Aims. Biofilm infections are among the most challenging complications in orthopaedics, as bacteria within the biofilms are protected from the host immune system and many antibiotics. Halicin exhibits broad-spectrum activity against many planktonic bacteria, and previous studies have demonstrated that halicin is also effective against Staphylococcus aureus biofilms grown on polystyrene or polypropylene substrates. However, the effectiveness of many antibiotics can be substantially altered depending on which orthopaedically relevant substrates the biofilms grow. This study, therefore, evaluated the activity of halicin against less mature and more mature S. aureus biofilms grown on titanium alloy, cobalt-chrome, ultra-high molecular weight polyethylene (UHMWPE), devitalized muscle, or devitalized bone. Methods. S. aureus-Xen36 biofilms were grown on the various substrates for 24 hours or seven days.
Aims. This study investigated vancomycin-microbubbles (Vm-MBs) and meropenem (Mp)-MBs with ultrasound-targeted microbubble destruction (UTMD) to disrupt biofilms and improve bactericidal efficiency, providing a new and promising strategy for the treatment of device-related infections (DRIs). Methods. A film hydration method was used to prepare Vm-MBs and Mp-MBs and examine their characterization.
Aim. “Implant associated Staphylococcus aureus or S. epidermidis infections are often difficult to treat due to the formation of biofilms on prosthetic material.
Aim. Rifampicin plays an important role in the treatment of staphylococcal prosthetic joint infection, as rifampicin-containing combinations have shown a high efficacy against S. aureus biofilm infections. However, the emergence of rifampin-resistant strains is a feared complication and the use of rifampicin in those cases seems unwarranted. Therefore, we evaluated the activity of bacteriophage Sb1 in combination with different antibiotics against the biofilm of four rifampicin-resistant MRSA strains as alternative therapeutic approach. Method. Four rifampicin-resistant MRSA strains were used in this study. The MIC for all tested antibiotics was determined by Etest.
Aim. Ciprofloxacin is recommended as anti-biofilm therapy for gram-negative periprosthetic joint infection. With ciprofloxacin monotherapy, resistance in gram-negative bacteria was observed. Therefore, we evaluated in vitro synergistic activity of fosfomycin, ciprofloxacin and gentamicin combinations against biofilms formed by E. coli and P. aeruginosa strains. Method. E. coli ATCC 25922, P. aeruginosa ATCC 27853 and 15 clinical isolates were used for this study. MIC values were determined by Etest.
Biofilm formation (BF) in wounds and on biomaterials is a severe complication in trauma and orthopaedic surgery. Maggot therapy is successfully applied in wounds, that are suspected for BF. This study investigated BF by Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella oxytoca, Enterococcus faecalis and Enterobacter cloacae on polyethylene, titanium and stainless steel and tested the effect on BF by maggot excretions/secretions (ES). Comb-forming models of the biomaterials were made to fit into a 96-well microtiter plate. In the wells, a suspension of 2.5 x 105 bacteria/ml and nutrient medium was pipetted. Combs were placed in the wells and incubated for 3, 5, 7, and 9 days at 37°C. The formed biofilms were stained in crystal-violet and eluted in ethanol. The optical density (OD 595 nm) was measured to quantify BF. Then, maggots excretions/secretions (ES) were collected according to a standardized method, added in different concentrations to (non-stained) mature biofilms (7 days), incubated another 24 hours and at last stained and measured. The results showed biofilm reduction by ES on all biomaterials.
The aim of the present study was to assess the antibiofilm activity of daptomycin- and vancomycin-loaded poly(methyl methacrylate) (PMMA) and PMMA-Eudragit RL100 (EUD) microparticles against mature biofilms of polysaccharide intercellular adhesin-positive S. epidermidis. The effect of plain, daptomycin- and vancomycin-loaded PMMA and PMMA-EUD microparticles on S. epidermidis biofilms was assessed by isothermal microcalorimetry (IMC) and fluorescence in situ hybridization (FISH).
Aim
Method
Using a methicillin-sensitive Abstract
OBJECTIVES
METHODS
Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, with cumulative treatment costs for all prosthetic joint infections estimated to be ∼ $1 billion per annum (UK and North America). Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Low intensity pulsed ultrasound is already licensed for clinical use in fracture management and thus could be translated quickly into a clinical treatment Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged with gentamicin +/− low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 180 minutes and 20 minutes, respectively. The primary outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. Assessment of cellular metabolism was conducted using a liquid-chromatography-mass spectrometry, as well as a triphenyltetrazolium chloride assay coupled with spectrophotometry. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was reduced to 64mg/L. Metabolic activity of biofilm-associated S. aureus was increased by 25% following ultrasound therapy (p < 0 .0001), with identification of key biosynthetic pathways activated by non-lethal dispersal. Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin, bringing the MBEC of gentamicin to within clinically achievable concentrations. The mechanism of action was due to partial disruption of the extracellular matrix which led to an increase of nutrient availability and oxygen tension within the biofilm. This metabolic stimulus was responsible for the reversal of gentamicin tolerance in the biofilm-associated S. aureus.
Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explores, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, bacterial biofilms were challenged by gentamicin +/− low-intensity ultrasound (1.5MHz, 30W/cm2, pulse duration 200µs/1KHz) for 20 minutes. The outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was 64mg/L. Low intensity pulsed ultrasound was associated with a four-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations
Staphylococcus aureus is responsible for 60–70% infections of surgical implants and prostheses in Orthopaedic surgery, costing the NHS £120–200 million per annum. Its ability to develop resistance or tolerance to a diverse range of antimicrobial compounds, threatens to halt routine elective implant surgery. One strategy to overcome this problem is to look beyond traditional antimicrobial drug therapies and investigate other treatment modalities. Biophysical modalities, such as ultrasound, are poorly explored, but preliminary work has shown potential benefit, especially when combined with existing antibiotics. Using a methicillin-sensitive S. aureus reference strain and the dissolvable bead assay, biofilms were challenged by a low-intensity ultrasound (1.5MHz, 30mW/cm2, pulse duration 200µs/1KHz) for 20 minutes and gentamicin. The outcome measures were colony-forming units/mL (CFU/mL) and the minimum biofilm eradication concentration (MBEC) of gentamicin. The mean number of S. aureus within control biofilms was 1.04 × 109 CFU/mL. There was no clinically or statistically significant (p=0.531) reduction in viable S. aureus following ultrasound therapy alone. The MBEC of gentamicin for this S. aureus strain was 256 mg/L. The MBEC of gentamicin with the addition of ultrasound was 64mg/L. Further studies confirmed that the mechanism of action was due to incomplete disruption of the extracellular matrix with subsequent metabolic stimulation of the dormant biofilm-associated bacteria due to increased nutrient availability and oxygen tension. Low intensity pulsed ultrasound was associated with a 4-fold reduction in the effective biofilm eradication concentration of gentamicin; bringing the MBEC of gentamicin to within clinically achievable concentrations.
Prosthetic joint infection (PJI) remains the most severe complication of arthroplasty. Failure of intensive, long-term antibiotic treatment for PJI often requires removal of the implant. Antibiotic failure is thought to be caused by biofilm and persister formation. Novel anti-biofilm and anti-persister strategies are urgently needed. Here, we investigated the effects of several antimicrobial peptides on the bacteria within antibiotic-treated biofilms in an On polystyrene, a mature (7 day-old) methicillin-resistant Aims
Methods
Irrigation is a major step during debridement surgery in the context of Prosthetic Joint Infections (PJI), but its effects on biofilms are poorly described. The present study aims at evaluating the effect of PW alone or followed by antibiotics on MSSA and MRSA biofilms grown on Ti6Al4V coupons in-vitro. Strains: 1 reference (MSSA: ATCC25923; MRSA: ATCC33591) and 2 clinical MSSA and MRSA isolated from PJI. Biofilm culture: Coupons were incubated for 24h at 37°C with bacteria (starting inoculum ∼6.6Log10CFU/mL in TGN [TSB + 1% glucose + 2% NaCl]), under shaking at 50rpm. Treatment: Half of the coupons were irrigated with 50mL physiological serum from 5cm using a Stryker Interpulse; the coupons were then either analysed (ControlT0 and PWT0) or reincubated for 24h in TGN or TGN containing flucloxacillin (MSSA) or vancomycin (MRSA) at MIC or 20mg/L. Analysis: Coupons were rinsed twice with PBS. Biomass was measured by crystal violet (CV) assay. CFUs were counted after recovering bacteria from coupons using sonication and TSA plating.Aim
Method
Antimicrobial resistance (AMR) is projected to result in 10 million deaths every year globally by 2050. Without urgent action, routine orthopaedic operations could become high risk and musculoskeletal infections incurable in a “post-antibiotic era.” However, current methods of studying AMR processes including bacterial biofilm formation are 2D in nature, and therefore unable to recapitulate the 3D processes within Within this study, 3D printing was applied for the first time alongside a custom-developed bioink to bioprint 3D bacterial biofilm constructs from clinically relevant species including In conclusion, mature bacterial biofilm constructs were reproducibly 3D bioprinted for the first time using clinically relevant bacteria. This methodology allows the study of antimicrobial biofilm penetration in 3D, and potentially aids future antimicrobial research, replicating joint infection more closely than current 2D culture models. Furthermore, by deploying Raman spectroscopy in a novel fashion, it was possible to diagnose 3D bioprinted biofilm infections within a joint replacement model.
Peri-prosthetic joint infection (PJI) is one the most devastating complications of joint arthroplasty. Although PJI is an infrequent complication (the reported incidence is 1%-2% in the United States), it is the most common indication for revision total knee arthroplasty in the Medicare population and the third most frequent indication for revision total hip arthroplasty. Moreover, the prevalence of PJI appears to be on the rise, with a projected number exceeding 60,000 to 70,000 cases in the United States by 2020. It is estimated that more than 25% of revision procedures annually are attributed to PJI and this number is expected to increase in the upcoming years. The increase in the prevalence of obesity, diabetes, and other comorbidities among the patient population and the emergence of resistant infecting organisms are some of the reasons for the expected rise in the number of infections that medical community will witness. The challenges that PJI present to the orthopaedic community are on many fronts. Prevention of PJI has proven to be a difficult task indeed. Effective strategies for prevention of PJI are being refined. The Center for Disease Control will be publishing its updated Surgical Site Prevention Guidelines in the next few months that consists of specific recommendations for prevention of PJI. In recent years, strides are made in introducing novel molecular techniques for diagnosis of PJI, which may stand to change our practices. The current surgical technique for management of PJI, besides the immense cost, fall short of delivering high success to the patients. The major problem in eradication of infection relates to formation of biofilm on the implant surface and internalization of the organisms by affected cells. Biofilm is a sophisticated structure comprising of organisms embedded in multiple layers of glycoccalyx that allows the organisms to evade host immunity and is impenetrable to antibiotics. These organisms are capable of communicating through molecular mechanisms such as quorum sensing that affords them advantage for survival in the host environment. In recent years strategies to prevent colonization of the implant surface, an essential first step in formation of biofilm, or biofilm disruption techniques have been introduced. A recent International Consensus meeting on PJI that assembled more than 350 experts identified some of the best practices in this field and identified areas in need of future research. Moving into the future, the field of orthopaedics in general and PJI in particular stand to benefit from the discoveries in the field of molecular diagnostics, metabolomics and epigenetics.
Determine the time concentration profile required to achieve vancomycin-mediated eradication of Mature Aim
Method
Cement is still in common usage in primary and revision arthroplasty surgery. Infection rates in cemented arthroplasties ranges from 1–4% and poses a huge problem for the revision arthroplasty surgeon. Infection in septic implants is biofilm based and almost completely resistant to conventional anti-microbial therapy. Recent papers have questioned the efficacy of using gentamicin-loaded cement in arthroplasty as staphylococcus aureus biofilms will develop on same. The focus of this study was to investigate the efficacy of antibiotic loaded cement in preventing initial bacterial adhesion and subsequent development of a bacterial biofilm in vitro. Three cements Simplex unloaded, Simplex with erythromycin and Simplex with tobramycin were mixed in a conventional manner, ie vacuum hand mixing in sterile conditions and then injected into pre-moulded PTFE coated cylinder moulds yielding 8 cylinders in each group. The cement cylinders were then removed and exposed to a known pathogenic strain of staphylococcus aureus ATCC—29213-NCTC 12973 in solution 3x106 Colony forming units CFH/ml) for 15 minutes. The cylinders were then removed and cultured for 24 hours at 37°C in RPMI with Glutamine. Cylinders were then removed and subjected to rinsing in PBS to remove any non-adherent bacteria. Cylinders were then sonicated at 50 Hz in Ringer’s solution and adherent biofilms were serially log diluted and plated on Columbia blood agar. Colonies were counted manually. Control cylinders of unloaded cement showed 120,000 CFU/cm2 of adherent bacteria whereas loaded cement erythromycin and tobramycin showed 500 and 80 CFU/cm2 respectively (p<
.0005 Student t-test). This study shows that loaded cement does not prevent biofilm adhesion in its initial reversible stages whereas unloaded cement does not. This is important since most infected implants are infected at time of primary operation and cements anti-bacterial role beyond the first 48 hours remains questionable, when inflammatory encapsulation of the implant begins. We would therefore question the usage of unloaded cement in primary arthroplasty surgery.
Post-operative surgical site infection following total joint arthroplasty occurs at rates between ~ 0.2–5 %, depending on the joint and the surgeon volume, as well as various patient risk factors. Given that an estimated over 700,000 knee and hip arthroplasties are performed in the US each year this translates to thousands of patients that are affected by this serious, costly and traumatic complication. In addition, it is now recognized that clinical culturing underestimates the infection rate and that a number of aseptic loosenings might actually have an infectious etiology. We have used a combination of non-culture based molecular methods to detect bacteria associated with hardware, antimicrobial impregnated cement, reactive tissue and pus collected during revision surgery in a total elbow arthroplasty (TEA) case and a total ankle revision (TAR) case. Confocal microscopy showed live cocci in biofilm cell clusters, and fluorescent in situ hybridization (FISH) demonstrated S. aureus biofilms. Reverse transcriptase (RT)-PCR, and multiplex PCR coupled with electrospray-ionization mass spectrometry (Ibis T5000) to identify S. aureus, S. epidermidis and genes for methicillin resistance. Together our complimentary techniques comprise compelling evidence that viable biofilm bacteria played an important role in the refractory infections in these cases.