Aim

Orthopedic implants play a tremendous role in fixing bone damages due to aging as well as fractures. However, these implants tend to get colonized by bacteria on the surface, leading to infections and subsequently prevention of healing and osteointegration. Recently, Roupie et al. showed that a nisin layer-by-layer based coating applied on biomaterials has both osteogenic and antibacterial properties. The Galleria mellonella larva is a well-known insect infection model that has been used to test the virulence of bacterial and fungal strains as well as for the high throughput screening of antimicrobial compounds against infections. Recently, we have developed an insect infection model with G. mellonella larvae to study implant-associated biofilm infections using Kirschner (K)-wires as implant material. Here, we would like to test the antibacterial capacity of nisin layer-by-layer based coatings on K-wires against Staphylococcus aureus in the G. mellonella larva implant infection model.

Aim

In trauma surgery, the development of biomaterial-associated infections (BAI) is one of the most common complications affecting trauma patients, requiring prolonged hospitalization and the intensive use of antibiotics. Following the attachment of bacteria on the surface of the biomaterial, the biofilm-forming bacteria could initiate a chronic implant-related infection. Despite the use of conventional local and systemic antibiotic therapies, persistent biofilms involve various resistance mechanisms that contribute to therapeutic failures. The development of in vivo chronic BAI models to optimize antibiofilm treatments is a major challenge. Indeed, the biofilm pathogenicity and the host response need to be finely regulated, and compatible with the animal lifestyle. Previously, a Galleria mellonella larvae model for the formation of an early-stage biofilm on the surface of a Kirschner (K)-wire was established. In the present study, two models of mature biofilm using clinical Staphylococcus aureus strains were assessed: one related to contaminated K-wires (in vitro biofilm maturation) and the second to hematogenous infections (in vivo biofilm maturation). Rifampicin was used as a standard drug for antibiofilm treatment.

Aims

Bacteriophages infect, replicate inside bacteria, and are released from the host through lysis. Here, we evaluate the effects of repetitive doses of the Staphylococcus aureus phage 191219 and gentamicin against haematogenous and early-stage biofilm implant-related infections in Galleria mellonella.

Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. To prevent biofilm formation using a non-antibiotic based strategy, we aimed to develop a novel permanently fixed antimicrobial coating for titanium devices based on stable immobilized quaternary ammonium compounds (QACs).

Medical grade titanium implants were dip-coated in subsequent solutions of hyperbranched polymer, polyethyleneimine and 10 mM sodium iodide, and ethanol. The QAC-coating was characterized using water contact angle measurements, scanning electron microscopy, FTIR, AFM and XPS. The antimicrobial activity of the coating was evaluated against S. aureus strain JAR060131 and S. epidermidis strain ATCC 12228 using the JIS Z 2801:2000 surface microbicidal assay. Lastly, we assessed the in vivo antimicrobial activity in a mouse subcutaneous implant infection model with S. aureus administered locally on the QAC-coated implants prior to implantation to mimic contamination during surgery.

Detailed material characterization of the titanium samples showed the presence of a homogenous and stable coating layer at the titanium surface. Moreover, the coating successfully killed S. aureus and S. epidermidis in vitro. The QAC-coating strongly reduced S. aureus colonization of the implant surface as well as of the surrounding tissue, with no apparent macroscopic signs of toxicity or inflammation in the peri-implant tissue at 1 and 4 days after implantation.

An antimicrobial coating with stable quaternary ammonium compounds on titanium has been developed which holds promise to prevent BAI. Non-antibiotic-based antimicrobial coatings have great significance in guiding the design of novel antimicrobial coatings in the present, post-antibiotic era.

Acknowledgements: This research was financially supported by the Health∼Holland/LSH-TKI call 2021–2022, project 25687, NACQAC: ‘Novel antimicrobial coatings with stable non-antibiotic Quaternary Ammonium Compounds and photosensitizer technology'.

Aim

The origin of surgical site and biomaterial-associated infection is still elusive. Microorganisms contaminating the wound may come from the air, the surgical team, or from the skin of the patient. Prior to surgery the skin of patients is disinfected, but bacteria deeper in the skin (e.g. in sweat glands or sebaceous glands), may not be reached. This study aims to assess a potential role of this intracutaneous bacterial reservoir in biomaterial-associated infection.

Aim

The use of medical devices has grown significantly over the last decades, and has become a major part of modern medicine and our daily life. Infection of implanted medical devices (biomaterials), like titanium orthopaedic implants, can have disastrous consequences, including removal of the device. For still not well understood reasons, the presence of a foreign body strongly increases susceptibility to infection. These so-called biomaterial-associated infections (BAI) are mainly caused by Staphylococcus aureus and Staphylococcus epidermidis. Formation of biofilms on the biomaterial surface is generally considered the main reason for these persistent infections, although bacteria may also enter the surrounding tissue and become internalized within host cells. To prevent biofilm formation using a non-antibiotic based strategy, we aimed to develop a novel permanently fixed antimicrobial coating for titanium devices based on stable immobilized quaternary ammonium compounds (QACs).

Staphylococcus aureus is the main cause of osteomyelitis and forms biofilm and staphylococcal abscess communities (SACs) in humans. While S. aureus has several toxins with specificity for human targets and working with human host cells would be preferred, for SACs no in vitro models, two-dimensional (2D) or three-dimensional (3D), have been described in literature to date. Advanced 3D in vitro cell culture models enable the incorporation of human cells and resemble in vivo tissue more closely than conventional 2D cell culture. Therefore, the aim of this study was to develop an in vitro model of SACs by using a 3D system. The model should allow for studies into antibiotic tolerance and S. aureus - human host cells interactions.

With a clinical isolate (S. aureus JAR) or a lab strain (S. aureus ATCC 49230-GFP), SACs were grown in a collagen gel (1.78 mg/ml, Gibco) supplemented with 200 µl human plasma at 37 °C. Transmission and scanning electron microscopy was used to obtain a detailed overview of SACs, whereas immunofluorescent stainings were done to determine whether the pseudocapsule around SACs consist of fibrin. Antibiotic tolerance of SACs was assessed with 100× the minimal inhibitory concentration (MIC) of gentamicin (Roth). Bacterial clearance of non-establised SACs and established SACs with or without pseudocapsule was determined by exposure to differentiated PLB neutrophil-like cells (differentiation with 1.25% DMSO and 5% FBS for 5 days; dPLB) or primary neutrophils isolated with lymphoprep from fresh heparin blood. Degradation of the pseudocapsule was done with 7.5 µl/ml plasmin (Sigma). Colony forming unit (CFU) counts were performed as quantification method. Statistical analysis was performed with the ANOVA multiple comparison test or, when data was not normally distributed, with a Mann-Whitney U test.

We have developed a 3D in vitro model of SACs which after overnight growth were on average 200 micrometers in diameter, consisted of 8 log10 CFUs and were surrounded by an inner and outer fibrin pseudocapsule. The in vitro grown SACs tolerated 100× the MIC of gentamicin for 24h and did not significantly differ from control SACs (p=0.1000). dPLB neutrophil-like cells or primary neutrophils did not clear established in vitro SACs (p=0.1102 and p=0.8767, respectively). When the fibrin pseudocapsule was degraded by the enzyme plasmin, dPLB neutrophil-like cells or primary neutrophils caused for a significant decrease in total CFU compared the SACs that did had a pseudocapsule (p=0.0333 and p=0.0272, respectively).

The in vitro SACs model offers a tool for host-pathogen interaction and drug efficacy assessments and is a valuable starting point for future research.