Prevention of perioperative vascular prosthetic infection with a novel triple antimicrobial- bonded arterial graft Ibrahim Aboshady, MD, Issam Raad, MD,

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Prevention of perioperative vascular prosthetic infection with a novel triple antimicrobial- bonded arterial graft Ibrahim Aboshady, MD, Issam Raad, MD, Deborah Vela, MD, Mohamed Hassan, MD, Yara Aboshady, BS, Hazim J. Safi, MD, L. Maximilian Buja, MD, Kamal G. Khalil, MD Journal of Vascular Surgery Volume 64, Issue 6, Pages 1805-1814 (December 2016) DOI: 10.1016/j.jvs.2015.09.061 Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 1 Flow chart of the pilot phase of the study. AM, Antimicrobials; Bact, bacteria; Gr, graft; IV, intravenous. Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 2 After repetitive sequence-based polymerase chain reaction tests, the dendrogram and virtual banding patterns showed that the inoculated Staphylococcus aureus (SA) strain (ATCC 29213) recovered from the specimens was the same as the strain in the initial inoculum. Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 3 Ex vivo aortic graft from one pig (group 3) that received a bonded graft with the triple antimicrobial agents. The graft shows no macroscopic signs of infection. Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 4 Grafted infrarenal aorta of pig 2 (group 2, unbonded grafts). A, Movat stain showing a cross-section of the aorta containing a Dacron graft (g). B, Higher magnification, hematoxylin and eosin stain of the inset area marked in (A), showing a region of the aortic wall (a) where the Dacron graft (g) was attached. A mixture of acute and lytic thrombus (*) covers the luminal surface of the graft. Neutrophilic infiltration is present within the thrombus and in the media of the adjacent aortic wall (white arrows). A circular space left by a removed suture can be seen at the aorta-graft junction (black arrow). C, Higher magnification Gram stain of the inset in (A), showing large clusters of bacterial colonies (stained dark blue) within the thrombus. Bar = 2 mm in (A), 1 mm in (B), and 100 μm in (C) (original magnification ×1, ×4, and ×40, respectively). Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 5 Histologic sample from pig 3 (group 2, unbonded grafts). A, The luminal surface of the Dacron graft (g) is covered by a thick layer of thrombus (t) seeded with abundant bacterial colonies (arrows). B, Higher magnification of the inset in (A) showing large clusters of bacterial colonies (stained blue) within the graft and in the adjacent thrombus. Stains: hematoxylin and eosin in (A), Gram stain in (B). Bar = 1 mm in (A) and 100 μm in (B) (original magnification ×4 and ×40, respectively). Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 6 Histogram of microbiologic results. Samples from pigs 2 and 3 (group 2, unbonded grafts) showed significantly greater bacterial growth than samples from pig 1 (group 1, control), pig 4 (group 2, unbonded grafts), and pigs 5 to 9 (group 3, bonded grafts). CFU, Colony-forming unit. Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 7 Aortic tissue sample from the control pig in group 1. A, Movat stain of a cross-section of a portion of the aortic defect repaired with a Dacron graft (arrow). The site of graft implantation shows disruption of the normal arterial wall architecture. The defect was filled by myofibrotic tissue (*). A foreign-body granulomatous response surrounded the graft. B, Gram stain of a portion of the graft (g) and adjacent myofibrotic tissue (mf) shows no bacterial growth. Bar = 2 mm in (A) and 100 μm in (B) (original magnification ×1 and ×40, respectively). Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 8 Photomicrographs of aortic tissue samples from a pig in group 3. A, Movat stain of a cross-section of a portion of infrarenal aorta repaired with a Dacron graft (arrows). The graft fragments are surrounded by marked foreign-body granulomatous inflammation (white asterisks); myofibrotic tissue growth filled the defect on the luminal side (black asterisks). The adventitial layer (a) displayed chronic inflammation and thickening, with marked collagen deposition. B, Gram stain of a portion of the graft (g) and adjacent chronic inflammation (c) revealed no bacteria. Bar = 2 mm in (A) and 100 μm in (B) (original magnification ×1 and ×40, respectively). Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions

Fig 9 Inflammatory response. A and B, Group two, unbonded graft: neutrophilic infiltration in the media and adventitia, respectively, in a region near the graft in pig 2. C, Group two, unbonded graft: abundant collection of degenerating neutrophils (n) within the thrombus overlying the graft (g) in pig 3. Note a large cluster of bacterial colonies (b). D-F, Inflammatory infiltrates surrounding the graft at 8 weeks in pigs from groups 2, 1, and 3, respectively, are composed mostly of neutrophils (n), mononuclear chronic inflammatory cells (c), and multinucleated giant cells (arrows). F, Chronically inflamed advanced granulation tissue. Hematoxylin and eosin stain. Bar = 200 μm in (B) (original magnification ×20). Journal of Vascular Surgery 2016 64, 1805-1814DOI: (10.1016/j.jvs.2015.09.061) Copyright © 2015 Society for Vascular Surgery Terms and Conditions