Gentamicin release from polymethylmethacrylate bone cements and Staphylococcus aureus biofilm formation

We measured the formation of a Staphylococcus aureus biofilm in vitro on unloaded and gentamicin-loaded bone cements (CMW3 and Palacos R) and related the formation to antibiotic release rates. All experiments were done in triplicate. Microbial growth on gentamicin-loaded cements occurred despite the release of antibiotic. Biofilm formation on gentamicin loaded CMW3 bone cement was one fourth to one fifth less than on the unloaded bone cement, while biofilm formation on Palacos R bone cement was not significantly affected by antibiotic loading. More gentamicin was released from CMW3 (79 mg) than from Palacos R (70 mg), but the percentage gentamicin released after one week relative to the total amount incorporated was significantly lower for CMW3 (4.7%) than for Palacos R (8.4%). After one day, subinhibitory concentrations of antibiotics were eluted from the cements. We concluded that antibiotic-loaded bone cement does not necessarily inhibit the formation of an infectious biofilm in vitro.     

Gentamicin release from polymethylmethacrylate bone cements and Staphylococcus aureus biofilm formation

We measured the formation of a Staphylococcus aureus biofilm in vitro on unloaded and gentamicin-loaded bone cements (CMW3 and Palacos R) and related the formation to antibiotic release rates. All experiments were done in triplicate. Microbial growth on gentamicin-loaded cements occurred despite the release of antibiotic. Biofilm formation on gentamicin loaded CMW3 bone cement was one fourth to one fifth less than on the unloaded bone cement, while biofilm formation on Palacos R bone cement was not significantly affected by antibiotic loading. More gentamicin was released from CMW3 (79 mg) than from Palacos R (70 mg), but the percentage gentamicin released after one week relative to the total amount incorporated was significantly lower for CMW3 (4.7%) than for Palacos R (8.4%). After one day, subinhibitory concentrations of antibiotics were eluted from the cements. We concluded that antibiotic-loaded bone cement does not necessarily inhibit the formation of an infectious biofilm in vitro.     

The effect of mixing on gentamicin release from polymethylmethacrylate bone cements

We compared the release of gentamicin from 6 different commercially available, antibiotic-loaded PMMA bone cements used for vacuum- and hand-mixed cement using a Cemvac vacuum mixing system. We also measured the release of gentamicin after manual addition of the antibiotic to different commercial, unloaded bone cements after hand-mixing. The porosity of cements was reduced in all vacuum-mixed cements, as compared with hand-mixed cements, concurrent with a statistically significant reduction (3 of 6) or increase (1 of 6) in the total amounts of gentamicin released. The total gentamicin release was studied in 3 of the brands after manual addition and mixing of the antibiotics. We found that the release of antibiotics was lower than in samples made from industrial mixing. In conclusion, the manual addition and mixing of gentamicin in PMMA bone cements leads to a lower release of antibiotics than that in corresponding commercially available antibiotic-loaded cements, while vacuum-mixing only leads to a minor reduction in antibiotic release, as compared to hand-mixing.     

The effect of mixing on gentamicin release from polymethylmethacrylate bone cements

We compared the release of gentamicin from 6 different commercially available, antibiotic-loaded PMMA bone cements used for vacuum- and hand-mixed cement using a Cemvac vacuum mixing system. We also measured the release of gentamicin after manual addition of the antibiotic to different commercial, unloaded bone cements after hand-mixing. The porosity of cements was reduced in all vacuum-mixed cements, as compared with hand-mixed cements, concurrent with a statistically significant reduction (3 of 6) or increase (1 of 6) in the total amounts of gentamicin released. The total gentamicin release was studied in 3 of the brands after manual addition and mixing of the antibiotics. We found that the release of antibiotics was lower than in samples made from industrial mixing. In conclusion, the manual addition and mixing of gentamicin in PMMA bone cements leads to a lower release of antibiotics than that in corresponding commercially available antibiotic-loaded cements, while vacuum-mixing only leads to a minor reduction in antibiotic release, as compared to hand-mixing.     

The effect of mixing on gentamicin release from polymethylmethacrylate bone cements

We compared the release of gentamicin from 6 different commercially available, antibiotic-loaded PMMA bone cements used for vacuum- and hand-mixed cement using a Cemvac vacuum mixing system. We also measured the release of gentamicin after manual addition of the antibiotic to different commercial, unloaded bone cements after hand-mixing. The porosity of cements was reduced in all vacuum-mixed cements, as compared with hand-mixed cements, concurrent with a statistically significant reduction (3 of 6) or increase (1 of 6) in the total amounts of gentamicin released. The total gentamicin release was studied in 3 of the brands after manual addition and mixing of the antibiotics. We found that the release of antibiotics was lower than in samples made from industrial mixing. In conclusion, the manual addition and mixing of gentamicin in PMMA bone cements leads to a lower release of antibiotics than that in corresponding commercially available antibiotic-loaded cements, while vacuum-mixing only leads to a minor reduction in antibiotic release, as compared to hand-mixing.     

Gentamicin release from commercially-available gentamicin-loaded PMMA bone cements in a prosthesis-related interfacial gap model and their antibacterial efficacy

Background  

Around about 1970, a gentamicin-loaded poly (methylmethacrylate) (PMMA) bone cement brand (Refobacin Palacos R) was introduced to control infection in joint arthroplasties. In 2005, this brand was replaced by two gentamicin-loaded follow-up brands, Refobacin Bone Cement R and Palacos R + G. In addition, another gentamicin-loaded cement brand, SmartSet GHV, was introduced in Europe in 2003. In the present study, we investigated differences in gentamicin release and the antibacterial efficacy of the eluent between these four cement brands.     

Sustained pressurization of polymethylmethacrylate: a comparison of low- and moderate-viscosity bone cements

There is at present great uncertainty relating to the fixation of joint implants. The deficiencies of acrylic bone cement are well documented, but the limitations of cementless fixation are as yet imcompletely identified. The purpose of this study was to investigate the potential of sustained external pressurization to improve the mechanical characteristics of conventional acrylic bone cement. The effect of serially increasing sustained pressurization of two commerically available acrylic bone cements (Simplex-P and LVC) was evaluated in human cadaver femora. A new method for determination of the shear strength of the bone-cement interface in place of the traditional pushout tests was used. In this model, there was a significant increase in the bone-cement interfacial shear strength with increasing pressure, but no difference in the shear strength was found between the two cements. At all pressure levels, the shear strength of the cement was greater than that previously reported. Increased cement penetration into the cortical bone was demonstrated with increasing pressure and low-viscosity cement, but the extent of cement penetration did not correlate with the shear strength of the bone-cement interface.     

Staphylococcus aureus capsular material promotes osteoclast formation

Osteomyelitis, which is most frequently due to infection by Staphylococcus aureus, commonly causes bone destruction. S. aureus is known to secrete a number of surface-associated proteins that are potent stimulators of bone resorption. The precise cellular and humoral mechanisms that mediate this stimulatory effect are uncertain. In this study, we have determined whether osteoclast formation and resorption is directly promoted by surface-associated proteins. Surface-associated material (SAM) obtained from a 24-hour culture of S. aureus was added to cultures of mouse and human monocytes. Human monocyte cultures were incubated in the presence and absence of a soluble receptor activator of nuclear factor kappa B ligand (RANKL) and macrophage colony stimulating factor (M-CSF). In cultures where M-CSF, RANKL, and SAM were added together, osteoclast formation did not exceed that seen in cultures with M-CSF and RANKL. In keeping with this finding, SAM did not increase osteoclast formation and resorption when mouse monocytes were cocultured with RANKL-expressing osteoblasts. In the absence of RANKL, however, SAM was capable of inducing osteoclast formation in cultures of human monocytes. This finding was evidenced by the generation of vitronectin receptor and tartrate-resistant acid phosphatasepositive multinucleated cells that were capable of lacunar resorption. Inhibitors of RANKL-dependent (RANK:Fc, OPG) and RANKL-independent (anti-TNF-alpha, gp130, IL-8, TGF-beta) osteoclast formation did not inhibit SAM-induced osteoclast formation. SAM did not stimulate mature osteoclast resorption activity. These findings indicate that RANKL, which is present in the circulation as a soluble factor, does not play a role in osteoclast formation in the presence of S. aureus SAM and that S. aureus SAM contains a soluble factor that promotes osteoclast formation by a RANKL-independent mechanism.     

Prevention of Staphylococcus aureus biofilm on dialysis catheters and adherence to human cells

BACKGROUND: Dialysis patients, often carriers of Staphylococcus aureus in their nares, are at high risk of S. aureus infections. METHODS: We examined whether RNAIII inhibiting peptide (RIP), which interferes with quorum sensing mechanisms, reduces adherence of S. aureus to host cells and to dialysis catheter polymers in vitro. Adherence was tested by spectroscopy using safranin staining, by confocal scanning laser microscopy and by atomic force microscopy. RESULTS: RIP inhibited bacterial adherence to HaCat and HEp-2 cells and reduced adherence and biofilm formation not only on polystyrene, but also on both polyurethane- and silicone-made dialysis catheters, with a preponderant effect on silicone, to which bacteria were more adherent. CONCLUSION: RIP opens a new perspective in anti-S. aureus prophylaxis, particularly in dialysis patients.     

Comparison of antibiotic release from polymethylmethacrylate beads and sponge collagen

The rates of elution of tobramycin in vitro were compared for polymethylmethacrylate beads impregnated with the powder form and an alternative biodegradable substance, sponge collagen. The impregnated polymethylmethacrylate beads initially had a lower zone of inhibition, but the rate of release was slow in comparison with that of the impregnated sponge collagen. The sponge collagen delivered a higher dose faster and with a shorter duration than the polymethylmethacrylate beads with the same antibiotic concentration in vitro, but the beads delivered a therapeutic concentration for longer periods. Because it deteriorates rapidly, sponge collagen may be unsatisfactory as an agent of antibiotic delivery in patients who have chronic osteomyelitis; however, it may be useful for patients who have acute trauma with highly contaminated bone or soft tissue, or during hemiarthroplasty revision, to deliver a high local concentration of antibiotic for a short period of time.     

Lugol's solution and Gentian violet eradicate methicillin-resistant Staphylococcus aureus biofilm in skin wound infections

The study aimed to evaluate the antibacterial efficacy of Lugol's solution 5% and Gentian violet 1% against methicillin-resistant Staphylococcus aureus (MRSA) biofilm in vivo. The bactericidal efficacy for treatment of MRSA-biofilm skin wound infection was tested in a murine model. Luciferase-tagged S. aureus Xen31, a MRSA-strain derived from S. aureus ATCC-3359130, was used for infection. Wounds were made in the skin of mice and infected with MRSA. The mice were treated with Lugol's solution and Gentian violet. Application of the antimicrobial agents started 24 hours post infection and was repeated daily for five-days. The antimicrobial effect on the biofilm bacteria was evaluated by measuring bioluminescence from MRSA daily for seven-days. Lugol's solution and Gentian violet showed a significant reduction in luminescent signals from the first assessment day to all subsequent days (P < .001). Lugol's solution and Gentian violet effectively eradicated MRSA in biofilm in vivo and could be alternatives or in addition to topical antibiotics when MRSA-biofilm wound infection is suspected.     

Vancomycin-Impregnated polymethylmethacrylate beads for methicillin-resistant Staphylococcus aureus (MRSA) infection: Report of two cases

Two patients with methicillin-resistant Staphylococcus aureus (MRSA) infection were treated with vancomycin (VCM)-impregnated polymethylmethacrylate (PMMA) beads. One patient, who had a history of polycystic kidney and diabetes mellitus, who was receiving hemodialysis due because of non-functional kidney, underwent resection of an intermediate grade chondrosarcoma in the pelvis. MRSA infection developed and curettage of the lesion was performed, but MRSA infection recurred. During the second revision surgery, VCM-impregnated PMMA beads were implanted. MRSA infection has not recurred for 16 months since the implantation of the VCM beads. The second patient had a history of total hip arthroplasty (THA) performed because of coxarthrosis. After the initial surgery, MRSA infection developed, recurring after the second revision surgery for THA. After curettage following removal of the prosthesis, VCM beads were implanted with a spacer composed of VCM-PMMA and a Luque rod. Infection did not recur and THA revision was performed 3 months after the VCM beads implantation. Fifteen months after the last revision surgery, infection has not recurred. Received for publication on July 24, 1997; accepted on Jan. 13, 1998     

The clearance of Gentamicin from antibiotic-loaded bone cement

Summary The concentrations of Gentamicin in the serum, drainage fluid and urine were determined by radioimmunoassay in 40 patients after total hip replacement and 10 after knee replacement. Palacos R bone cement with added Gentamicin had been used to fix the prostheses in all cases. Two types of response were observed; in Group I the Gentamicin levels rose very rapidly, whereas in Group II a more gradual increase was seen. In all cases the level of Gentamicin had fallen to a negligible amount by the fifth day after operation.

---

Résumé La concentration de la Gentamycine dans le sérum, le liquide de drainage et l'urine a été déterminée par dosage radioimmunologique chez 40 malades après arthroplastie totale de la hanche et chez 10 malades après le arthroplastie du genou. Le ciment Palacos R additionné de Gentamycine a été employé dans chaque cas pour fixer la prothèse. Deux sortes réactions ont été observées: dans le premier groupe le taux de Gentamycine s'est élevé très rapidement, tandis que dans le second groupe l'élévation a été plus progressive. Dans chaque cas le taux de Gentamycine était tombé à un niveau négligeable dès le cinquième jour après l'opération.

     

Granulation tissue formation in experimental wounds inoculated with Staphylococcus aureus

The effects of inoculated Staphylococcus aureus microorganisms on developing granulation tissue were studied in rats. Hollow cylindrical viscose cellulose sponges were implanted subcutaneously as an inductive matrix for the growth of granulation tissue. The control implants were injected immediately after implantation with 1 ml of physiological saline while the experimental implants were injected with a corresponding volume of saline solution containing live staphylococci in the following concentrations: 10(2), 10(3), 10(4) or 10(5) microorganisms per milliliter. Analyses of wound fluid and granulation tissue were carried out two weeks after implantation. Implants inoculated with 10(3) or more organisms reproducibly developed infection which delayed healing while implants inoculated with 10(2) bacteria showed no infection and were usually able to clear themselves from the organisms. In the latter implants the number of wound fluid monocytes and macrophages was markedly elevated and the mean amount of collagen hydroxyproline exceeded the control level by 55%. This demonstrates that wound healing can be accelerated to a certain extent by inoculation of live staphylococci. The acceleration concerns only such bacterial concentrations which are able to promote a local inflammatory response but which can be governed by wound defence mechanisms.     

Pseudomonas aeruginosa biofilm formation and slime excretion on antibiotic-loaded bone cement

Background Infection is an infrequent but serious complication of prosthetic joint surgery. These infections will usually not clear until the implant is removed and re-implantation has a high failure rate, especially when Pseudomonas aeruginosa is involved.

Material and methods We examined Pseudomonas aeruginosa biofilm formation on plain and gentami-cin-loaded bone cement with confocal scanning laser microscopy (CSLM). Two different stains were applied in order to visualize and quantify the distribution of bacterial cells and extracellular polymeric substances (slime) from the bone cement surface to the top of the biofilm. Staining with LIVE/DEAD viability stain differentiated between live and dead bacteria within the biofilm, and slime production was evaluated after staining with Calcofluor white.

Results CSLM showed that the biofilm was a nonuniform structure of variable thickness, with differences in local bacterial cell and slime densities. Incorporation of gentamicin in bone cement resulted in a 44% reduction in bacterial viability, while the slime density increased significantly. In addition, conventional plate counting showed the development of small-colony variants on gentamicin-loaded bone cement with a decreased sensitivity for gentamicin (MIC: 8 mg/L), as compared with normal-sized colonies taken from plain and gentamicin-loaded bone cement (MIC: 3 mg/L). The enhanced slime production on antibiotic-loaded bone cement, together with the formation of small-colony variants, resulted in decreased susceptibility to antibiotics—probably concomitant with the onset of persistent and relapsing infections.

Interpretation In the clinical situation, our findings help to explain the frequent re-implantation failure of joint replacements infected with P. aeruginosa when the procedure has been performed using antibiotic-loaded bone cement.