In vitro efficacy of antibiotic beads in treating abdominal vascular graft infections

From the Society for Vascular Surgery

In vitro efficacy of antibiotic beads in treating abdominal vascular graft infections Kristofer M. Charlton-Ouw, MD, FACS,a,b Fernando Kubrusly, MD,a Harleen K. Sandhu, MD, MPH,a Michelle C. Swick, PhD,c Samuel S. Leake, BS,a Brian E. Gulbis, PharmD,b Theresa M. Koehler, PhD,c and Hazim J. Safi, MD, FACS,a,b Houston, Tex Objective: Abdominal aortic vascular graft infection often involves several different organisms. Antibiotic polymethyl methacrylate (PMMA) beads may be effective in controlling infection after débridement, but bacterial species identification and antibiotic susceptibility are often not available at the time of operation, generating a need for a broad-spectrum drug combination for empirical use. We sought to determine an effective antibiotic in PMMA beads for use in abdominal vascular graft infection. Methods: PMMA beads were impregnated with combinations of antibiotics, consisting of daptomycin, tobramycin, and meropenem. Antibiotics were selected on the basis of activity spectrum and heat stability. Beads were placed on separate agar plates with vancomycin-resistant Enterococcus faecalis, Klebsiella pneumoniae, Staphylococcus epidermidis, and methicillin-resistant Staphylococcus aureus. Antibiotic inhibition was recorded by use of a modified agar-based diskdiffusion method. Results: Daptomycin alone was not active against K. pneumoniae (average [ 0 mm). Tobramycin alone was not active against vancomycin-resistant E. faecalis, K. pneumoniae, or methicillin-resistant S. aureus. Tobramycin and daptomycin in combination had moderate broad-spectrum activity with 8- to 14-mm mean inhibition halos. Meropenem showed strong activity against all tested organisms with >15-mm mean inhibition halos. The addition of daptomycin to meropenem provided improved coverage of gram-positive organisms. The presence of tobramycin reduced the efficacy of meropenem. Conclusions: Antibiotic PMMA beads containing 10% meropenem with 2.5% daptomycin had excellent in vitro activity against typical bacterial species associated with abdominal vascular graft infections. The addition of antibiotic beads may be a useful adjunct in managing such cases. Further studies are required to determine efficacy in clinical practice. (J Vasc Surg 2014;-:1-6.) Clinical Relevance: Regardless of revascularization technique or conduit, repair of infected abdominal aortic aneurysms and infected prosthetic grafts has nearly 50% 5-year mortality despite improved in-hospital mortality rates. We believe that this is mostly due to local reinfection. Antibiotic beads have the potential to help sterilize the contaminated field and have been successfully used in infections involving extremity vascular grafts, orthopedic hardware, and ventricular assist devices. This technique may also be useful in trauma and military settings, where vascular anastomoses may be required in contaminated fields. This report describes the formulation of cement beads for use against organisms commonly found in abdominal vascular graft infections.

Abdominal aortic vascular graft infection is uncommon. Nevertheless, when infections occur, there are high rates of limb loss and death. The consequences of persistent virulent organisms include graft reinfection, anastomotic and aortic stump disruptions, sepsis, limb loss, and death.1 Techniques to combat graft reinfection have focused on débridement and revascularization with either extra-anatomic From the Department of Cardiothoracic and Vascular Surgerya and Department of Microbiology and Molecular Genetics,c University of Texas Medical School at Houston; and the Memorial Hermann Heart and Vascular InstituteeTexas Medical Center.b Author conflict of interest: none. Presented at the 2013 Vascular Annual Meeting of the Society for Vascular Surgery, San Francisco, Calif, May 30-June 1, 2013. Reprint requests: Kristofer M. Charlton-Ouw, MD, FACS, Department of Cardiothoracic and Vascular Surgery, University of Texas Medical School at Houston, 6400 Fannin St, Ste 2850, Houston, TX 77030 (e-mail: [email protected]). The editors and reviewers of this article have no relevant financial relationships to disclose per the JVS policy that requires reviewers to decline review of any manuscript for which they may have a conflict of interest. 0741-5214/$36.00 Copyright Ó 2014 by the Society for Vascular Surgery. http://dx.doi.org/10.1016/j.jvs.2014.03.241

bypass or infection-resistant conduit material, such as native femoropopliteal vein,2 cadaveric arteries,3,4 and antibiotic5 or silver bonding to prosthetic grafts.6 Despite these measures, 20% of patients with abdominal graft infection will have recurrent infection.7-9 Diffusion of a high-concentration antibiotic into the infected field from implanted beads may be a useful adjunct to prevent graft reinfection. Use of antibiotic polymethyl methacrylate (PMMA) cement beads was first described in 1970 for treatment of infected orthopedic prostheses.10 Recently, several authors reported success with use of antibiotic PMMA beads in extremity vascular graft infections.11 The perigraft tissues are débrided, and antibiotic beads are wrapped around the prosthetic graft. The wound is temporarily closed, usually with a negative-pressure dressing, and the wound is reexplored after a couple of days. The beads are removed before final closure. To date, there are no reports of use of antibiotic PMMA beads in abdominal vascular graft infections. Current commercially available PMMA beads in the United States contain gentamicin, tobramycin, and gentamicin plus clindamycin. Such antibiotics do not always appropriately treat resistant bacteria, such as 1

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vancomycin-resistant Enterococcus faecalis (VRE) and methicillin-resistant Staphylococcus aureus (MRSA), and may not be appropriate for use in abdominal vascular graft infections, especially in the setting of infection with enteric organisms. The choice of antibiotics in PMMA requires careful consideration. The antibiotics must be heat stable because the PMMA cement can reach up to 70
C during the curing process.12 In addition, the antibiotics should be active in the supplied form without requiring in vivo metabolic activation. On the basis of our experience of the broad microbial spectrum found on abdominal aortic graft cultures,9 we favor antibiotics that provide maximal antibacterial coverage, including anaerobes. Tobramycin is an aminoglycoside antibiotic with bactericidal activity against gram-negative bacteria, including Pseudomonas. Vancomycin is commonly added to PMMA beads in extremity and superficial wounds but does not treat VRE. However, daptomycin is a lipopeptide antibiotic that is highly active against drug-resistant, grampositive bacteria, including VRE and MRSA,13 as well as gram-positive anaerobic bacteria. In combination, tobramycin- and daptomycin-laden PMMA beads are effective against a broad spectrum of bacteria.14 Meropenem is a carbapenem antibiotic that is effective against both grampositive and gram-negative bacteria, including anaerobes. It is theoretically less effective against some resistant bacteria, such as VRE and MRSA. The elution of meropenem from PMMA has been shown to be effective for up to 3 weeks.15 The combination of meropenem and daptomycin provides broad-spectrum antimicrobial coverage. The aim of our study was to test in vitro efficacy of tobramycin, daptomycin, and meropenem, alone and in combination, in PMMA cement beads used against the most common organisms that cause abdominal aortic graft infection. The most common organisms isolated from infected abdominal aortic grafts in the literature and from our experience include MRSA, Staphylococcus non-aureus sp, Enterococcus sp, Streptococcus sp, and Klebsiella sp.8,9,16 Fungus was isolated in 13% to 18% of reported cases.8,9 The addition of an antifungal agent should be considered especially in cases of graft-enteric connections. The clinical rationale for combining meropenem with tobramycin was to provide additional enhanced antimicrobial activity for drug-resistant Pseudomonas and other gram-negative bacteria. The addition of daptomycin provides antimicrobial activity for drug-resistant gram-positive bacteria. Although fungal isolates were not tested in this study, we added voriconazole to make sure that it did not adversely affect antibacterial properties of the other agents. Because data on the infecting species and antibiotic susceptibilities are often not available at the time of surgical treatment for abdominal vascular graft infection, we sought to find an antimicrobial combination that provided empirical, broad-spectrum coverage in PMMA beads. METHODS The experiments were divided into stages. In the first stage, eight different sets of PMMA beads were made

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combining 1.25% tobramycin, 1.25% daptomycin (Cubicin; Cubist Pharmaceuticals, Lexington, Mass), and 5% meropenem (Merrem; AstraZeneca, Wilmington, Del). Control beads contained no antibiotics. Antibiotics were selected on the basis of information in the current literature, review of the microbiology reports in our cases of infected abdominal aortic grafts, susceptibility testing, and antibiotic heat stability. The initial concentration of antibiotic was selected on the basis of recommended 24hour dosing for each antibiotic. Dosing was increased in subsequent experiments. We added an antifungal agent to make sure that antibacterial efficacy was not adversely affected. Antibiotic PMMA beads. Each sterile bone cement kit (Surgical Simplex P; Stryker, Kalamazoo, Mich) contained 40 g of cement, including 75% methyl methacrylateestyrene, 15% PMMA, and 10% barium sulfate. Under sterile conditions, the cement was combined with the antibiotic powder by a commercial mixer (Mixevac; Stryker). Initial antibiotic concentrations were made by mixing 1.25% (500 mg) tobramycin and/or 1.25% (500 mg) daptomycin and/or 5% (2 g) meropenem in 40 g PMMA cement. Concentrations were later increased to 2.5% daptomycin and 10% meropenem. An antifungal agent, 1% voriconazole (400 mg per 40 g PMMA), was added only to the high-dose cement mix. Although fungal isolates were not tested, we wanted to make sure that an antifungal agent would not reduce the antibacterial activity of the most efficacious combination. After mixing to a uniform consistency for 20 seconds, the cement was transferred to a sterile 8-mm silicone mold (Bead Maker; CK Products, Fort Wayne, Ind). After hardening, the beads were removed and placed in a sterile container. Beads containing daptomycin were stored at 3
C to 5
C. Bacterial cultures and inhibition halos. Four separate bacterial cultures were made according to American Type Culture Collection (ATCC) specifications: VRE (E. faecalis, ATCC 51299), Klebsiella (K. pneumoniae, ATCC 700603), MRSA (S. aureus, ATCC 43300), and Staphylococcus non-aureus (S. epidermidis, ATCC 12228). From each culture, two glycerol cryopreserved vials were made to facilitate same-strain reproduction. For VRE, we used brain heart infusion broth (BD 237500) containing 4 mg vancomycin per milliliter. For MRSA, we used tryptic soy broth (BD 211825). For both S. epidermidis and K. pneumoniae, we used nutrient broth (BD 234000) and yeast extract (NBY). Broth cultures were allowed to incubate for 24 hours. Sterile swabs were used to transfer the bacteria to 150-mm Mueller-Hinton 0.8% agar plates. Templates were affixed under each plate, and antibiotic PMMA beads containing each drug combination were pressed 2 mm into the agar (Fig 1). Beads were placed at least 24 mm apart from each other. Plates with cracked agar were discarded and replaced. The plates were incubated at 37
C for 24 hours. Each assay was performed in triplicate, and experiments were repeated at least three times. After removal of the plates from the incubator, the inhibition halos surrounding each PMMA bead were

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Fig 1. Polymethyl methacrylate (PMMA) beads placed on culture plates.

immediately measured according to the disk diffusion method (Fig 2). Statistical analyses were carried out with STATA 12.1 (StataCorp, College Station, Tex). Values of inhibition halos were expressed as mean 6 standard deviation. Statistical significance was assumed for P # .05 by unpaired Student t-test. To assess interactions between types of microbial cultures, drug/drug combinations, and drug dose, an analysis of variance and Tukey method of multiple comparisons was used to determine statistically significant interactions. RESULTS Average measurements of the inhibition halos for each strain are listed in Table I. As expected, daptomycin alone was not effective against the gram-negative bacterium (Klebsiella), and tobramycin showed weak activity against all tested gram-positive bacteria (VRE, MRSA, S. epidermidis). The combination of daptomycin and tobramycin demonstrated moderate activity against most organisms, with average inhibition halos of 8 mm with VRE, 11 mm with Klebsiella, 14 mm with S. epidermidis, and 10 mm with MRSA. Low-dose meropenem alone was highly effective against all bacteria, with an average inhibition halo of 17 mm with VRE, 15 mm with Klebsiella, 21 mm with S. epidermidis, and 17 mm with MRSA. The addition of daptomycin to meropenem did not significantly alter the average inhibition halos compared with meropenem alone in VRE, S. epidermidis, and MRSA cultures. However, the combination of daptomycin and meropenem increased the average inhibition halos with Klebsiella by 0.8 mm (5%; 95% confidence interval [CI], 4.6-10.7; P < .001). Combining tobramycin and meropenem decreased the

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Fig 2. Inhibition halos surround polymethyl methacrylate (PMMA) beads on culture plates after incubation.

average inhibition halos in all bacterial cultures compared with meropenem alone. Inhibition halos were reduced by 11.5% against Klebsiella (95% CI, 8.6 to 2.4; P ¼ .001) and by 21% against S. epidermidis (95% CI, 15.2 to 4.2; P ¼ .001). A combination of daptomycin, tobramycin, and meropenem did not produce significant change in the average inhibition halos compared with meropenem alone. High-dose antibiotic inhibition halo results are shown in Table II. Increasing the dose of meropenem to 10% and daptomycin to 2.5% did not significantly change the inhibition halos in VRE, Klebsiella, or MRSA cultures. However, the high-dose meropenem and daptomycin combination increased the average inhibition halo against S. epidermidis from 22 to 27 mm, an increase of 23% (95% CI, 2.2-8.4; P ¼ .001). The addition of voriconazole did not adversely affect the efficacy of high-dose meropenem and daptomycin. DISCUSSION Abdominal vascular graft infection is uncommon but is associated with limb loss and death. Standard therapy includes systemic antibiotics, débridement of dead tissue, repair of enteric connections, and resection and replacement of the infected graft. Regardless of the surgical technique used, reinfection affects up to 20% of patients treated for graft infection.9 We postulate that antibiotic beads are adjunctive and should not replace standard treatment of infected abdominal vascular grafts. Antibiotic-laden cement beads deliver high local concentrations without systemic drug toxicities. Antibiotic beads have proved to be effective for orthopedic, soft tissue, extremity arterial graft, and ventricular assist device infections,11,17-20 whereas local

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Table I. Average inhibition halo radius (in millimeters and with standard deviation) of low-dose, antibiotic polymethyl methacrylate (PMMA) beads in bacterial culture Antibiotic DA TO DA þ TO ME ME þ DA ME þ TO ME þ DA þ TO No antibiotic

VRE 9.8 2.0 8.0 17.0 17.2 13.8 16.9 3.2

6 6 6 6 6 6 6 6

1.5 3.1 1.1 0.9 1.0 3.4 2.0 3.5

Klebsiella sp 10.7 10.8 15.3 16.0 13.5 15.0 6.9

0 6 6 6 6 6 6 6

0.8 0.8 0.6 1.3 2.6 1.6 3.9

Staphylococcus epidermidis 10.2 14.0 14.3 21.0 21.7 16.7 21.0 8.7

6 6 6 6 6 6 6 6

0.4 1.3 1.0 2.5 2.3 6.2 5.2 3.1

MRSA 9.6 4.7 9.9 17.3 17.7 13.4 17.0 4.2

6 6 6 6 6 6 6 6

0.5 3.8 1.1 1.5 2.1 4.2 2.1 6.5

DA, Daptomycin; ME, meropenem; MRSA, methicillin-resistant Staphylococcus aureus; TO, tobramycin; VRE, vancomycin-resistant Enterococcus faecalis.

Table II. Average inhibition halo radius (in millimeters and with standard deviation) of high-dose, antibiotic polymethyl methacrylate (PMMA) beads in bacterial culture Antibiotic ME þ V DA þ V ME þ DA ME þ DA þ V

VRE 19.0 13.8 17.4 18.6

6 6 6 6

1.6 2.7 2.7 2.0

Klebsiella sp 17.2 12.0 16.8 17.7

6 6 6 6

0.8 1.3 1.3 0.8

Staphylococcus epidermidis 25.7 18.7 27.2 27.5

6 6 6 6

2.2 1.5 1.8 3.1

MRSA 21.7 14.3 19.7 20.3

6 6 6 6

2.7 1.6 1.9 2.1

DA, Daptomycin; ME, meropenem; MRSA, methicillin-resistant Staphylococcus aureus; V, voriconazole; VRE, vancomycin-resistant Enterococcus faecalis.

antibiotic irrigations and dressings have been relatively unsuccessful for prevention of deep wound infections after vascular procedures.21,22 The composition of antibiotic beads for use against organisms found in abdominal vascular graft infections has not been previously described. Although there is increasing evidence of the efficacy of dissolvable bone cement, such as calcium sulfate,23 we prefer permanent bone cement with PMMA. We believe that the residue from the bioabsorbable cement may prevent adhesion of a pedicled flap to the graft or aortic stump. In addition, antibiotic elution proceeds faster than bead absorption so that the residue could theoretically provide a nidus for reinfection. The disadvantage of PMMA beads is that the beads must be removed. In clinical practice, this is somewhat less relevant because we prefer serial débridement and inspection of the infected area. We typically resect and replace infected abdominal aortic grafts, and we envision a prophylactic role for antibiotic beads to prevent reinfection of new in situ grafts or the aortic stump. The abdomen is temporarily closed with a vacuum-assisted dressing. Most antibiotics show decreased elution from PMMA cement after 2 days.24 Antibiotic beads are removed before coverage of the graft or aortic stump with an omental flap. In our experiments, 10% meropenem and 2.5% daptomycin PMMA beads showed excellent in vitro activity against bacteria commonly found in abdominal vascular graft infection. We were surprised that meropenem provided such strong activity against VRE and MRSA. We attributed this to our use of E. faecalis instead of E. faecium for the VRE cultures.25 We cannot explain the strong activity of meropenem against MRSA. Although our experiments suggested that meropenem alone provided broad-spectrum

coverage, we recommend adding daptomycin for drugresistant, gram-positive organisms. On its own, tobramycin had good activity against Klebsiella and S. epidermidis. Together, daptomycin and tobramycin beads showed moderate broad-spectrum activity and would be an acceptable alternative to meropenem and daptomycin. However, tobramycin decreased the efficacy of meropenem with every bacterium tested. Whereas other authors reported enhanced activity against Pseudomonas with combined meropenem and tobramycin,26 it is unclear if there are unfavorable drug interactions only when mixed in PMMA, if these effects persist in vivo when administered systemically, or if the reduced efficacy occurs only against certain bacterial strains. We postulate that tobramycin interferes with meropenem activity in PMMA and should not be used together until additional studies are performed. Increasing the ratio of antibiotics to PMMA made it increasingly difficult to mix and to set the cement. The 10% meropenem and 2.5% daptomycin with or without 1% voriconazole in PMMA formulation was easy to handle and to form into beads. In clinical practice, these beads can be strung onto suture and wrapped around contaminated abdominal prosthetic vascular grafts, such as in the example in Fig 3. We do not recommend routine use of antibiotic beads in clean cases. On the basis of studies in extremity vascular graft infection, local delivery of highconcentration antibiotics may facilitate graft sterilization in contaminated fields. We postulate that there may also be a prophylactic role for antibiotic beads in abdominal trauma cases with bowel injury and requiring vascular grafts. In patients with enteric graft contamination, the addition of an antifungal agent, such as voriconazole, should be considered. In vivo efficacy requires additional study.

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Fig 3. Intraoperative picture of antibiotic-laden polymethyl methacrylate (PMMA) beads around a new aortobi-iliac Dacron graft after resection of previous infected prosthetic vascular graft.

CONCLUSIONS Antibiotic PMMA beads containing 10% meropenem with 2.5% daptomycin had excellent in vitro activity against typical bacteria found in abdominal vascular graft infections. Tobramycin reduced the efficacy of meropenem. The addition of antibiotic beads may be a useful adjunct in managing such cases. Further studies are required to determine efficacy in clinical practice. The authors thank Ali Azizzadeh, MD, Anthony Estrera, MD, and Charles C. Miller III, PhD, for guidance and article review; Chris Akers and Troy Brown for assistance with manuscript preparation; and Shayan Izaddoost, MD, PhD, for assistance in conception and bead formulation. AUTHOR CONTRIBUTIONS Conception and design: KC, FK, HKS, MS, SL, BG, TK Analysis and interpretation: KC, FK, HKS Data collection: FK, SL Writing the article: KC Critical revision of the article: KC, MS, TK Final approval of the article: KC, TK, HJS Statistical analysis: FK, HKS Obtained funding: Not applicable Overall responsibility: KC REFERENCES 1. Reilly LM, Altman H, Lusby RJ, Kersh RA, Ehrenfeld WK, Stoney RJ. Late results following surgical management of vascular graft infection. J Vasc Surg 1984;1:36-44. 2. Clagett GP, Bowers BL, Lopez-Viego MA, Rossi MB, Valentine RJ, Myers SI, et al. Creation of a neo-aortoiliac system from lower extremity deep and superficial veins. Ann Surg 1993;218:239-48. 3. Snyder SO, Wheeler JR, Gregory RT, Gayle RG, Zirkle PK. Freshly harvested cadaveric venous homografts as arterial conduits in infected fields. Surgery 1987;101:283-91.

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Submitted Jan 27, 2014; accepted Mar 7, 2014.