• View in gallery

    Schematic representation of the placement of PMMA beads for each of 6 treatment groups on large (150-mm-diameter) Mueller-Hinton plates inoculated with Staphylococcus epidermidis, Staphylococcus aureus, MRSA, Escherichia coli, Enterobacter cloacae, or Pseudomonas aeruginosa during an experiment conducted to determine the effect of bead sterilization method on antimicrobial efficacy (experiment 1). The agar surface was divided into sixths, and 1 bead from each treatment group was placed in the middle of each sixth in a sequential manner. Treatment group 1 consisted of PMMA beads that were not sterilized. Group 2 consisted of M-PMMA beads that were not sterilized. Group 3 consisted of PMMA beads that were autoclaved. Group 4 consisted of M-PMMA beads that were autoclaved. Group 5 consisted of PMMA beads that underwent VHP sterilization. Group 6 consisted of M-PMMA beads that underwent VHP sterilization. A 6-mm paper disc (indicated by a black circle) was placed in the center of each plate and covered with 10 μg of meropenem to serve as a positive antimicrobial control. The experiment was performed in triplicate and replicated 3 times for each bacterial species, with the same order of bead placement used for all replicates.

  • View in gallery

    Mean ± SD diameter of the zone of bacterial inhibition by beads in treatment groups 2 (black bars), 5 (gray bars), and 6 (white bars) and the meropenem positive control (striped bars) for each of the 6 bacterial species described in Figure 1. Following bead and control placement, each plate was incubated at 37 °C for 24 hours. For each bead-bacterial species combination, the zone of bacterial inhibition was measured in 2 dissecting planes, and the mean diameter was calculated and used for statistical analysis. *Mean differs significantly (P < 0.05) from that for group 2. †Mean differs significantly (P < 0.05) from that for group 6. ‡Mean differs significantly (P < 0.05) from that for the meropenem positive control. See Figure 1 for remainder of key.

  • View in gallery

    Mean ± SD percentage change in the zone of S aureus growth inhibition over time on solid agar relative to that on day 1 for beads of treatment groups 2 (circles), 5 (triangles), and 6 (squares) described in Figure 1 (experiment 2). For each treatment group, 1 bead was serially transferred to a new S aureus– inoculated Mueller-Hinton agar plate every 24 hours for a period of 14 days. The agar plates were prepared and incubated, and the zone of bacterial growth inhibition was measured for each bead as described in Figure 2. The experiment was replicated 3 times. See Figures 1 and 2 for remainder of key.

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  • 17. MacDonald DG, Morley PS, Bailey JV, et al. An examination of the occurrence of surgical wound infection following equine orthopaedic surgery (1981–1990). Equine Vet J 1994;26:323326.

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  • 18. Trencart P, Elce YA, Rodriguez Batista E, et al. Sterilization by gamma radiation of antibiotic impregnated polymethylmethacrylate and plaster of Paris beads. A pilot study. Vet Comp Orthop Traumatol 2014;27:97101.

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  • 19. Báez LA, Langston C, Givaruangsawat S, et al. Evaluation of in vitro serial antibiotic elution from meropenem-impregnated polymethylmethacrylate beads after ethylene oxide gas and autoclave sterilization. Vet Comp Orthop Traumatol 2011;24:3944.

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  • 20. Ramos JR, Howard RD, Pleasant RS, et al. Elution of metronidazole and gentamicin from polymethylmethacrylate beads. Vet Surg 2003;32:251261.

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  • 21. McDonnell G. The use of hydrogen peroxide for disinfection and sterilization applications. In: Greer A, Liebman JF, eds. The chemistry of peroxides. Volume 3. Part 1. PATAI'S chemistry of functional groups. Chichester, England: John Wiley & Sons Ltd, 2014;713746.

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Evaluation of vaporized hydrogen peroxide sterilization on the in vitro efficacy of meropenem-impregnated polymethyl methacrylate beads

Myra E. Durham DVM, MS1 and Johanna R. Elfenbein DVM, PhD2
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  • 1 Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.
  • | 2 Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606.

Abstract

OBJECTIVE To evaluate the effects of vaporized hydrogen peroxide (VHP) sterilization on the in vitro antimicrobial efficacy of meropenem-impregnated polymethyl methacrylate (M-PMMA) beads.

SAMPLE 6-mm-diameter polymethyl methacrylate beads that were or were not impregnated with meropenem.

PROCEDURES Meropenem-free polymethyl methacrylate and M-PMMA beads were sterilized by use of an autoclave or VHP or remained unsterilized. To determine the antimicrobial efficacy of each bead-sterilization combination (treatment), Mueller-Hinton agar plates were inoculated with 1 of 6 common equine pathogens, and 1 bead from each treatment was applied to a sixth of each plate. The zone of bacterial inhibition for each treatment was measured after 24 hours. To estimate the duration of antimicrobial elution into a solid or liquid medium, 1 bead from each treatment was transferred every 24 hours to a new Staphylococcus aureus–inoculated agar plate or a tube with PBS solution, and an aliquot of the eluent from each tube was then applied to a paper disc on an S aureus–inoculated agar plate. All agar plates were incubated for 24 hours, and the zone of bacterial inhibition was measured for each treatment.

RESULTS In vitro antimicrobial efficacy of M-PMMA beads was retained following VHP sterilization. The duration of antimicrobial elution in solid and liquid media did not differ significantly between unsterilized and VHP-sterilized M-PMMA beads.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that M-PMMA beads retained in vitro antimicrobial activity and eluted the drug for up to 2 weeks after VHP sterilization.

Abstract

OBJECTIVE To evaluate the effects of vaporized hydrogen peroxide (VHP) sterilization on the in vitro antimicrobial efficacy of meropenem-impregnated polymethyl methacrylate (M-PMMA) beads.

SAMPLE 6-mm-diameter polymethyl methacrylate beads that were or were not impregnated with meropenem.

PROCEDURES Meropenem-free polymethyl methacrylate and M-PMMA beads were sterilized by use of an autoclave or VHP or remained unsterilized. To determine the antimicrobial efficacy of each bead-sterilization combination (treatment), Mueller-Hinton agar plates were inoculated with 1 of 6 common equine pathogens, and 1 bead from each treatment was applied to a sixth of each plate. The zone of bacterial inhibition for each treatment was measured after 24 hours. To estimate the duration of antimicrobial elution into a solid or liquid medium, 1 bead from each treatment was transferred every 24 hours to a new Staphylococcus aureus–inoculated agar plate or a tube with PBS solution, and an aliquot of the eluent from each tube was then applied to a paper disc on an S aureus–inoculated agar plate. All agar plates were incubated for 24 hours, and the zone of bacterial inhibition was measured for each treatment.

RESULTS In vitro antimicrobial efficacy of M-PMMA beads was retained following VHP sterilization. The duration of antimicrobial elution in solid and liquid media did not differ significantly between unsterilized and VHP-sterilized M-PMMA beads.

CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that M-PMMA beads retained in vitro antimicrobial activity and eluted the drug for up to 2 weeks after VHP sterilization.

Osteomyelitis and infections associated with orthopedic implants are common complications following fracture repair and are often difficult to treat successfully. In 1 study,1 53 of 192 (28%) horses developed a postoperative infection following internal fixation of long bone fractures and arthrodesis, of which only 31 of 53 (58%) survived to be discharged from the hospital. Those statistics demonstrate the life-threatening consequences of orthopedic implant infections in horses. As the incidence of multidrug-resistant bacterial infections increases, clinicians may use alternative methods to manage infections including treatment with natural antibacterial compounds such as honey or local application of antimicrobials in PMMA beads.2 Newer-generation broad-spectrum antimicrobials such as meropenem should be reserved for use only in patients with life-threatening infections that have been determined to be susceptible to only those agents by bacterial culture and susceptibility testing.3 Meropenem retains efficacy against common multidrug-resistant bacteria, but its cost typically makes systemic use in horses prohibitive. Local delivery of antimicrobials such as meropenem by elution from AI-PMMA beads can provide a high concentration of the antimicrobial at the site of infection, minimize the risk of systemic complications such as antimicrobial-induced colitis, and decrease the antimicrobial volume used, thereby decreasing the total cost of treatment.4

Antibiotic-impregnated PMMA beads have been used for the treatment of soft tissue and bone infections for > 30 years.5 Gentamicin-impregnated PMMA beads were first demonstrated to be effective in canine femurs with experimentally induced osteomyelitis in 1978.6 Since then, multiple reports7–10 have described the use of AI-PMMA beads in clinical veterinary practice. One problem with the use of AI-PMMA beads is that clinicians frequently prepare the beads by hand at the time of surgery, which results in beads of varying shapes and sizes and prolongs surgery duration. Antimicrobial elution from PMMA beads is highly dependent on bead surface area5,11; therefore, variability in bead size and shape leads to uneven elution of the anti-microbial in infected tissues. Additionally, the methyl methacrylate monomer persists for 2 hours after bead preparation and is toxic to musculoskeletal tissue.12 Therefore, advantages associated with the preparation of AI-PMMA beads prior to surgery include the production of beads with a uniform shape and size for implantation, which facilitates rapid initiation of antimicrobial infusion into the site of infection and a decrease in surgery duration. However, AI-PMMA beads prepared prior to surgery require sterilization before use, and the type of sterilization used requires special attention to ensure that the antimicrobial is not degraded.

Many antimicrobials lose activity following exposure to the high temperatures associated with steam sterilization in an autoclave.13 Low-temperature sterilization methods rely on vaporization of a bactericidal compound for sterilization. Traditionally, ethylene oxide vaporization has been the standard low-temperature sterilization method used for AI-PMMA beads to retain antimicrobial activity, but accessibility to ethylene oxide has become challenging owing to an increase in its cost and awareness of its potential hazards to human health.14,15 Vaporized hydrogen peroxide is an alternative for low-temperature sterilization, but information regarding the efficacy of AI-PMMA beads following VHP sterilization is lacking. Therefore, the purpose of the study reported here was to evaluate the effects of VHP sterilization on the in vitro antimicrobial efficacy of M-PMMA beads. We hypothesized that VHP sterilization would not affect the antimicrobial efficacy of M-PMMA beads against bacteria commonly associated with orthopedic implant infections of horses.

Materials and Methods

PMMA beads

All PMMA beads were prepared in a class IIA biosafety cabinet by 1 investigator (MED). A commercially manufactured polypropylene bead molda was used to produce beads with a uniform diameter of 6 mm. For the control beads, PMMA powderb was prepared in accordance with the manufacturer's instructions. For M-PMMA beads, 2 g of meropenem powderc was added to 20 g of PMMA powder prior to mixing with the liquid component and then placed into the bead mold and allowed to set for 12 hours.11

Beads were transferred to 1 of 6 sterile plastic containers immediately following removal from the mold. Each container represented a treatment group (bead-sterilization method combination). Group 1 consisted of PMMA beads that were not sterilized. Group 2 consisted M-PMMA beads that were not sterilized. Group 3 consisted of PMMA beads that were autoclaved. Group 4 consisted of M-PMMA beads that were autoclaved. Group 5 consisted of PMMA beads that underwent VHP sterilization. Group 6 consisted of M-PMMA beads that underwent VHP sterilization.

The beads for groups 1 and 2 (controls) remained in their respective sterile plastic containers until antimicrobial efficacy was assessed. Beads in groups 3 and 4 were transferred to separate autoclave sterilization pouches and autoclaved at 121°C for 15 minutes. Beads from groups 5 and 6 were transferred to separate VHP sterilization pouches and processed by means of a nonlumen cycle for 28 minutes in a commercial VHP system.d The antimicrobial efficacy of all beads in groups 3 through 6 was assessed within 24 hours after completion of the sterilization process.

Bacteria

For beads within each of the 6 treatment groups, the antimicrobial efficacy against the following bacteria was assessed: Staphylococcus epidermidis, Staphylococcus aureus, MRSA, Escherichia coli, Enterobacter cloacae, and Pseudomonas aeruginosa. All bacterial isolates were stored frozen in 30% glycerol at −80°C until use and grown on Luria-Bertani (Miller) agare or in Luria-Bertani brothf at 37°C with agitation.

Experiment 1

For each bacterial species assessed, bacteria that had been cultured overnight (approx 16 to 18 hours) were diluted to 0.5 McFarland standard in sterile water as described.16 A sterile cotton swab was used to spread the diluted bacterial suspension evenly across the entire surface of a large (diameter, 150 mm) Petri dish containing Mueller-Hinton agar.f The agar surface was divided into sixths, and 1 bead from each treatment group was placed in the middle of each sixth in a sequential manner (Figure 1). A 6-mm-diameter paper discg was placed in the center of the Petri dish and covered with 10 μg of meropenem to serve as a positive control for meropenem sensitivity. Each plate was incubated at 37°C for 24 hours. For each bead–bacterial species combination, the zone of bacterial inhibition was measured in 2 dissecting planes, and the mean diameter was calculated and used for statistical analysis. The experiment was performed in triplicate and replicated 3 times, and the same bead-placement pattern was used for each replicate.

Figure 1—
Figure 1—

Schematic representation of the placement of PMMA beads for each of 6 treatment groups on large (150-mm-diameter) Mueller-Hinton plates inoculated with Staphylococcus epidermidis, Staphylococcus aureus, MRSA, Escherichia coli, Enterobacter cloacae, or Pseudomonas aeruginosa during an experiment conducted to determine the effect of bead sterilization method on antimicrobial efficacy (experiment 1). The agar surface was divided into sixths, and 1 bead from each treatment group was placed in the middle of each sixth in a sequential manner. Treatment group 1 consisted of PMMA beads that were not sterilized. Group 2 consisted of M-PMMA beads that were not sterilized. Group 3 consisted of PMMA beads that were autoclaved. Group 4 consisted of M-PMMA beads that were autoclaved. Group 5 consisted of PMMA beads that underwent VHP sterilization. Group 6 consisted of M-PMMA beads that underwent VHP sterilization. A 6-mm paper disc (indicated by a black circle) was placed in the center of each plate and covered with 10 μg of meropenem to serve as a positive antimicrobial control. The experiment was performed in triplicate and replicated 3 times for each bacterial species, with the same order of bead placement used for all replicates.

Citation: American Journal of Veterinary Research 80, 1; 10.2460/ajvr.80.1.45

Experiment 2

To evaluate the duration of antimicrobial elution from the beads onto a solid surface, beads were serially transferred to a new S aureus–inoculated Mueller-Hinton agar plate every 24 hours over a period of 14 days. The agar plates were prepared and incubated, and the zone of bacterial inhibition was measured for each bead as described for experiment 1. The experiment was performed in triplicate and replicated 3 times.

Experiment 3

To evaluate the duration of antimicrobial elution from the beads into a liquid, 1 bead from each treatment group was serially transferred into a tube containing 5 mL of PBS solution every 24 hours until bacterial inhibition was no longer detected. The tubes were incubated at 37°C in an atmosphere with 5% CO2 for 24 hours. From each tube following incubation, a 10-μL aliquot was spotted onto a 6-mm-diameter paper discg centrally placed on a small (diameter, 100 mm) Mueller-Hinton agar plate inoculated with S aureus. A 6-mm-diameter paper discg spotted with 10 μg of meropenem was also placed onto a plate to serve as a positive control. The small agar plates were prepared and incubated, and the zone of bacterial growth inhibition was measured for each bead and control as described for experiment 1. The experiment was replicated 3 times.

Statistical analysis

All outcomes of interest were continuous variables (ie, diameter of the zone of bacterial inhibition [experiments 1 and 3] or change in the diameter of the zone of bacterial inhibition [experiment 2]), and the data distribution for those variables was assessed by means of a Shapiro-Wilk normality test and found to be normally distributed. For experiment 1, a 2-way repeated-measures ANOVA was used to assess the effect of sterilization method on bead antimicrobial efficacy. Independent variables included in the model were treatment group (1 through 6) and replicate (1 through 3). A Tukey correction was used when multiple pairwise comparisons were necessary. For experiment 2, the change in the zone of bacterial inhibition relative to that on day 1 was calculated as the diameter of the zone of bacterial inhibition on the day in question divided by the diameter of the zone of bacterial inhibition on day 1 and was expressed as a percentage. Unpaired t tests were used to compare the mean change in the diameter of the zone of bacterial inhibition between groups 2 and 6 on each day. For experiment 3, a 2-way repeated-measures ANOVA with a Tukey correction for multiple comparisons was performed to evaluate the effect of sterilization on liquid elution each day. A χ2 analysis was performed to evaluate the effect of sterilization on the maximal duration of liquid antimicrobial elution for groups. Values of P < 0.05 were considered significant. All analyses were performed with a commercially available software package.h

Results

Beads from groups 1 (PMMA beads that were not sterilized), 3 (PMMA beads that were autoclaved), and 4 (M-PMMA beads that were autoclaved) did not provide detectable bacterial inhibition in any of the 3 experiments. Therefore, those treatment groups were not mentioned further in the results.

Experiment 1

Beads from groups 2 (M-PMMA beads that were not sterilized), 5 (PMMA beads that underwent VHP), and 6 (M-PMMA beads that underwent VHP) inhibited growth of all 6 bacterial species evaluated (Figure 2). For the gram-negative organisms (E coli, E cloacae, and P aeruginosa), the zone of bacterial inhibition produced by beads from group 5 was significantly smaller than the zones produced by beads from group 6 and the meropenem control. There were no differences between groups 2 and 6. For S aureus and S epidermidis, the zone of bacterial inhibition did not differ significantly among beads from groups 2, 5, and 6 and the meropenem positive control. For MRSA, the zone of bacterial inhibition for group 6 was significantly larger than that for group 2, and the zones of bacterial inhibition for groups 5 and 6 were significantly larger than that for the meropenem control.

Figure 2—
Figure 2—

Mean ± SD diameter of the zone of bacterial inhibition by beads in treatment groups 2 (black bars), 5 (gray bars), and 6 (white bars) and the meropenem positive control (striped bars) for each of the 6 bacterial species described in Figure 1. Following bead and control placement, each plate was incubated at 37 °C for 24 hours. For each bead-bacterial species combination, the zone of bacterial inhibition was measured in 2 dissecting planes, and the mean diameter was calculated and used for statistical analysis. *Mean differs significantly (P < 0.05) from that for group 2. †Mean differs significantly (P < 0.05) from that for group 6. ‡Mean differs significantly (P < 0.05) from that for the meropenem positive control. See Figure 1 for remainder of key.

Citation: American Journal of Veterinary Research 80, 1; 10.2460/ajvr.80.1.45

Experiment 2

Growth of S aureus was inhibited by beads from groups 2 and 6 for all 14 days of the observation period, and the percentage change in the zone of bacterial inhibition did not differ significantly between groups 2 and 6 on any day (Figure 3). For both groups 2 and 6, the zones of bacterial inhibition decreased by < 25% for day 11 relative to that on day 1. For group 5, the zone of bacterial inhibition decreased rapidly after day 1 and was absent by day 3.

Figure 3—
Figure 3—

Mean ± SD percentage change in the zone of S aureus growth inhibition over time on solid agar relative to that on day 1 for beads of treatment groups 2 (circles), 5 (triangles), and 6 (squares) described in Figure 1 (experiment 2). For each treatment group, 1 bead was serially transferred to a new S aureus– inoculated Mueller-Hinton agar plate every 24 hours for a period of 14 days. The agar plates were prepared and incubated, and the zone of bacterial growth inhibition was measured for each bead as described in Figure 2. The experiment was replicated 3 times. See Figures 1 and 2 for remainder of key.

Citation: American Journal of Veterinary Research 80, 1; 10.2460/ajvr.80.1.45

Experiment 3

The median duration of antimicrobial elution from beads into a PBS solution was 15 days (range, 15 to 19 days) for group 2 and 11 days (range, 10 to 15 days) for group 6. The duration of antimicrobial elution into a liquid solution did not differ significantly between groups 2 and 6. Culture of PBS solution exposed to beads from group 5 failed to yield a zone of bacterial inhibition at any time.

Discussion

Results of the present study indicated that VHP sterilization did not alter the in vitro antimicrobial efficacy of M-PMMA beads against bacterial species (S epidermidis, S aureus, MRSA, E coli, E cloacae, and P aeruginosa) commonly isolated from orthopedic infections of horses.17 For MRSA specifically, the zone of bacterial inhibition for M-PMMA beads that underwent VHP sterilization (group 6) was significantly larger than that for both M-PMMA beads that did not undergo sterilization and the meropenem positive control. That finding suggested that hydrogen peroxide may act synergistically with meropenem against MRSA. Perhaps most importantly, the results of the present study indicated that VHP sterilization did not significantly affect in vitro meropenem elution from PMMA beads on a solid surface or within a liquid.

Most antimicrobials are degraded when exposed to heat; therefore, low-temperature sterilization methods are necessary for AI-PMMA beads to preserve antimicrobial efficacy. Historically, ethylene oxide was the disinfectant commonly used for low-temperature sterilization, but its use is falling out of favor owing to evolving emissions regulations, the fairly long aeration time required to dissipate ethylene oxide residues, and the potential health risks for personnel exposed to ethylene oxide.14 Occupational exposure to ethylene oxide is associated with the development of neoplasia.14,15 Moreover, ethylene oxide residue remains on the surface of items such as PMMA beads for some time following sterilization and may dissipate into tissues when those items are placed in the body.15 Results of another study18 indicate that γ radiation is an effective alternative for sterilization of AI-PMMA beads, but radiation safety concerns for personnel involved with its use have limited its adoption. Vaporized hydrogen peroxide can also be used for low-temperature sterilization, and its by-products (water and oxygen) are environmentally friendly and have a low risk for inducing adverse effects in medical personnel or patients.14 Although VHP is an accepted method for low-temperature sterilization, it is important to note that we did not directly assess the efficacy of the VHP sterilization process in the present study. The PMMA beads did not undergo bacterial culture following sterilization, which was a study limitation.

During experiment 3 of the present study, meropenem was eluted into PBS solution for a median of 11 days from PMMA beads that underwent VHP sterilization (group 6), compared with a median of 15 days from PMMA beads that were not sterilized (group 2); however, that difference was not significant. In another study,19 the meropenem concentration in bead eluent remained greater than the breakpoint sensitivity limit (4 μg/mL) for 15 days for unsterilized M-PMMA beads and 18 days for M-PMMA beads that underwent low-temperature sterilization with ethylene oxide. Results of the present study are not directly comparable with those of that study19 owing to the different low-temperature sterilization methods used in the 2 studies. Nevertheless, results of the present study suggested that meropenem was eluted effectively from PMMA beads and maintained its antimicrobial efficacy following VHP sterilization. Therefore, we believe VHP sterilization may be a viable alternative for low-temperature sterilization of M-PMMA beads.

We found it interesting that meropenem-free PMMA beads that underwent VHP sterilization (group 5) had some antimicrobial efficacy for 24 hours after sterilization. The PMMA beads that were not sterilized (group 1) did not inhibit the growth of any of the bacterial species evaluated, which led us to conclude that PMMA alone had no antimicrobial activity, a finding that was consistent with results of other studies.19,20 Hydrogen peroxide is widely used for disinfection and sterilization, and although its method of action is not completely understood, it reacts with ferrous iron to damage DNA and metalloproteins.21,22 We suspect that hydrogen peroxide either was absorbed into the PMMA during or remained on the surface of the beads after VHP sterilization, and it was the residual hydrogen peroxide that was responsible for the antimicrobial effect observed for the beads of group 5. Certain products, such as paper, absorb hydrogen peroxide during processing and are therefore not compatible with VHP sterilization.14 It is unclear whether PMMA absorbs hydrogen peroxide, and because the antimicrobial efficacy decreased rapidly and disappeared by day 3 for group 5 beads, we believe it was most likely that residual hydrogen peroxide remained on the surface of the group 5 beads following VHP sterilization.

Another interesting finding of the present study was that, for MRSA, the zone of bacterial inhibition for M-PMMA beads that underwent VHP sterilization (group 6) was significantly larger than that for the M-PMMA beads that were not sterilized (group 2). This suggested that VHP sterilization had a synergistic effect with meropenem against MRSA in vitro. Preincubation of MRSA with hydrogen peroxide increases the bactericidal effect of meropenem, which further supports a synergistic effect between hydrogen peroxide and meropenem for that particular organism.23 The potential for VHP-sterilized AI-PMMA beads to improve treatment response of patients with drug-resistant infections warrants further investigation. Also, hydrogen peroxide is cytotoxic in vivo; therefore, further research is necessary to determine whether residual hydrogen peroxide on the surface of PMMA beads following VHP sterilization is detrimental to in vivo tissues that come into contact with the beads after they are implanted in patients.

The elution rate of an antimicrobial from PMMA is dependent on a number of factors. Elution is biphasic, with a high elution rate early followed by a sustained slower release as the antimicrobial diffuses from pores, cracks, and voids in the PMMA.24 For AI-PMMA beads in general, approximately 5% of the antimicrobial is eluted within the first 24 hours, and elution continues over the next 6 to 8 weeks until approximately 10% of the total antimicrobial content within individual beads is released.5,11,25 The antimicrobial elution potential can be increased by the use of uniformly sized beads with a small volume so that the available bead surface area is increased.5 The antimicrobial elution properties of AI-PMMA beads are also affected by the type of PMMA used and the method of preparation.11,26 In the present study, 1 investigator used a commercially available bead mold to create uniformly sized and shaped beads, which minimized the variability of the antimicrobial elution rate among experiments and replicates. We also ensured that the surface area of the beads in contact with the agar plates remained consistent for all treatment groups in all replicates of experiments 1 and 2. Additionally, we used a commercially available veterinary bone cement to mimic clinical use; therefore, we believe the findings of this study have direct application to the clinical use of VHP-sterilized M-PMMA beads.

In the present study, VHP sterilization did not negatively affect the antimicrobial efficacy of M-PMMA beads in vitro for up to 2 weeks. Additionally, PMMA beads that were not impregnated with an antimicrobial appeared to have antibacterial activity for 24 hours after VHP sterilization, which we attributed to the antibacterial effects of hydrogen peroxide residues on the bead surface. Further research is warranted to evaluate the effect of VHP sterilization residues and determine the in vivo antimicrobial efficacy and safety of VHP-sterilized M-PMMA beads before their use in clinical practice can be recommended.

Acknowledgments

Supported by the Department of Clinical Sciences, College of Veterinary Medicine, North Carolina State University.

The authors thank Mitsu Suyemoto for providing the bacterial isolates used in the study and for critical discussion of the work.

ABBREVIATIONS

AI-PMMA

Antimicrobial-impregnated polymethyl methacrylate

M-PMMA

Meropenem-impregnated polymethyl methacrylate

MRSA

Methicillin-resistant Staphylococcus aureus

PMMA

Polymethyl methacrylate

VHP

Vaporized hydrogen peroxide

Footnotes

a.

Bone Cement Bead Mold System, EXCELEN, Minneapolis, Minn.

b.

Veterinary bone cement, BioMedtrix, Boonton, NJ.

c.

ArkPharm Inc, Libertyville, Ill.

d.

VHP MD 140X, Steris Life Sciences, Mentor, Ohio.

e.

Fisher Scientific, Hampton, NH.

f.

BD Biosciences, San Jose, Calif.

g.

Whatman paper, GE Healthcare Bio-Sciences Corp, Marlborough, Mass.

h.

Prism, version 7.0, GraphPad Software, La Jolla, Calif.

References

  • 1. Ahern BJ, Richardson DW, Boston RC, et al. Orthopedic infections in equine long bone fractures and arthrodeses treated by internal fixation: 192 cases (1990–2006). Vet Surg 2010;39:588593.

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Contributor Notes

Address correspondence to Dr. Elfenbein (jrelfenb@ncsu.edu).