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• W2022183438 abstract "Infection of a prosthetic graft is the most serious complication of vascular reconstructive surgery. Although graft sepsis is uncommon, it significantly increases the risk of loss of life or limb.1Szilagvi DE Smith RF Elliot JP Vrandecic MP. Infection in arterial reconstruction with synthetic grafts.Ann Surg. 1972; 176: 321-333Crossref PubMed Scopus (644) Google Scholar, 2Goldstone J Moore WS. Infection in vascular prostheses.Am J Surg. 1974; 118: 225-233Abstract Full Text PDF Scopus (292) Google Scholar, 3Bunt TJ. Synthetic vascular graft infections. I. Graft infections.Surgery. 1983; 93: 733-746PubMed Google Scholar Therefore, the prevention of prosthetic graft infection is an important concern of vascular surgeons. The three main postulated mechanisms of vascular graft infection are operative contamination, mechanical erosion of intestine, and bacteremia.3Bunt TJ. Synthetic vascular graft infections. I. Graft infections.Surgery. 1983; 93: 733-746PubMed Google Scholar Strict aseptic techniques and prophylactic antibiotics should obviate operative contamination and separation of the intestine from the graft can be achieved surgically, but the prevention of bacteremic infection would require methods applicable throughout the period that grafts are vulnerable to colonization by circulating bacteria. Although proven bacteremic graft infection in humans has been uncommon, canine experiments suggest that grafts remain vulnerable to bacteremia for months after insertion and that resistance is related to the rate of neointimal formation, with healed grafts resistant, and 95% of unhealed grafts infected after a bacteremic challenge.4Malone JM Moore WS Campagna G Bean B. Bacteremic infectability of vascular grafts: The influence of pseudointimal integrity and duration of graft function.Surgery. 1975; 78: 211-216PubMed Google Scholar, 5Moore WS Malone JM Keown K. Prosthetic arterial graft material: Influence of neointimal healing and bacteremic infectability.Arch Surg. 1980; 115: 1379-1383Crossref PubMed Scopus (62) Google Scholar We report the effects of bacteremia studied in a new rabbit aortic graft model, which challenges this conventional point of view. Adult New Zealand white rabbits weighing 4 to 5 kg were used for aortic grafting. Animal care complied with the “Principles of Laboratory Animal Care” and the “Guide for the Care and the Use of Laboratory Animals” (NIH Publication No. 80-23, revised 1978). Anesthesia was induced with a regimen of intramuscular xylazine (Rompun, Haver-Lockhart) (5 mg/kg) and ketamine (Ketalar, Parke-Davis) (50 mg/kg). During operation, an average of 200 ml of Ringer's solution was infused via a 22-gauge cannula inserted in a marginal ear vein. A midline abdominal incision was performed and the infrarenal aorta exposed. Heparin (500 units) was given intravenously before aortic cross-clamping and an additional 300 units was injected directly into the distal aorta after clamping. The distal clamp was placed just above the aortic bifurcation and the aorta was divided. Retraction of the ends facilitated graft placement without resection of native vessel. Two different graft materials, polytetrafluoroethylene (PTFE) (Impra, Impra Inc.) and microporous silicone rubber (Replamineform, Interpore Inc.)6Hiratzka LF Goeken JA White RA Wright CB In vivo comparison of Replamineform, Silastic and Bioelectric Polyurethane arterial grafts.Arch Surg. 1979; 114: 698-702Crossref PubMed Scopus (15) Google Scholar all with an inner diameter of 3 mm and a length of 10 mm and were inserted as interposition grafts with continuous 7-0 polypropylene sutures (Prolene, Ethicon Inc.). Immediate patency of the graft was assessed by palpation and visualization of pulsatile flow in the distal vessel. Cross-clamp time ranged from 15 to 35 minutes. The abdominal wall was closed in two layers with 3-0 polyglycolic acid sutures (Dexon, Davis & Geck). No antibiotics were used and routine postoperative care was provided. A strain of methicillin-resistant Staphylococcus aureus, obtained from a human wound infection, was used for the bacteremia experiments. This was provided by the Division of Infectious Diseases, Harbor-UCLA Medical Center, where the strain had proved pathogenic in a rabbit endocarditis model. Supplies were maintained in slush culture at room temperature. For inoculation, a loopful of the culture was added to 5 ml of brain heart infusion (BHI, Difco Laboratories, Inc.) and incubated overnight at 37° C. This had previously been shown to produce a bacterial concentration of around 108 per milliliter of culture medium by the following day. The overnight culture was administered either undiluted or after serial dilutions with BHI down to 102 organisms per milliliter of solution. Quantitation of the actual inoculum given was by the colony counts method.7Meynell GG Meynell E. Theory and practice in experimental bacteriology.2nd ed. : C U Press, Cambridge1970Google Scholar Bacteremia was induced via a marginal ear vein either at anastomotic completion or from 1 to 14 days postoperatively. Two additional groups of animals received control infusions of 1 ml of BHI only, either at anastomotic completion or 2 weeks after graft insertion. Grafts were removed 7 days after bacterial or control inoculation, although any animal that had a significant postoperative complication was killed immediately for graft removal. Under sterile conditions and with the same anesthetic agents used for graft insertion, a paramedian incision was made, avoiding the previous wound. The posterior peritoneum was opened and patency of the graft assessed by palpation and the presence of downbleeding when the aorta distal to the graft was divided. The rabbit was then killed by a rapid intravenous injection of 60 mg/kg of pentobarbital sodium (Nembutal, Abbott Laboratories). The graft was removed, opened, and patency assessed by visual inspection. The graft was then divided into two longitudinal sections, one placed in fixative for histologic evaluation, the other trimmed of all host tissues and placed in a sterile test tube containing 5 ml of BHI broth, and incubated for 48 to 72 hours at 37° C. Bacterial growth was suggested by turbidity of the broth and confirmed by inoculation of a loopful of media onto BHI agar plates. Cultures positive for S. aureus were characterized by the presence of typical white colonies, which was confirmed by Gram staining and coagulase testing with a standard commercial coagulase kit (coagulase plasma, rabbit with EDTA, BBL Microbiology Systems). Specimens for light microscopy were fixed in 10% formol saline solution cut into 4 μm sections, stained with hematoxylin and eosin or Mallory's phosphotungstic acid—hematoxylin (PTAH) and mounted on a glass slide. Specimens for scanning electron microscopy (SEM) were fixed in 2.5% glutaraldehyde in 0.1 mol/L phosphate buffer. After dehydration in acetone, critical-point drying, and sputtercoating with gold palladium, the specimens were examined with a scanning electron microscope (Hitachi S405A) at 25 kV. All the grafts from the eight animals given control infusions were sterile on cultures. In PTFE grafts, 25 rabbits were infused with 4 × 102 to 6 × 107 colony forming units (CFUs) of S. aureus at anastomotic completion. All the grafts from animals receiving 104 CFUs or more had cultures positive for S. aureus. Three of four grafts from animals challenged with 103 CFUs, and two of four grafts from animals challenged with 102 CFUs showed cultures positive for S. aureus. Bacterial challenges lower than 102 CFUs were not given because of the difficulty in accurately preparing such low doses. From these results an ID50 for PTFE was calculated (Fig. 1).Four of six animals receiving the highest dose of 107 CFUs died early and thrombosed grafts were found at autopsy. Therefore, it was decided that a lesser dose of 106 CFUs should be used for the delayed bacteremia studies. In Replamineform grafts, 12 rabbits had infusions of 0.5 × 104 to 1 × 106 CFUs at anastomotic completion. All the grafts from the eight animals receiving 106 CFUs were infected compared with two grafts from four animals infused with 104 CFUs, giving an approximate ID50 of 104 CFUs. No additional animals were tested to give a more precise estimate. With PTFE grafts, 26 rabbits received infusion of approximately 106 CFUs of S. aureus between 1 and 14 days after PTFE graft insertion (Table I).Table IGraft infection rates after bacteremiaPTFEReplamineformControlDay of infusionNo.InfectedNo.InfectedNo.Infected0101088401-4181084——76140——14404040NOTE Bacteremia was produced by 106 CFUs of S. aureus infused intravenously at anastomotic completion (day 0) and at intervals up to 14 days after grafts were inserted. Open table in a new tab The actual doses given were within a relatively narrow range, 0.8 to 3.5 × 106 CFUs of S. aureus. Ten of 18 grafts infused between 1 and 4 days after insertion had cultures positive for S. aureus. Only one of six grafts infused at 7 days was infected and none of four grafts infused at 2 weeks had positive cultures. NOTE Bacteremia was produced by 106 CFUs of S. aureus infused intravenously at anastomotic completion (day 0) and at intervals up to 14 days after grafts were inserted. With Replamineform grafts, 16 rabbits received infusions of 0.6 to 0.9 × 106 CFUs between 1 and 14 days after insertion of Replamineform grafts. Four of six animals infused between 1 and 4 days had infected grafts whereas all eight animals infused between 7 and 14 days had sterile grafts. Thus both materials achieved a rapid resistance to bacteremia with significantly more infections after immediate (time of implant) compared with delayed infusion (X2 = 19.7, p < 0.001). Light microscopy showed that within the first few days after insertion, the graft interstices became infiltrated with a relatively acellular protein coagulum. Specific staining with PTAH showed that fibrin was a major component of this infiltrate. Some areas of the luminal surface were covered with a layer of neointima, whereas in others, graft materials was exposed to the bloodstream (Fig. 2). The SEM examination showed varying rates of healing but in all specimens, infected and noninfected, there were areas with exposed graft material (Fig. 3).At higher magnification with PTFE grafts, the typical node and fibril pattern of the prosthetic material is clearly recognizable (Fig. 4).Fig. 4Scanning electron micrograph (× 1000) of PTFE 3 weeks after insertion shows fibrin and red cell deposition between exposed PTFE nodes and fibrils.View Large Image Figure ViewerDownload Hi-res image Download (PPT) These studies lend no credence to the current and widely held views that (1) vascular grafts are extremely vulnerable to bacteremic infection for months or years after transplantation and (2) infectability is related to neointimalization. That grafts can be infected by low doses of circulating bacteria (as low as 102 CFUs) when challenged immediately after insertion has previously been reported.8Bennion RS Williams RA Wilson SE. Comparison of infectability of vascular prosthetic materials by quantitation of median infective dose.Surgery. 1984; 95: 22-26PubMed Google Scholar This has obvious clinical relevance and lends strong support to the administration of systemic antibiotics during the perioperative period. Yet, 24 hours after implantation, infectability is already reduced and after 7 days bacteremic infection did not occur. Graft healing as judged by light microscopy and SEM was minimal during this early postoperative period. At this time coverage of the luminal surface of the graft was minimal, although gradual filling of the graft interstices with a fibrin-containing protein coagulum had occurred. These results are clearly at odds with previous findings in the canine model of bacteremia,4Malone JM Moore WS Campagna G Bean B. Bacteremic infectability of vascular grafts: The influence of pseudointimal integrity and duration of graft function.Surgery. 1975; 78: 211-216PubMed Google Scholar, 5Moore WS Malone JM Keown K. Prosthetic arterial graft material: Influence of neointimal healing and bacteremic infectability.Arch Surg. 1980; 115: 1379-1383Crossref PubMed Scopus (62) Google Scholar but there are several important differences between the present and earlier work. The previous canine studies used very large bacterial challenges derived from overnight cultures and no details of repeated colony counting were presented. We found that repeated colony counts derived from overnight cultures varied considerably, often by a factor of 10. Moreover, the lower bacterial challenges used in these present studies are much closer to the bacteremic episodes encountered in clinical practice and it is suggested that inappropriately large bacterial challenges will prevent an appreciation of any alteration in the resistance of grafts to infection. The previous conclusions on graft healing related to bacteremic infection were derived from simple inspection of the graft lining and no detailed data, such as SEM, were presented.4Malone JM Moore WS Campagna G Bean B. Bacteremic infectability of vascular grafts: The influence of pseudointimal integrity and duration of graft function.Surgery. 1975; 78: 211-216PubMed Google Scholar, 5Moore WS Malone JM Keown K. Prosthetic arterial graft material: Influence of neointimal healing and bacteremic infectability.Arch Surg. 1980; 115: 1379-1383Crossref PubMed Scopus (62) Google Scholar Our SEM studies consistently showed that neointimalization was incomplete even months after graft insertion and therefore we conclude that the rapid development of resistance to bacteremic infection is unrelated to neointimal formation. We suggest that it is the initial interstitial filling of microporous grafts that prevents bacteremic infection. Furthermore, we propose that this occurs because of the simple physical barrier of the coagulum, which prevents the entry of the bacteria into the graft interstices. Bacteria that make contact with the surface of the grafts after the initial interstitial filling has occurred are presumably dealt with by standard defense mechanisms, such as macrocytic phagocytosis, such that graft infection does not occur. Although this concept may well apply to less microporous graft materials such as Dacron, this must be regarded as speculative until Dacron grafts of small diameter become available and are tested in this model. Of course, there are difficulties in comparing results between different animals. It has been proposed that the dog is the most appropriate animal for studying prosthetic grafts because of similarities in the healing characteristics.9Sauvage LR Berger KE Wood SJ Yates SG Smith JC Mansfield PE. Interspecies healing of porous arterial prostheses.Arch Surg. 1974; 109: 698-705Crossref PubMed Scopus (215) Google Scholar However, the authors concerned did not study the rabbit and the more sophisticated methods for assessing graft healing, such as SEM, were not applied. Rabbit and human aortic tissue have strikingly similar thromboplastic and fibrinolytic properties; both have marked thromboplastic activity yet weak fibrinolytic activity, whereas the reverse is true for the dog.10Astrup T Buluk K. Thromboplastic and fibrinolytic activity in vessels of animals.Circ Res. 1963; 13: 253-260Crossref PubMed Scopus (29) Google Scholar Thus the rabbit is an appropriate model for studying small-diameter vascular grafts and should be applied more often in the future. In summary, microporous vascular grafts, although initially vulnerable to low circulating doses of bacteria, rapidly develop resistance to bacteremic infection. This resistance is independent of neointimal formation and may be caused by simple filling of the graft interstices with protein coagulum. These studies reinforce the administration of antibiotics at the time of implantation but lend no support to the concept of giving antibiotics to cover potential bacteremic episodes at later intervals. Moreover, alterations in graft design to produce improved healing and techniques such as endothelial seeding should not be promoted as potential methods to prevent late bacteremic infection." @default.
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• W2022183438 title "Microporous vascular grafts do not require neointima for resistance to bacteremic infection" @default.
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