An Enzybiotic Regimen for the Treatment of Methicillin-Resistant Staphylococcus aureus Orthopaedic Device-Related Infection

Orthopaedic device-related infection (ODRI) presents a significant challenge to the field of orthopaedic and trauma surgery. Despite extensive treatment involving surgical debridement and prolonged antibiotic therapy, outcomes remain poor. This is largely due to the unique abilities of Staphylococcus aureus, the most common causative agent of ODRI, to establish and protect itself within the host by forming biofilms on implanted devices and staphylococcal abscess communities (SACs). There is a need for novel antimicrobials that can readily target such features. Enzybiotics are a class of antimicrobial enzymes derived from bacteria and bacteriophages, which function by enzymatically degrading bacterial polymers essential to bacterial survival or biofilm formation. Here, we apply an enzybiotic-based combination regimen to a set of in vitro models as well as in a murine ODRI model to evaluate their usefulness in eradicating established S. aureus infection, compared to classical antibiotics. We show that two chimeric endolysins previously selected for their functional efficacy in human serum in combination with a polysaccharide depolymerase reduce bacterial CFU numbers 10,000-fold in a peg model and in an implant model of biofilm. The enzyme combination also completely eradicates S. aureus in a SAC in vitro model where classical antibiotics are ineffective. In an in vivo ODRI model in mice, the antibiofilm effects of this enzyme regimen are further enhanced when combined with a classical gentamicin/vancomycin treatment. In a mouse model of methicillin-resistant S. aureus (MRSA) ODRI following a fracture repair, a combined local enzybiotic/antibiotic treatment regimen showed a significant CFU reduction in the device and the surrounding soft tissue, as well as significant prevention of weight loss. These outcomes were superior to treatment with antibiotics alone. Overall, this study demonstrates that the addition of enzybiotics, which are distinguished by their extremely rapid killing efficacy and antibiofilm activities, can enhance the treatment of severe MRSA ODRI.

Clinical Metagenomic Sequencing for Species Identification and Antimicrobial Resistance Prediction in Orthopaedic Device Infection

Background Diagnosis of orthopaedic device-related infection is challenging, and causative pathogens may be difficult to culture. Metagenomic sequencing can diagnose infections without culture, but attempts to detect antimicrobial resistance (AMR) determinants using metagenomic data have been less successful. Human DNA depletion may maximise the amount of microbial DNA sequence data available for analysis. Methods Human DNA depletion by saponin was tested in 115 sonication fluid samples generated following revision arthroplasty surgery, comprising 67 where pathogens were detected by culture and 48 culture-negative samples. Metagenomic sequencing was performed on the Oxford Nanopore Technologies GridION platform. Filtering thresholds for detection of true species versus contamination or taxonomic misclassification were determined. Mobile and chromosomal genetic AMR determinants were identified in Staphylococcus aureus-positive samples. Results Of 114 samples generating sequence data, species-level sensitivity of metagenomic sequencing was 49/65 (75%; 95%CI 63-85%) and specificity 103/114 (90%; 95%CI 83-95%) compared with culture. Saponin treatment reduced the proportion of human bases sequenced in comparison to 5um filtration from a median (IQR) 98.1% (87.0%-99.9%) to 11.9% (0.4%-67.0%), improving reference genome coverage at 10-fold depth from 18.7% (0.30%-85.7%) to 84.3% (12.9%-93.8%). Metagenomic sequencing predicted 13/15 (87%) resistant and 74/74 (100%) susceptible phenotypes where sufficient data were available for analysis. Conclusions Metagenomic nanopore sequencing coupled with human DNA depletion has the potential to detect AMR in addition to species detection in orthopaedic device-related infection. Further work is required to develop pathogen-agnostic human DNA depletion methods, improving AMR determinant detection and allowing its application to other infection types.

The non-steroidal anti-inflammatory drug carprofen negatively impacts new bone formation and antibiotic efficacy in a rat model of orthopaedic-device-related infection

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for pain management during recovery from orthopaedic surgery. NSAID use is associated with increased risk of bone healing complications but it is currently unknown whether NSAIDs increase the risk of developing an orthopaedic-device-related infection (ODRI) and/or affects its response to antibiotic therapy. The present study aimed to determine if administration of the NSAID carprofen [a preferential cyclooxygenase-2 (COX-2) inhibitor] negatively affected Staphylococcus epidermidis (S. epidermidis) bone infection, or its subsequent treatment with antibiotics, in a rodent ODRI model. Sterile or S. epidermidis-contaminated screws (~ 1.5 x 106 CFU) were implanted into the proximal tibia of skeletally mature female Wistar rats, in the absence or presence of daily carprofen administration. A subset of infected animals received antibiotics (rifampicin plus cefazolin) from day 7 to 21, to determine if carprofen affected antibiotic efficacy. Bone changes were monitored using in vivo µCT scanning and histological analysis. The risk of developing an infection with carprofen administration was assessed in separate animals at day 9 using a screw contaminated with 102 CFU S. epidermidis. Quantitative bacteriological analysis assessed bacterial load at euthanasia. In the 28-day antibiotic treatment study, carprofen reduced osteolysis but markedly diminished reparative bone formation, although total bacterial load was not affected at euthanasia. Antibiotic efficacy was negatively affected by carprofen (carprofen: 8/8 infected; control: 2/9 infected). Finally, carprofen increased bacterial load and diminished bone formation following reduced S. epidermidis inoculum (102 CFU) at day 9. This study suggests that NSAIDs with COX-2 selectivity reduce antibiotic efficacy and diminish reparative responses to S. epidermidis ODRI.

Orthopaedic device innovation in South Africa: A study of patenting activity

We assessed knowledge development and exchange among actors who patent orthopaedic devices in South Africa over the period 2000–2015. A social network analysis was performed on bibliometric data using co-inventorship on patents as an indicator of collaboration between different organisations, with a focus on the spatial and sectoral contexts. Network metrics and innovation system indices are used to describe knowledge development and exchange. The results show that university, healthcare and industry organisations have primarily been responsible for increased patenting over time. The key actors were a set of industry actors – a national actor and its US partner – who have patented many devices jointly. National universities were found to make a small contribution, and science councils were found to be absent, despite the efforts in the changing innovation landscape to encourage publicly financed research organisations to protect their intellectual property. The collaboration networks were found to be sparse and disjointed, with many actors – largely from the private healthcare sector – patenting in isolation.

Temporal Changes in Patient-Matched Staphylococcus epidermidis Isolates from Infections: towards Defining a ‘True’ Persistent Infection

Staphylococcus epidermidis is found naturally on the skin but is a common cause of persistent orthopaedic device-related infections (ODRIs). This study used a pan-genome and gene-by-gene approach to analyse the clonality of whole genome sequences (WGS) of 115 S. epidermidis isolates from 55 patients with persistent ODRIs. Analysis of the 522 gene core genome revealed that the isolates clustered into three clades, and MLST analysis showed that 83% of the isolates belonged to clonal complex 2 (CC2). Analysis also found 13 isolate pairs had different MLST types and less than 70% similarity within the genes; hence, these were defined as re-infection by a different S. epidermidis strain. Comparison of allelic diversity in the remaining 102 isolates (49 patients) revealed that 6 patients had microevolved infections (>7 allele differences), and only 37 patients (77 isolates) had a ‘true’ persistent infection. Analysis of the core genomes of isolate pairs from 37 patients found 110/841 genes had variations; mainly in metabolism associated genes. The accessory genome consisted of 2936 genes; with an average size of 1515 genes. To conclude, this study demonstrates the advantage of using WGS for identifying the accuracy of a persistent infection diagnosis. Hence, persistent infections can be defined as ‘true’ persistent infections if the core genome of paired isolates has ≤7 allele differences; microevolved persistent infection if the paired isolates have >7 allele differences but same MLST type; and polyclonal if they are the same species but a different MLST type.

Getting your devices ready for MDR compliance – a clinical approach and orthopaedic device manufacturers’ perspective

On 26 May 2017, the Medical Devices Regulation (MDR 2017/745) was published, with the aim of replacing the current Medical Devices Directive (MDD 93/43/EC). Following a transition period of 3 years, this regulation will be applied in full from 26 May 2020. This article describes and discusses the impact of MDR on the clinical evaluation and gives advice to manufacturers on the preparation, planning and implementation of processes to support compliance with the new regulation. (HTA & Market Access)