A pilot study of a triple antimicrobial-bonded Dacron graft for the prevention of aortic graft infection

A pilot study of a triple antimicrobial-bonded Dacron graft for the prevention of aortic graft infection Ibrahim Aboshady, MD,a,b Issam Raad, MD,b,c,d Aamir S. Shah, MD,b,e Deborah Vela, MD,a,b Tanya Dvorak, PhD,c Hazim J. Safi, MD,b,d,e L. Maximilian Buja, MD,a,b and Kamal G. Khalil, MD,b,d,e Houston, Tex Objective: Perioperative infection of an aortic graft is one of the most devastating complications of vascular surgery, with a mortality rate of 10% to 30%. The rate of amputation of the lower limbs is generally >25%, depending on the graft material, the location of the graft and infection, and the bacterial virulence. In vitro studies suggest that an antibioticimpregnated graft may help prevent perioperative graft infection. In a pilot animal study, we tested a locally developed technique of bonding Dacron aortic grafts with three antimicrobial agents to evaluate the ensuing synergistic preventive effect on direct perioperative bacterial contamination. Methods: We surgically implanted a 6-mm vascular knitted Dacron graft in the infrarenal abdominal aorta of six Sinclair miniature pigs. Two pigs received unbonded, uninoculated grafts; two received unbonded, inoculated grafts; and two received inoculated grafts that were bonded with chlorhexidine, rifampin, and minocycline. Before implantation, the two bonded grafts and the two unbonded grafts were immersed for 15 minutes in a 2-mL bacterial solution containing 1 to 2 ⴛ 107 colony-forming units (CFU)/mL of Staphylococcus aureus (ATCC 29213). Two weeks after graft implantation, the pigs were euthanized, and the grafts were surgically excised for clinical, microbiologic, and histopathologic study. Results: The two bonded grafts treated with S aureus showed no bacterial growth upon explant, whereas the two unbonded grafts treated with S aureus had high bacterial counts (6.25 ⴛ 106 and 1.38 ⴛ 107 CFU/graft). The two control grafts (unbonded and untreated) showed bacterial growth (1.8 ⴛ 103 and 7.27 ⴛ 103 CFU/graft) that presumably reflected direct, accidental perioperative bacterial contamination; S cohnii ssp urealyticus and S chromogenes, but not S aureus, were isolated. The histopathologic and clinical data confirmed the microbiologic findings. Only pigs that received unbonded grafts showed histopathologic evidence of a perigraft abscess. Conclusions: Our results suggest that bonding aortic grafts with this triple antimicrobial combination is a promising method of reducing graft infection resulting from direct postoperative bacterial contamination for at least 2 weeks. Further studies are needed to explore the ability of this novel graft to combat one of the most feared complications in vascular surgery. ( J Vasc Surg 2012;56:794-801.) Clinical Relevance: This study introduces a novel bonded Dacron aortic graft for the prevention of perioperative aortic graft infection. The quantitative results of our experimental studies showed that the bonding of three antimicrobial agents (rifampin, minocycline, and chlorhexidine) to aortic grafts nearly prevented aortic graft infection by synergistically prolonging antistaphylococcal activity. Use of an antibiotic-bonded prosthetic graft with antistaphylococcal activity should be considered in patients who have arterial infections caused by Staphylococcus aureus. After the preventive effect of this graft and its safety have been further assessed, its use may be recommended for the in situ replacement of infected grafts and possibly for routine primary cases, especially in immunocompromised patients, patients with hostile abdomen, and patients undergoing redo procedures.

Infection of an aortic graft is a devastating perioperative complication of arterial surgery. Although the incidence of graft infection is low (0.2%-5%), particularly in the descending thoracic/thoracoabdominal aorta (0.5%1.9%), urgent attention is often required.1 Despite improvements in preventive measures, including atraumatic

and sterile surgical technique and routine antibiotic prophylaxis, graft infection has a high rate of mortality (10%-30%) and lower limb amputation (usually ⬎25%), depending on the anatomic location of the infected graft, the graft material, and the virulence of the bacteria.2,3

From the Texas Heart Institute at St. Luke’s Episcopal Hospital,a The University of Texas Health Science Center,b The University of Texas MD Anderson Cancer Center,c Baylor College of Medicine,d and Memorial Hermann Heart and Vascular Institute.e This study was supported by The University of Texas Health Science Center, Houston, Texas. Author conflict of interest: none. Portions of the data described in this manuscript were presented at the 2011 Scientific Sessions of the Transcatheter Cardiovascular Therapeutics (TCT), San Francisco, Calif, November 7-11, 2011.

Reprint requests: Ibrahim Aboshady, MD, Texas Heart Institute, 6700 Bertner Ave, Houston, TX 77030 (e-mail: [email protected]. edu). 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 © 2012 by the Society for Vascular Surgery. doi:10.1016/j.jvs.2012.02.008

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Table I. Microbiologic findings of cultured aortic grafts Group/pig

Graft

CFU/graft

Organism cultured

Pig 1 Pig 2

Unbonded, uninoculated Unbonded, uninoculated

1.8 ⫻ 103 7.27 ⫻ 103

Staphylococcus cohnii ssp urealyticus Staphylococcus chromogenes

Pig 3 Pig 4

Unbonded, inoculated Unbonded, inoculated

6.25 ⫻ 106 1.38 ⫻ 107

Staphylococcus aureus Staphylococcus aureus

Pig 5 Pig 6

Bonded, inoculated Bonded, inoculated

A B C

0 0

None None

CFU, Colony-forming units.

The most common pathogenic organism to cause early (⬍4 months) graft infections is Staphylococcus aureus (32%), whereas S epidermidis causes most late (⬎4 months) vascular graft infections.1 Recently, the incidence of vascular infections with methicillin-resistant S aureus (MRSA) has increased considerably (2%), especially in residents of long-term facilities (23%-49%). MRSA infections are more invasive and are accompanied by septicemia.4 Antibiotic-impregnated grafts may be useful in preventing and decreasing the severity of vascular infections. Rifampin is reportedly effective because it has a high affinity for collagen- and gelatin-coated grafts, resulting in stable bonding.5 In clinical studies, rifampin-bonded grafts have shown promising results against microorganisms of low virulence, such as S epidermidis, but have been less effective against virulent bacteria, including MRSA.6-9 Our group has shown that the impregnation of central venous catheters (CVCs) with minocycline and rifampin is efficacious and safe in reducing catheter-related bloodstream infections in cancer patients and in patients who are critically ill.10-12 Moreover, our recent studies have shown that CVCs impregnated with a triple combination of chlorhexidine, rifampin, and minocycline (M/RCHX) enhance activity against MRSA and broaden the anti-biofilm and antiadherence activities to include Pseudomonas aeruginosa and Candida organisms.13,14 In the current study, we examined the benefits of simultaneously bonding M/RCHX to aortic grafts to assess the consequential effect on reducing the risk of perioperative aortic graft infection by direct operative bacterial contamination in an animal model. METHODS These animal studies were performed according to the guidelines set by the Animal Welfare Committee at The University of Texas Health Science Center, Houston, Texas. Animal model. The study comprised six Sinclair (Sinclair Bio Resources, Columbia, Mo) miniature pigs (weight, 30-35 kg) that were divided into three groups (Table I). Two pigs received unbonded, uninoculated grafts (group A; controls); two pigs received unbonded, inoculated grafts (group B); and two pigs received inoculated grafts bonded with M/RCHX (group C). Before implantation, groups B and C pigs received grafts that were

immersed for 15 minutes in 2-mL bacterial solution containing 1 to 2 ⫻ 107 colony-forming units (CFU)/mL of S aureus (ATCC 29213). The pigs did not receive preoperative or postoperative antibiotics or blood transfusions. Ketoprofen and morphine were administered for postoperative analgesia. The pigs were placed in individual cages, examined daily, and monitored for symptoms of systemic toxicity, which could be caused by the graft or the antimicrobials. The pigs were euthanized 2 weeks after graft implantation, and the grafts were surgically excised for study. Surgical protocol. Animals were anesthetized initially by an intramuscular injection of a 1-mL mixture of zolamine (5 mg/mL), butorphanol (1 mg/mL), ketamine (10 mg/mL), and xylazine (2 mg/mL), followed by isoflurane (0.5%-3%) throughout the operation. For analgesia, we used 0.25% bupivacaine (⬍1 mL/kg) before the incision and buprenorphine (0.01-0.02 mg/kg, subcutaneously or intramuscularly) postoperatively. A fentanyl transdermal patch (75-100 ␮g/kg/h) was placed immediately after surgery. Strict aseptic conditions and techniques were used for all procedures, including the use of sterile protective covering, instruments, and drapes. The abdomen and left flank were clipped and surgically prepared. The infrarenal aorta and its bifurcation were exposed through a retroperitoneal incision. The infrarenal aorta was carefully dissected from the surrounding tissues by sharp, blunt dissection of the periaortic tissues. The lumbar arterial branches were spared to avoid spinal cord ischemia. Heparin (100-300 U/kg) was administered intravenously, and the pigs were maintained on heparin (100-200 U/kg every 45 minutes) during the operation. The abdominal aorta was clamped with atraumatic vascular clamps, and a 2.0- ⫻ 1.0-cm Dacron roof patch was sutured to a 2.0-cm aortotomy with 5-0 double-ended polypropylene vascular suture to create a roof-patch angioplasty in a standard fashion. The total anastomotic time was ⬍20 minutes per pig. The vascular clamps were released, and the suture line was observed for leakage. The wound was irrigated with sterile saline, the retractors were removed, and the wound was closed in layers in the usual fashion. The skin was closed with subcuticular sutures of 4-0 Vicryl (Monosof, Syneture, Norwalk, Conn) to help

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prevent infection. The surface of the closed incision and the entire surgical area were cleaned and sprayed with 2% chlorhexidine. The pigs were allowed to recover from anesthesia before they were returned to their cages. Antiplatelet therapy (10 mg/kg) was administered daily for 1 week after surgery. Graft preparation. Dacron vascular grafts (Terumo Cardiovascular Systems Corp, Ann Arbor, Mich) were impregnated with M/RCHX by using a modified proprietary method.15 This solvent-based impregnation method involves precoating the grafts with a chlorhexidine solution and then soaking them in a solvent solution of minocycline and rifampin. The doses of rifampin (30 mg/mL) and minocycline (15 mg/mL) were selected to ensure that the largest graft would contain less than the normal daily dose of both drugs used clinically. Uncoated grafts were used as controls to show the ability of the bacterial challenge to establish infection. Bacterial challenge preparation. To examine the efficacy of the M/RCHX vascular grafts, impregnated (group C) and uncoated grafts (group B) were inoculated with S aureus (ATCC strain 29213). Fresh challenge bacteria were streaked for isolation from frozen stock onto trypticase soy agar and 5% sheep’s blood and incubated overnight. A uniform colony was inoculated into Muller Hinton II broth (25 mL) and incubated overnight at 37°C. The resulting inoculum was diluted to a 0.5 McFarland standard and further diluted in saline to a concentration of 1 ⫻ 106 CFU/mL for use in the bacterial challenge. Antibioticcoated and uncoated grafts were soaked in 25 mL of the S aureus inoculum for 20 minutes before surgical implantation. Graft excision. Pigs were given a lethal dose of Beuthanasia (0.22 mL/kg; Merck, Summit, NJ) at the time of euthanasia. The same sterile technique that was used for graft implantation was used to reopen the retroperitoneal incision. The grafts and attached segments of aortic wall were excised for microbiologic and histopathologic studies. Microbiologic assays. Samples of vascular tissue surrounding the graft were homogenized for 2 minutes in 0.9% sterile saline (5 mL) by using a Stomacher Lab Blender (Tekmar Corp, Cincinnati, Ohio). The resulting homogenate was quantitatively cultured to determine the efficacy of the antimicrobial grafts. All samples were cultured in triplicate on trypticase soy agar with 5% sheep’s blood following the Clinical and Laboratory Standards Institute (formerly, National Committee for Clinical Laboratory Standards) procedures. Microbes grown from the homogenate were identified by using the API Staph Identification kit (bioMérieux, Marcy l’Etoile, France). Samples identified as S aureus were further typed genetically as described below for molecular comparison. Samples from other areas with signs of infection (abscesses) were cultured and typed for comparison with the inoculated strain. Genetic strain typing from collected S aureus isolates. Isolates identified as S aureus that were recovered from inoculated pigs were genetically typed by using repetitive sequence-based polymerase chain reaction (rep-PCR)

by using the DiversiLab rep-PCR strain typing system (bioMérieux), according to the manufacturer’s instructions. Strains were compared with the inoculated strain (ATCC 29213) to determine whether the recovered S aureus isolates were the same as the inoculated ATCC strain. Histopathologic studies. Samples from the site where the graft was attached to the aorta (range, 1-2.5 cm) were sliced every 3 to 4 mm and were formalin-fixed and paraffin-embedded. Sections (5-␮m thick) were stained with hematoxylin and eosin, Movat pentachrome, and Gram stain. Histopathologic and microbiologic examinations were performed by using Olympus MicroSuite software connected to an Olympus BX61 microscope (Olympus America, Melville, NY). For each 5-␮m section of the paraffin-embedded specimens, histopathologic sections were examined for signs of inflammation in two different low-power (original magnification, ⫻4) fields of the vascular wall. Statistical analysis. The number of positive cultureinfected grafts observed in the three groups of pigs was compared by using the t-test. A value of P ⬍ .05 was considered statistically significant. RESULTS The six pigs survived the surgery and completed the protocol. For at least 7 days after graft implantation, all pigs showed reduced food intake and altered behavior. Signs of postoperative infection (eg, fever, malaise, increased total leukocyte count) were seen in the group B pigs (unbonded, inoculated grafts; Table II). One pig in group C (bonded, inoculated grafts) also showed clinical signs of infection due to the formation of a deep wound abscess. Body temperatures were elevated in group B pigs for 1 week after graft implantation but not in group A or group C pigs (Table II). The pigs that received bacterial inoculations showed moderate leukocytosis with low serum albumin levels for 2 weeks after graft implantation. Operative signs of infection were seen on the excised grafts of group B pigs, such as perigraft exudates, abscess, and necrotic tissue, which were not seen in the other pigs. Microbiologic assays were performed on matched specimens of aortic tissue samples surrounding the aortic grafts. Quantification of the culture results showed significantly lower bacterial counts for tissues from group A (control) pigs (1.8 ⫻ 103 and 7.27 ⫻ 103 CFU/graft) than for specimens from group B (S aureus–treated, unbonded grafts) pigs (6.25 ⫻ 106 and 1.38 ⫻ 107 CFU/graft; P ⬍ .001; Table I). Specimens obtained from group C (S aureus–treated, bonded grafts) pigs showed no bacterial growth. The organisms isolated from the grafts of group A (control) pigs were S cohnii ssp urealyticus and S chromogenes, but not S aureus, indicating that the bacterial growth had resulted from direct accidental perioperative bacterial contamination of the unbonded, uninoculated grafts. Thus, the synergistic effect of the bonded graft in group C pigs may have prevented direct bacterial contamination with non-S aureus bacteria.

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Table II. Postoperative clinical and laboratory data after graft implantation in the abdominal aorta Group A Variable Fever, °C Heart rate, beats/min SPO2, mm Hg PCO2, mm Hg TLC Hematocrit Hemoglobin Platelet count Cholesterol ALT ALP Glucose Albumin Total protein

Group B

Group C

Pig 1

Pig 2

Pig 3

Pig 4

Pig 5

Pig 6

No 108 N 24 9 (PNL) 2 N 2 N 2 2 2 N 2

No 126 N 13 8.4 (PNL) N 2 N N 1 2 2 N N

Yes (38.9) 99 99 27 14.5 (PNL, Ly) N N N High 1 2 2 N N

Yes (38.3) 115 94 20 13.9 (PNL, Ly, Eo) 2 N 2 High 1 2 2 1 2

No 92 96 23 8 (PNL, Ly) N N N High N 2 2 1 2

No 78 93 36 6.25 (PNL, Ly) 2 N N High N 2 2 N 2

ALP, Alkaline phosphatase; ALT, alanine aminotransferase; Eo, eosinophils; Ly, lymphocytes; N, within normal range; PCO2, partial pressure of carbon dioxide; PNL, neutrophils; SPO2, saturation of peripheral oxygen; TLC, total leukocyte count.

Fig 1. The dendrogram and virtual banding patterns after repetitive sequence-based polymerase chain reaction (rep-PCR) showed that the inoculated Staphylococcus aureus (SA) strain (ATCC 29213) recovered from the specimens was the same as the strain in the initial inoculum.

Aortic graft specimens from both group B pigs were infected with the same S aureus strain that was inoculated onto the uncoated graft. For group C pigs, no staphylococci were recovered from the culture of the M/RCHXcoated Dacron grafts inoculated with S aureus or from the surrounding tissues. However, S aureus cultured from a deep subcutaneous wound abscess of pig 5 in group C was the same strain as the inoculated strain. The dendrogram and virtual banding patterns in Fig 1 show that all S aureus isolates recovered from the inoculated pigs are the same strain used in the initial inoculum. Results from the histopathologic analysis were concordant with the microbiologic findings. Table III summarizes

the main histopathologic outcomes for all samples. For all three groups, microscopic examination of aortic specimens adjacent to the graft implant showed marked adventitial thickening, consisting predominantly of fibrosis and chronic inflammation. Multinucleated giant cells were also present in all samples, often associated with the presence of minute graft remnants. Specimens from the medial layer of all groups displayed sites, at irregular intervals, of mild-tomoderate disruption of the lamellar structure, consisting mostly of smooth muscle cell loss and increased matrix deposition. This finding appeared to be related to the placement of sutures. The intimal layer was absent from all samples at the site of graft implantation.

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Table III. Comparison of histologic outcomes of perigraft aortic tissues Group A

Media Adventitia thickening Inflammation Abscess Neutrophils Macrophages Lymphocytes MNG Gram stain

Group B

Group C

Pig 1

Pig 2

Pig 3

Pig 4

Pig 5

Pig 6

Focal fibrosis Yes Chronic No ⫾ ⫹⫹⫹ ⫹⫹ ⫹ ?/negative

Focal fibrosis Yes Chronic No ⫹ ⫹⫹⫹ ⫹⫹ ⫹ Gram ⫹ cocci

Focal fibrosis Yes Mixed (acute foci) Yes ⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ Gram ⫹ cocci

Focal fibrosis Yes Mixed (acute foci) Yes ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ Abundant Gram ⫹ cocci

Focal fibrosis Yes Chronic No ⫾ ⫹⫹⫹ ⫹⫹ ⫹ Negative

Focal fibrosis Yes Chronic No ⫾ ⫹⫹⫹ ⫹⫹ ⫹ Negative

?, Questionable; MNG, multinucleated giant cells.

Important differences were observed among the three groups, mostly in the inflammatory infiltrate in the aortic wall at the site of graft implantation (Table III). Specimens from group A and group C predominantly showed chronic inflammatory cell infiltrates (macrophages and lymphocytes). Gram stain yielded negative results in these two groups, except for pig 4 in group A, which showed a few gram-positive cocci near the graft implantation site (Fig 2), with minimal to mild neutrophilic infiltration. In contrast, specimens from pigs that received unbonded grafts with bacterial inoculations (group B) showed abundant neutrophilic infiltration with perigraft abscess formation (Figs 2-4 and Table III). In this same group, Gram stain revealed the presence of gram-positive cocci in the aortic wall, near the graft (Figs 2 and 3). In the specimen from pig 4, these gram-positive cocci were quite numerous and arranged in multiple clusters (Fig 3). At the time of euthanasia, an anterior abdominal abscess was observed in pig 4 in group B pig and in pig 5 in group C, confirming that the inoculum was pathogenic. DISCUSSION Periprosthetic arterial graft infection is a rare but devastating complication of arterial reconstructive surgery, with a high incidence of mortality and limb loss.1 Few clinical studies have described the results of managing prosthetic graft infection after descending thoracic/thoracoabdominal aortic aneurysmectomy.16-18 Nevertheless, in most series, operative mortality has been 15% to 30%, and limb loss has been ⬃30%. Hence, it is extremely desirable to use every conceivable strategy to minimize or prevent bacterial seeding and subsequent graft infection. In this study, we have introduced a novel bonded aortic graft impregnated with three antimicrobial agents— chlorhexidine, rifampin, and minocycline—and have examined its ability to prevent and reduce perioperative aortic graft infections with the most common causative organism, S aureus. Our results showed that the synergistic effect of this antimicrobial combination helps reduce and prevent direct perioperative bacterial contamination. We used Sinclair miniature pigs in this study because this strain is free of many common infectious diseases and is

Fig 2. Representative images of aortic specimens and adjacent graft material stained with (I) hematoxylin and eosin, (II) Movat pentachrome, and Gram stain. A and B, Images from pig 2 (group A) show a small cluster of gram-positive cocci and mild neutrophilic infiltration near the graft implantation site. C and D, Images from pig 4 (group B) show multiple clusters of gram-positive cocci (arrows) and abscess formation (asterisk). E and F, Images from pig 6 (group C) show negative Gram staining. Bar ⫽ 200 ␮m in B and F and 1 mm in D.

often used in cardiovascular research, especially in studies of aortic disease.19 Although dog models have been used to study perioperative graft infections,20,21 using large animals is costly and time-consuming because general anesthesia with tracheal intubation and large experimental facilities are required.22 To minimize these expenses, we implanted the grafts in the abdominal aorta rather than in the thoracic aorta and used roof-patch angioplasty rather than a tube-

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Fig 3. High-power magnification image (bar ⫽ 20 ␮m) shows dense clusters of gram-positive cocci in the perigraft tissue of pig 4 (group B).

Fig 4. Images of abscess formation in specimens from group B pigs. Low-power magnification of aortic specimens are shown from (A) pig 3 and (B) pig 4. Corresponding high-power magnification images of aortic specimens are shown from (C) pig 3 and (D) pig 4. C, Neutrophilic infiltration is seen surrounding a core of graft and gram-positive bacterial remnants (br). D, An accumulation of mostly dead neutrophils, fibrin, and loose connective tissue is shown. A large cluster of gram-positive cocci is seen in the upper left corner (bc). Bar ⫽ 2 mm in A and 100 ␮m in C.

graft replacement to simplify the technique and reduce operative and postoperative morbidities. Our group has previously evaluated the efficacy of long-term, nontunneled silicone CVCs impregnated with minocycline and rifampin (M-R) in reducing catheterrelated bloodstream infections in cancer patients.23 In this prospective, randomized, double-blind trial, 17 catheterrelated infections were detected in the bloodstream of 174 patients with a malignancy. Three of the 17 infections were associated with the use of M-R catheters, and 14 were associated with the nonimpregnated CVCs; the rate of

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catheter-related bloodstream infection was 0.25 and 1.28/ 1000 catheter-days, respectively (P ⱕ .003). The etiologic agent for most infections was gram-positive cocci. No allergic reactions were associated with the M-R catheters. Also, in prospective, randomized clinical studies and metaanalyses, CVCs impregnated with M-R were safe and reduced catheter-related bloodstream infections in cancer patients and in critically ill patients.13,14,24-26 Because of these encouraging results, we assessed the synergistic effect of M/RCHX in reducing perioperative infections associated with aortic graft implantation, especially given that the primary etiologic agent is the same in both situations, and recent data have shown that the addition of chlorhexidine to M-R enhances antimicrobial activity against resistant bacteria and fungi.13,14 We recently showed that CVCs and peripherally inserted central catheters impregnated with M/RCHX completely inhibited biofilm colonization of MRSA, vancomycin-resistant Enterococcus faecium, P aeruginosa, and Candida spp in a manner superior to that of catheters treated with M-R and chlorhexidine (P ⱕ .003).27 Furthermore, M/RCHX– coated CVCs had significantly more effective and prolonged antimicrobial activity for up to 3 weeks against MRSA and P aeruginosa than uncoated CVCs or those coated with M-R or chlorhexidine/silver sulfadiazine (P ⬍ .0001). No side effects were reported.27 Although rifampin is well known to have excellent bactericidal activity against MRSA,28 its use as a single agent could result in the rapid development of resistance to this antibiotic. Rifampin has been widely used as part of a triple combination of antimicrobial agents, in which each agent works by a different mechanism of action to reduce the potential for developing resistance. The M-R combination has been used to successfully treat various infections, including staphylococcal infections.29 Moreover, infectionresistant grafts have been effective in combating perioperative infections of aortic grafts.30 In studies involving a quorum-sensing inhibitor or a sustained-release vancomycin sheet placed on a graft, communication between bacterial cells was prevented.31 Infection-resistant vascular grafts have considerable appeal. However, several important issues must be considered when antibiotic-loaded grafts are designed. It is essential that the bonding or sterilization process does not alter the chemical nature of the drugs or the substrate used as a carrier. Grafts containing silver and collagen are used more frequently in Europe20,32 but are less resistant to bacteremic infection with S aureus than most other pathogens that cause early perioperative graft infections.21 The use of silver compounds bonded to the sewing ring of prosthetic heart valves has been shown to significantly impair the ingrowth of tissue into the fabric and is therefore thought to predispose to subsequent valve dehiscence.33 Many groups have used minocycline to treat vascular graft infections because of the increased prevalence of S aureus infections; however, its use has been questioned because of the development of resistant S aureus

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strains.34,35 This issue is especially important in patients who undergo repeat arterial-grafting procedures after problems with healing of the implant site or who have medical conditions, such as diabetes or immunosuppression, that increase the risk of infection, including MRSA infection. In patients considered at higher risk for prosthetic implant infection, empiric use of an antibioticbonded vascular graft may be beneficial. Rifampin-soaked polyester grafts have been used successfully as in situ replacement grafts in clinical and experimental models of vascular graft infection caused by Staphylococcus spp, especially S epidermidis and S aureus.20 However, the antibiotic is promptly leached out—within minutes or hours— because the arterial blood rapidly leaches out the surface adherent chemical, hence the need for long-term stable bonding of the antibiotic within the graft fabric. Our study focused on early or perioperative graft infections. About 53% of infections occur within the first 30 days, and 70% occur in the first 60 days.36,37 Preventing perioperative infection of an aortic graft is obviously preferable to surgical treatment of an infected graft, especially in the case of late infections. An effective approach to preventing early graft infection is to provide antibiotic coverage during the early postoperative period that is strong enough to last through this crucial, vulnerable period. Our previous studies10,38 indicated that the M-R combination remains active for 6 weeks, which can be extended by adding chlorhexidine.13 Also, the doses used in this study appear to be sufficient, because no growth of bacteria was detected in pigs treated with bonded grafts. In a study in dogs, Koshiko et al39 showed that rifampingelatin grafts (implanted after soaking the graft in a 1-mg/mL rifampin solution) were not efficacious against virulent organisms such as S aureus; however, these researchers used a higher organism concentration (1 ⫻ 108 CFU/mL) than we did (2 mL of a bacterial solution containing 1 to 2 ⫻ 107 CFU/mL). One of the advantages of locally delivering antibiotics is the slow, continuous release of antibiotics from the graft. Among the different bonding techniques available, a glue has been used that allows antibiotic release over the duration of 1 to 2 months; however, the glue poses issues such as tissue toxicity and the lack of resorption by the body.40 The limitations of our study include a small sample size of six pigs; however, this was a pilot study, and additional experiments are in progress. In addition, our study focused on early graft infection rather than late graft infection; this was partly because the graft infection occurs early in 53% to 70% of infections.36,37 Future studies will assess the ability of our graft to prevent late graft infection with a follow-up period of ⬎2 weeks. Finally, the animals in our study did not receive preoperative antibiotics. This was done so that we could subject our hypothesis to the maximum testing challenge. Although perioperative antibiotics are widely used and may reduce the number and severity of graft infections, they do not provide complete protection from early graft infections.41

CONCLUSIONS Our results suggest that bonding of aortic grafts with three antimicrobial agents— chlorhexidine, minocycline, and rifampin—is a promising method of reducing direct postoperative bacterial contamination for at least 2 weeks. Further studies are needed to explore the ability of this novel graft to combat one of the most formidable complications in vascular surgery. AUTHOR CONTRIBUTIONS Conception and design: IA, HS, LB, KK Analysis and interpretation: IA, IR, HS, LB, KK Data collection: IA, AS, DV, TD, KK Writing the article: IA, IR, HS, LB, KK Critical revision of the article: IR, HS, LB, KK Final approval of the article: ALL Statistical analysis: IA Obtained funding: LB Overall responsibility: KK REFERENCES 1. Cronenwett JL, Johnston KW, Rutherford RB. Local complications: graft infection. In: Cronenwett JL, Johnston, KW, editors. Rutherford’s vascular surgery. Vol 7. Philadelphia: Saunders/Elsevier; 2010. p. 64361. 2. O’Hara PJ, Hertzer NR, Beven EG, Krajewski LP. Surgical management of infected abdominal aortic grafts: review of a 25-year experience. J Vasc Surg 1986;3:725-31. 3. Reilly L. Aortic graft infection: evolution in management. Cardiovasc Surg 2002;10:372-7. 4. Bandyk DF. Vascular surgical site infection: risk factors and preventive measures. Semin Vasc Surg 2008;21:119-23. 5. Chervu A, Moore WS, Chvapil M, Henderson T. Efficacy and duration of antistaphylococcal activity comparing three antibiotics bonded to Dacron vascular grafts with a collagen release system. J Vasc Surg 1991;13:897-901. 6. Bandyk DF, Novotney ML, Johnson BL, Back MR, Roth SR. Use of rifampin-soaked gelatin-sealed polyester grafts for in situ treatment of primary aortic and vascular prosthetic infections. J Surg Res 2001;95: 44-9. 7. Coggia M, Goëau-Brissonnière O, Leflon V, Nicolas MH, Pechère JC. Experimental treatment of vascular graft infection due to Staphylococcus epidermidis by in situ replacement with a rifampin-bonded polyester graft. Ann Vasc Surg 2001;15:421-9. 8. Goëau-Brissonnière O, Mercier F, Nicolas MH, Bacourt F, Coggia M, Lebrault C, et al. Treatment of vascular graft infection by in situ replacement with a rifampin-bonded gelatin-sealed Dacron graft. J Vasc Surg 1994;19:739-41. 9. Hayes PD, Nasim A, London NJ, Sayers RD, Barrie WW, Bell PR, et al. In situ replacement of infected aortic grafts with rifampicin-bonded prostheses: the Leicester experience (1992 to 1998). J Vasc Surg 1999;30:92-8. 10. Hanna H, Bahna P, Reitzel R, Dvorak T, Chaiban G, Hachem R, et al. Comparative in vitro efficacies and antimicrobial durabilities of novel antimicrobial central venous catheters. Antimicrob Agents Chemother 2006;50:3283-8. 11. Raad I, Darouiche R, Hachem R, Mansouri M, Bodey GP. The broadspectrum activity and efficacy of catheters coated with minocycline and rifampin. J Infect Dis 1996;173:418-24. 12. Chatzinikolaou I, Hanna H, Darouiche R, Samonis G, Tarrand J, Raad I. Prospective study of the value of quantitative culture of organisms from blood collected through central venous catheters in differentiating between contamination and bloodstream infection. J Clin Microbiol 2006;44:1834-5.

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Submitted Nov 9, 2011; accepted Feb 5, 2012.