a Department of Orthopaedic Surgery, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen; b Department of Biomedical Engineering, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen; c Department of Cell Biology and Electron Microscopy, University of Groningen, Oostersingel 69-II, 9713 EZ Groningen, The Netherlands
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Abstract |
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Introduction |
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The introduction of gentamicin-loaded bone cements for the primary fixation of total hip arthroplasties in orthopaedic surgery was followed within 10 years by reports of the first gentamicin-resistant staphylococci.5 Whereas the application of gentamicin-loaded bone cements for the primary fixation remains a matter of controversy, its use is more accepted in revision surgery after infection of a primary implant. Before a successful re-implantation, the infection should be fully eradicated through surgical debridement and removal of the implant. Subsequently, high local levels of antibiotics are achieved in the compromised tissue through the implantation of chains of gentamicin-loaded polymethylmethacrylate (PMMA) beads, before a second prosthesis is actually implanted. Gentamicin-loaded PMMA beads constitute an effective drug delivery system for local antibiotic therapy in bone and soft-tissue infections, and gentamicin concentrations at the site of the infection can exceed the MIC for the infecting organisms.6 Usually, these beads are removed after 1014 days, and the absence of cultivable microorganisms in excised soft-tissue samples by routine culture is taken as a sign that the infection is cured and implantation of a new prosthesis can take place. Although the use of gentamicin-loaded PMMA beads is accepted in clinical practice, the beads themselves can act as a biomaterial surface that microorganisms preferentially adhere to and grow on, and potentially develop antibiotic resistance. Several in vitro studies have demonstrated bacterial adhesion and growth on antibiotic-loaded cement, despite the release of antibiotics.7 Recently, Van de Belt et al.8 described a patient complaining of pain associated with his right hip prosthesis; a left hip prosthesis had previously been cemented with gentamicin-loaded bone cement. As the right hip turned out to be infected, gentamicin-loaded PMMA beads were inserted to cure the infection before implantation of a new prosthesis. Beads were retrieved and examined for the presence of infectious microorganisms in an extensive laboratory procedure, revealing the presence of gentamicin-resistant staphylococci growing on the beads themselves.
In this study, gentamicin-loaded PMMA beads were retrieved from patients requiring orthopaedic revision surgery because of infection of a primary implant with the aim of comparing routine culture of excised tissue, as applied during two-stage revision surgery of infected orthopaedic prostheses, with a more extensive microbiological analysis of the beads themselves. Gentamicin susceptibility of all strains isolated was also determined.
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Materials and methods |
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Gentamicin-loaded PMMA beads were retrieved from 20 patients of the Department of Orthopaedic Surgery, University Hospital Groningen, with suspected prosthesis-related infection, based on loosening as observed by X-ray and infection parameters, including erythrocyte sedimentation rate, C-reactive protein and white blood cell count. After thorough debridement of all necrotic and contaminated tissue, four chains each carrying 30 gentamicin-loaded PMMA beads (Septopal, E Merck, Darmstadt, Germany) were implanted in the joint cavity and patients were given systemic cefuroxime therapy. Each bead contained 4.5 mg of gentamicin and had a diameter of 7 mm. After 2 weeks, the chains were removed operatively and tissue cultures were taken. Based upon a per-operative visual inspection of the wound area, the orthopaedic surgeon then decided to perform a second insertion of gentamicin-loaded PMMA beads in some patients (patients 3, 4, 5 and 12). After removal, gentamicin-loaded beads were sent to the research laboratory for an extensive laboratory culture procedure and tissue samples were sent for routine culture.
Culture techniques
Routine hospital culture.
A sterile swab was passed per-operatively across the suspected infected area and/or tissue samples were taken. Within 2 h, tissue samples and swabs were transported to the hospital laboratory. Samples were incubated for 2 days on blood agar and chocolate agar and for 3 days in liquid medium (thioglycolate) under aerobic conditions and anaerobically for 5 days on brucella agar (DSMZ, Braunschweig, Germany). Subsequently, Gram's stain was performed and strains were identified by growth on selective agar and by API identification systems (bioMérieux SA, Marcy l'Étoile, France).
Extensive laboratory procedure.
Chains with gentamicin-loaded PMMA beads were put in sterile organ bags and transported in cooled (4°C) reduced transport fluid [RTF; NaCl 0.9 g/L (NH4)2SO4 0.9 g/L, KH2PO4 0.45 g/L, Mg2SO4 0.19g/L, K2HPO4 0.45 g/L, Na2EDTA 0.37 g/L, l-cysteine HCl 0.2 g/L, pH 6.8] to the research laboratory within 224 h for immediate handling. Gentamicin-loaded PMMA chains were cut into pieces of three beads with sterile scissors. The beads were streaked over blood agar, enriched with 0.5% haemin and 0.1% menadion and immersed in tryptone soya broth (TSB; Oxoid, Basingstoke, UK). The tubes were vortexed for 10 s at 66 W. Blood agar and liquid cultures were incubated for 7 days at 37°C, both aerobically and anaerobically. Gram's stain and a catalase test (hydrogen peroxide solution 3%) identified staphylococci. The distinction between Staphylococcus aureus and coagulase-negative staphylococci (CNS) was made with a DNase test (DNase agar; Oxoid). Other bacteria were identified with Biolog (Automated MicroLog System, Stag N.V., St Katelijne Waver, Belgium).
MICs for bacteria isolated from gentamicin-loaded PMMA beads
The MICs of gentamicin for the isolated bacteria and for possible sub-populations were determined by Etest (AB Biodisk, Dälvagen, Sweden).
Scanning electron microscopy
For scanning electron microscopy (SEM), beads from three patients were fixed with 2% glutaraldehyde in 100 mM cacodylate buffer. After fixation, the beads were exposed overnight to an osmium tetroxide vapour. Samples were dehydrated in air and sputter-coated with gold/palladium (~3 nm). Examination was done at 5.0 kV in a JEOL field emission scanning electron microscope type 6301F.
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Results |
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Table I presents the results of the routine hospital and extended research laboratory culture of retrieved beads. Routine culture showed bacterial growth in excised tissue after the first insertion of beads in three patients only (numbers 2, 14 and 15). Extensive culture demonstrated bacterial growth on gentamicin-loaded beads retrieved from 18 of the 20 patients after the first insertion, while after the second insertion of gentamicin-loaded beads growth was still observed for patients 3 and 4. Routine culture indicated that these patients were free of infection. The strains isolated in the extensive laboratory procedure were identified as S. aureus (n = 2), CNS (n = 15), Enterococcus (n = 1), Pseudomonas (n = 6), Stenotrophomonas maltophilia (n = 1), Comamonas acidovorans (n = 1), Streptococcus sanguinis (n = 1) and one unidentified anaerobe.
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Table II lists the MICs for the bacteria isolated from the gentamicin-loaded PMMA beads. Bacteria for which the MIC was
4 mg/L were considered gentamicin resistant and accordingly 19 of the 28 isolates were considered resistant, including S. aureus (n = 1), CNS (n = 9), Pseudomonas (n = 5), S. maltophilia (n = 1), C. acidovorans (n = 1), S. sanguinis (n = 1) and one anaerobe. Interestingly, CNS and Pseudomonas aeruginosa isolated after the second insertion of gentamicin-loaded beads from patients 3 and 4 had higher MICs than the same bacteria isolated after the first insertion, indicating the development of an increasing antibiotic resistance during treatment. Bacteria isolated from gentamicin-loaded beads in three patients (patients 5, 6 and 15) showed gentamicin-resistant sub-populations with MICs of 12, 32, 96 and >256 mg/L.
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The Figure shows electron micrographs of gentamicin-loaded beads after 2 weeks' implantation. The bacteria (presumably staphylococci, as the beads were retrieved from patient 10) on the bead surface are embedded in a layer of slime (Figure, a
), whereas the bacteria on the beads shown in (b) are more clearly discernible. Cocci can be seen colonizing the bead surface in (c) with slimy threads connecting individual bacteria.
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Discussion |
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The follow-up of the 12 patients who were considered free of infection by the routine culture method (while analysis of the bead surface still indicated infection) is of interest (see Tables I and II). Patient 1 returned to the hospital 15 months after revision of an infected right hip prosthesis complaining of pain in a left hip prosthesis. X-rays demonstrated loosening. The infecting organism on the left hip was found to be a gentamicin-resistant CNS. Patient 6 returned to the hospital with a severe wound infection 1 month after revision surgery and is now scheduled for excision due to septic loosening. Patient 15 returned to the hospital 2 months after revision surgery and was diagnosed with a CNS-infected prosthesis. This patient died of sepsis 2 months after surgery.
Resistant sub-populations with extremely high MICs were found on gentamicin-loaded beads from three patients (patients 5, 6 and 15). In routine culture such sub- populations are ignored, but their presence may have consequences in the further treatment of a patient after revision surgery. In the treatment of chronic osteomyelitis the use of gentamicin-loaded beads may contribute to the occurrence of re-infection with resistant variants. Likewise, the slow release of gentamicin from the beads into the local environment is an efficient way to select for antibiotic-resistant strains.12
In conclusion, in order to reduce the infection rate in orthopaedic revision surgery, it is suggested that routine bacterial cultures in orthopaedic revision surgery should include analysis of the gentamicin-loaded PMMA beads where the biofilm mode of growth firmly anchors and protects the infecting organisms. Prolonged incubation and the use of enrichment broth could also increase the detection rates.
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Notes |
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References |
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Received 1 November 2000; returned 22 January 2001; revised 7 March 2001; accepted 12 March 2001