to be introduced into the genome of an organism before the enzyme can be practically employed. Engineered SSRs hold great promise for biotechnology and advanced biomedical applications, as they promise to extend the usefulness of SSRs to allow efficient and specific recombination of pre-existing, natural genomic sequences. However, the generation of enzymes with desired properties remains challenging. Here, we use substrate-linked directed evolution in combination with molecular modeling to rationally engineer an efficient and specific recombinase (sTre) that readily and specifically recombines a sequence present in the HIV-1 genome. We elucidate the role of key residues implicated in the molecular recognition mechanism and we present a rationale for sTre's enhanced specificity. Combining evolutionary and rational approaches should help in accelerating the generation of enzymes with desired properties for use in biotechnology and biomedicine.