Identification of Staphylococcus epidermidis vascular graft infections: a comparison of culture techniques

Culture of prosthetic material is routinely used to exclude or implicate infection in the pathogenesis of late-appearing graft complications. In a canine model of aortic graft infection caused by a bacterial biofilm, the influence of culture media (blood agar and tryptic soy broth) and mechanical surface biofilm disruption (tissue grinding and ultrasonic oscillation) on microorganism recovery was determined. Dacron prostheses colonized in vitro with Staphylococcus epidermidis were implanted in the infrarenal aortas of 36 dogs. After 3 weeks an infection with anatomic characteristics of late graft infection in humans was present. Explantation (+/- surface biofilm disruption) of infected grafts showed broth culture was superior (p less than 0.001) to agar media in confirming infection. The recovery rate of S. epidermidis was 30% with agar media, was 72% with broth media alone, and was 83% with broth media plus biofilm disruption. In situ replacement of infected grafts plus parenteral antibiotics resulted in early (1 month) healing of 31 grafts without signs of infection. All replacement grafts were sterile when cultured in broth media alone, but the addition of biofilm disruption isolated the study strain from eight (22%) of 36 grafts (p less than 0.01). Biofilm disruption by tissue grinding or sonication increased bacteria recovery equally. When biofilm bacterial concentration was less than 100 colony-forming units/cm2 of graft, only culture in broth media reliably recovered microorganisms. In the absence of perigraft inflammation, microbiologic recovery techniques that identify bacterial biofilms are necessary to exclude infection in studies concerning the pathogenesis of late graft complications or the treatment of S. epidermidis prosthetic infections.     

Infection of vascular prostheses caused by bacterial biofilms

A canine model was developed to study the efficacy of graft replacement as treatment for vascular prosthesis infections from Staphylococcus epidermidis. Infrarenal aortic graft infections were established in 18 dogs by implantation of Dacron prostheses colonized in vitro with a slime-producing strain of S. epidermidis to form an adherent bacteria-laden biofilm (5 X 10(6) colony-forming units/cm2 graft). Study animals developed a graft infection with anatomic and microbiologic characteristics typical of late prosthetic graft infections in humans (sterile perigraft exudate, absent graft incorporation, and normal serum leukocyte count and sedimentation rate). The S. epidermidis study strain was isolated from 14 of 18 explanted grafts (78%) by mechanical disruption of the graft surface biofilm and culture in broth media. Four dogs with sterile graft cultures had histologic evidence of bacterial infection. The established prosthetic surface biofilm infection was treated by graft excision, parenteral cefazolin, and graft replacement with a Dacron or polytetrafluoroethylene (PTFE) vascular prosthesis. One month after graft replacement, no PTFE graft had signs of infection, but perigraft exudate and inflammation involved three of nine Dacron grafts (33%). The study strain was recovered from four of nine PTFE grafts (44%) and two of nine Dacron (22%) replacement grafts (p greater than 0.05). Prosthetic replacement of Dacron prostheses infected by S. epidermidis as a bacteria-laden surface biofilm can result in early graft healing, but persistent colonization of one third of replacement grafts signify that recurrent clinical infection remains a risk.     

Anastomotic femoral pseudoaneurysm: an investigation of occult infection as an etiologic factor

Occult infection was investigated as an etiologic factor in the formation of femoral anastomotic pseudoaneurysms associated with prosthetic vascular grafts. Forty-five femoral pseudoaneurysms with no clinical evidence of infection 10 to 173 months after prosthetic graft placement were consecutively studied. The explanted Dacron or explanted polytetrafluoroethylene graft material was cultured in trypticase soy broth and ultrasonically oscillated to remove adherent bacteria. All patients were treated by excision of the pseudoaneurysm and surrounding perigraft capsule and in situ replacement with an interposition prosthetic graft. Thirty-two bacterial isolates were recovered from 27 (60%) of the specimens, with coagulase negative staphylococci (Staphylococcus epidermidis S. warneri, S. hominis, S. capitis) accounting for 24 of the recovered species. No infection of the replacement graft developed in any patient and no recurrent pseudoaneurysms were observed. Bacterial colonization may occur at implantation or during subsequent procedures when the prosthetic graft is exposed. This chronic infection can be diagnosed by means of sensitive culture techniques that dislodge adherent bacteria from the graft surface. On grounds of the observations reported in this study, there appears to be suggestive evidence that an occult infectious process may be one of the factors that play a role in the development of some femoral anastomotic pseudoaneurysms.     

Prophylactic antibiotics prevent bacterial biofilm graft infection.

Bacterial biofilm graft infection is due to prostheses colonization by Staphylococcus epidermidis, a pathogen frequently recovered from perigraft tissues of man during vascular procedures despite the use of asepsis and prophylactic antibiotics. The effect of preoperative intraperitoneal cefazolin, administered at a standard (15 or 30 mg/kg) and high (120 mg/kg) dose, on the prevention of bacterial biofilm infection was studied in a rat model. Seventy-four Dacron grafts, colonized in vitro with S. epidermidis to produce an adherent biofilm (3.19 +/- 0.71 x 10(7) colony-forming units/cm2 graft), were implanted in the dorsal subcutaneous tissue at 0.5, 2, and 4 hr after antibiotic administration. The study strain was a slime-producing clinical isolate with minimum inhibitory concentration (MIC) of 15-30 micrograms/ml to cefazolin. Subcutaneous tissue antibiotic levels were determined at each time interval. One week after implantation, the concentration of bacteria in the surface biofilm by quantitative agar culture was significantly decreased (P less than 0.05) only for grafts implanted when antibiotic tissue levels were greater than or equal to the MIC of the study strain. The result of no growth by biofilm broth culture was significantly achieved (P less than 0.01) only for grafts implanted 0.5 hr after high dose cefazolin, in which the tissue antibiotic level was above the MIC of the study strain. Antibiotics can markedly reduce the bacteria concentration of a prosthetic surface biofilm. The effectiveness of prophylactic antibiotics on the prevention of graft infection is dependent upon maintaining an adequate antibiotic level in the perigraft tissues for the duration of the procedure.     

Differential effects of a gram-negative and a gram-positive infection on autogenous and prosthetic grafts

A canine model was developed to study the differential response of a gram-negative and a gram-positive bacterial infection on autogenous and prosthetic grafts. After replacing segments of the femoral arteries of 15 dogs with autogenous vein in one groin and polytetrafluoroethylene in the contralateral groin, 10(8) colony-forming units of nonmucin-producing Staphylococcus epidermidis (five dogs), Pseudomonas aeruginosa (five dogs), or sterile saline solution (five dogs) were directly inoculated onto the grafts. The grafts were examined 7 to 10 days after implantation. None of the control dogs exhibited inflammatory signs, and no grafts or anastomoses disrupted. S. epidermidis was unrecoverable from either graft material in any of the animals, although histologic evaluation confirmed neutrophils and bacteria in four of five animals in the vein and polytetrafluoroethylene groups. No dog inoculated with S. epidermidis had graft or anastomotic disruption. By contrast, P. aeruginosa was recovered from both types of grafts in all inoculated animals. Neutrophils, bacteria, and microabscesses were observed in all of these animals. In addition, three of five polytetrafluoroethylene grafts and all five vein grafts disrupted either at the anastomoses or in the body of the vein graft. Therefore S. epidermidis is a less virulent organism that may persist in graft walls despite negative cultures, whereas P. aeruginosa is a highly virulent organism that can disrupt native artery, vein grafts, and anastomoses. The graft material appears to be less important than the bacteria in determining the outcome of infection.     

Long-term complication and patency rates of Vectra and IMPRA Carboflo vascular access grafts with aggressive monitoring, surveillance and endovascular management

The purpose of this study is to compare infection, pseudoaneurysm formation and patency rates during long-term follow-up of polyurethane and polytetrafluoroethylene (PTFE) vascular access grafts maintained with contemporary endovascular methods. During a 34-month period, 239 polyurethane and 125 carbon-impregnated PTFE vascular access grafts were placed in 324 consecutive patients. Thirty-six patients (9.9%) developed a pseudoaneurysm (anastomotic, n = 6 or at the needle-stick site, n = 30). An additional 19 patients (5.2%) required graft excision for infection. Three-year graft infection and pseudoaneurysm formation (at needle-stick site) rates were similar in polyurethane and PTFE grafts (11% versus 8%, P = 0.61, and 17% versus 23%, P = 0.72, respectively). Three-year secondary patency was better in polyurethane than PTFE grafts (69% versus 57%, respectively, P = 0.012). Straight upper arm polyurethane grafts had the best secondary patency (P = 0.001). Contemporary long-term secondary patency of vascular access grafts is satisfactory. Further follow-up is necessary to compare late infection and pseudoaneurysm formation rates.     

Gelatin-sealed polyester resists Staphylococcus epidermidis biofilm infection.

BACKGROUND: The purpose of this study was to show that gelatin-impregnated polyester grafts inhibit Staphylococcus epidermidis biofilm infection in a canine model of aortic graft interposition. A clinically native species and two engineered strains, which differed in slime and adhesin antigen components, were compared to determine differential gelatin and slime interactions. METHODS: In vitro bacterial graft colonization was validated by immersion of graft segments in inoculating solutions (10(6) colony forming units/ml) of a clinically native species RP62A and two genetically engineered S. epidermidis species, M187sn3 (SN3: slime and adhesin negative) or M187sp11 (SP11: slime and adhesin positive), for 18 h at 23 degrees C. The grafts were washed, sonicated, and cultured to assess in vitro bacterial graft adherence. Grafts similarly inoculated were placed as aortic interposition grafts in dogs. Three sterile grafts were implanted as controls. Grafts were excised after 6 weeks and cultured for bacterial growth as in the in vitro study. Infection was defined by a positive culture in the excised grafts. Data were analyzed with nonparametric statistical methods. RESULTS: In vitro bacterial graft adherence in colony forming units per milliliter was similar at 18 h postsonication for RP62A (8 x 10(4) +/- 1 x 10(4)), SN3 (7 x 10(4) +/- 2 x 10(4)), and SP11 (6 x 10(4) +/- 2 x 10(4)) (P = NS). Only one of five grafts inoculated with RP62A was culture positive after 6 weeks. No grafts inoculated with the engineered strains SN3 or SP11 were culture positive after explanation. CONCLUSION: In vitro bacterial inoculation of gelatin-impregnated polyester was similar among the species and not dependent upon the presence of slime and adhesin components. Gelatin-impregnated polyester grafts demonstrated in vivo resistance to coagulase-negative staphylococcal biofilm infection.     

Adherence of mucin and non-mucin-producing staphylococci to preclotted and albumin-coated velour knitted vascular grafts

The mechanisms involved in bacterial adherence to vascular grafts are important in understanding prosthetic infections. Albumin-coated Dacron (ACD) is a new development in vascular graft fabrication. However, albumin acts as a receptor for certain gram-positive bacterial adhesions. Five pathogenic, coagulase-negative Staphylococcus epidermidis strains were used to measure the differential microbial adherence to ACD versus untreated velour-knitted Dacron (VKD) vascular prostheses. Specimens of VKD, preclotted VKD, and ACD were inoculated with each of the five strains (10(7) colony-forming units/ml) for 2, 4, 8, 12, and 24 hours. After incubation, graft specimens were washed to remove nonadherent organisms and oscillated ultrasonically to remove adherent organisms. The sonication effluent was plated to trypticase soy agar to quantitate the adherent organisms. Adherence was significantly greater (p less than 0.01) to VKD compared with preclotted VKD and ACD at 2, 4, 8, and 24 hours. Four of the five study strains demonstrated significantly greater adherence to VKD than to either ACD or preclotted VKD. Adherence of S. epidermidis increased with exposure time. Albumin bonded to velour-knitted Dacron does not increase coagulase-negative staphylococcal adherence compared with the noncoated vascular prostheses. Binding albumin to vascular prostheses does not increase the risk of staphylococcal colonization.     

Assessment of the antimicrobial activity of acellular vascular grafts

Several studies have reported biological vascular grafts to be more resistant to microbial infection than synthetic counterparts in?vivo. Indeed, small intestinal submucosa (SIS) materials have previously been reported to be antimicrobial. The aim of this study was to assess the antimicrobial activity and the ability to resist biofilm formation of a novel acellular vascular graft and compare it to commercially available alternatives using a range of organisms: MRSA, MSSA, Staphylococcus epidermidis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa and Candida albicans. This was achieved using a modified disk diffusion assay and extraction of the materials into solution followed by minimum inhibitory concentration assays. To assess resistance to biofilm formation a novel biofilm assay was developed which compared the total colony forming units (CFU) recovered from each material and identification of the percentage of CFU which were loosely attached, residing within the biofilm or attached to the biomaterial. The results indicated a lack of antimicrobial activity for all materials tested, including SIS. The biological materials were more resistant to the formation of a biofilm compared to Dacron.     

In situ replacement of arterial prosthesis infected by bacterial biofilms: Long-term follow-up

Purpose: Bacterial biofilm infections of vascular prostheses are characterized by an absence of systemic sepsis, a fluid-filled cavity surrounding the graft, a draining sinus tract, and microorganisms that must be removed from the fabric prosthesis for bacterial culture.Methods: Since 1987 we have treated 20 infected grafts with prosthetic excision and in situ replacement in 14 men and 6 women. The time from initial graft implantation to diagnosis of graft infection ranged from 3 months to 14 years (mean 4.5 years). The original graft (Dacron-17, polytetrafluoroethylene-3) was an aortobifemoral in 14, axillofemoral femorofemoral in 3, iliofemoral in 2, and femoropopliteal in 1 patient. Presenting symptoms were groin false aneurysm with perigraft fluid in 10, inflammatory mass in 6, and sinus tract in 4. At surgery all unincorporated graft material and the perigraft capsule were excised from a point where the proximal graft was incorporated, including debridement of vessels at the distal anastomosis. Of the 14 aortobifemoral grafts, only the femoral limbs were excised at the initial presentation of biofilm infection. The conduit was replaced with an in situ polytetrafluoroethylene interposition graft, which was covered with a gracilis or sartorius muscle flap when possible.Results: All surgical sites healed, all grafts remained patent, and there was no limb loss. After ultrasonic oscillation of the explanted graft, bacterial cultures recovered coagulase-negative Staphylococcus species in 14, coagulase-positive Staphylococcus species in one, both species in three, with no growth from two specimens. During follow-up, two patients have had clinical involvement in the proximal intraabdominal portion of the graft that had not been previously resected. In all grafts, the in situ replacement graft remained well incorporated.Conclusion: In situ graft replacement is effective treatment for biofilm infections of vascular prostheses. Because of the indolent nature of these infections, subsequent infection of previously uninvolved graft segments may be expected. (J VASC SURG 1994;19:226-35.)     

Laser-induced shock waves enhance sterilization of infected vascular prosthetic grafts

BACKGROUND AND OBJECTIVE: Bacteria that cause infection of vascular prosthetic grafts produce an exopolysaccharide matrix known as biofilm. Growth in biofilms protects the bacteria from leukocytes, antibodies and antimicrobial drugs. Laser-generated shock waves (SW) can disrupt biofilms and increase drug penetration. This study investigates the possibility of increasing antibiotic delivery and sterilization of vascular prosthetic graft. STUDY DESIGN/MATERIALS AND METHODS: Strains of Staphylococcus epidermidis and S. aureus were isolated from infected prosthetic grafts obtained directly from patients. Dacron grafts were inoculated with the isolated bacteria, which were allowed to form adherent bacterial colonies. The colonized grafts underwent the following treatments: (a) antibiotic (vancomycin) alone; (b) antibiotic and SW (c) saline only; and (d) saline and SW. Six hours after treatment, the grafts were sonicated, the effluent was cultured and the colony forming units (CFU) were counted. RESULTS: CFU recovered from control grafts colonized by S. epidermidis were comparable: saline, 3.05 x 10(8) and saline+SW 3.31 x 10(8). The number of S. epidermidis CFU diminished to 7.61 x 10(6) after antibiotic treatment but the combined antibiotic+SW treatment synergistically decreased CFU number to 1.27 x 10(4) (P<0.001). S. aureus showed a higher susceptibility to the antibiotic: 2.26 x 10(6) CFU; antibiotic +SW treatment also had an incremental effect: 8.27 x 10(4) CFU (P<0.001). CONCLUSIONS: This study demonstrates that laser-generated shock waves have no effects alone, but can enhance the effectiveness of antibiotics against bacteria associated with prosthetic vascular graft biofilms, suggesting that this treatment may be of value as adjunctive therapy for prosthetic graft infections.     

Histological comparison of autogenous canine fascia lata, Gore-Tex, lyophilized human fascia lata, and autogenous canine vein for vascular patch graft material in a canine arteriotomy model.

Autogenous fascia lata has found little clinical use as a vascular patch graft material. Previous experience, however, suggests that it possesses attributes that might make it useful in this regard. To assess its efficacy as a vascular patch graft, nine adult mongrel dogs each underwent four arteriotomies with placements of patch grafts. The four sites included both carotid arteries and both femoral arteries. In each animal, one of four patch graft materials (autogenous canine fascia lata, Gore-Tex, lyophilized human fascia lata, and autogenous canine vein) were placed as patch material at the arteriotomy site utilizing 7-0 running sutures and loop magnification. The site for placement of each graft material was rotated serially in the animals so that each site would have equal numbers of all four graft materials applied. The animals were killed at either 6 to 8 weeks or 11 to 12 weeks after angiography of all four vessels. The specimens were then evaluated histologically. No difference was observed among any of the patch graft materials with regard to myofibroblast plaque formation. Inflammatory responses were noted to be substantially less in the canine fascia lata group than in the other three groups. Granuloma formation, however, appeared to be most significant in the autogenous canine vein group. Only one vessel was occluded. Aneurysm or pseudoaneurysm formation was not noted in any specimen. It appears from the above results that autogenous fascia lata may be an appropriate alternative to currently utilized arterial patch graft materials and that it should be evaluated further for this purpose.     

Nitric oxide-releasing biopolymers inhibit thrombus formation in a sheep model of arteriovenous bridge grafts

OBJECTIVES: Nitric oxide (NO), produced by normal vascular endothelial cells, reduces platelet aggregation and thrombus formation. NO-releasing biopolymers have the potential to prolong vascular graft and stent patency without adverse systemic vasodilation. METHODS: 5-mm polyurethane vascular grafts coated with a polymer containing the NO-donor dialkylhexanediamine diazeniumdiolate were implanted for 21 days in a sheep arteriovenous bridge-graft model. RESULTS: Eighty percent (4/5) of grafts coated with the NO-releasing polymer remained patent through the 21 day implantation period, compared to fifty percent (2/4) of sham-coated grafts and no (0/3) uncoated grafts. Thrombus-free surface area (+/-SEM) of explanted grafts was significantly increased in NO-donor coated grafts (98.2% +/- 0.9%) compared with sham-coated (79.2% +/- 8.6%) and uncoated (47.2% +/- 5.4%) grafts ( P = .00046). Examination of the graft surface showed no adherent thrombus or platelets and no inflammatory cell infiltration in NO-donor coated grafts, while control grafts showed adherent complex surface thrombus consisting of red blood cells in an amorphous fibrin matrix, as well as significant red blood cell and inflammatory cell infiltration into the graft wall. CONCLUSION: In this study we determined that local NO release from the luminal surface of prosthetic vascular grafts can reduce thrombus formation and prolong patency in a model of prosthetic arteriovenous bridge grafts in adult sheep. These findings may translate into improved function and improved primary patency rates in small-diameter prosthetic vascular grafts.     

Sonication provides maximal recovery of staphylococcus epidermidis from slime-coated vascular prosthetics.

Staphylococcus epidermidis (S. epidermidis) prosthetic vascular graft infections are often difficult to detect. A reliable culture method is needed so that appropriate therapeutic decisions can be initiated in a timely fashion. Sonication of graft material has been proposed as a method of enhancing bacterial recovery. Theoretically, however, this could cause cell lysis and false-negative culture results. Several methods of obtaining bacterial cultures were compared to determine the best method of quantitative bacterial recovery from infected graft material. Polytetrafluoroetehylene (PTFE) and knitted Dacron graft segments (n = 192) were exposed to a slime-producing strain of S. epidermidis or Escherichia coli (E. coli) at four different bacterial concentrations. Recovery of bacteria from the segments was then compared using three different methods to obtain organisms. One-third of the segments were pressed directly onto the agar to transfer bacteria while the other two-thirds were placed in broth and then either sonicated or vortexed prior to plating onto agar. The direct technique was significantly less effective (P less than 0.01) at recovering bacteria than either sonicating or vortexing for both graft materials at bacterial concentrations of 10, 10(2), and 10(4) CFU/ml of S. epidermidis. Sonication was significantly better at recovery from either material at 10(2) and 10(4) CFU/ml of S. epidermidis than vortexing (P less than 0.05). E. coli graft adherence was poor, and significant recovery occurred only at 10(8) CFU/ml. Sonication is necessary to maximally recover S. epidermidis from infected prosthetic grafts.     

The prophylactic efficacy of rifampicin-soaked graft in combination with systemic vancomycin in the prevention of prosthetic vascular graft infection: an experimental study

OBJECTIVE: To investigate the prophylactic efficacy of systemic, topical, or combined antibiotic usage in the prevention of late prosthetic vascular graft infection caused by methicillin-resistant Staphylococcus epidermidis (MRSE) and the differential adherence of S. epidermidis to Dacron and ePTFE grafts in a rat model. MATERIALS AND METHODS: Graft infections were established in the back subcutaneous tissue of 120 adult male Wistar rats by implantation of 1-cm(2) Dacron/ePTFE prosthesis followed by topical inoculation with 2 x 10(7) CFU of clinical isolate of MRSE. Each of the series included one group with no graft contamination and no antibiotic prophylaxis (uncontaminated control), one contaminated group that did not receive any antibiotic prophylaxis (untreated control), one contaminated group in which perioperative intraperitoneal prophylaxis with vancomycin (10 mg/kg) was administered, two contaminated groups that received rifampicin-soaked (5 mg/1 ml) or vancomycin-soaked (1 mg/1 ml) grafts, and one contaminated group that received a combination of rifampicin-soaked (5 mg/1 ml) graft with perioperative intraperitoneal vancomycin prophylaxis (10 mg/kg). The grafts were removed sterilely 7 days after implantation and evaluated by using sonication and quantitative blood agar culture. RESULTS: MRSE had significantly greater adherence to Dacron than ePTFE grafts in the untreated contaminated groups (P < 0.001). Rifampicin had better efficacy than vancomycin in topical application, but the difference was not statistically significant (P > 0.05). Intraperitoneal vancomycin showed a significantly higher efficacy than topical vancomycin or rifampicin (P < 0.001). The best results were provided by a combination of intraperitoneal vancomycin in rifampicin-soaked graft groups (P < 0.001). CONCLUSIONS: The combination of rifampicin and intraperitoneal vancomycin seems to be the best choice for the prophylaxis of late prosthetic vascular graft infections caused by MRSE.     

Nuclide imaging of vascular graft-platelet interactions: comparison of indium excess and technetium subtraction techniques

Indium-111-labeled platelet adherence to ePTFE thoracoabdominal vascular prostheses in a canine model (n = 10) was quantitated by (1) an indium-111 excess technique, contrasting graft radioactivity to that in a reference region, and (2) a technetium-99m subtraction technique, with radioactivity of circulating platelets eliminated by discounting background blood activity. Variation in graft thrombogenicity was provided by seeding six prostheses with enzymatically derived autologous endothelial cells, and implanting four prostheses without seeding. Grafts were imaged at 1, 4, and 6 weeks postimplantation, with platelet labeling using indium-111-oxine and red blood cell labeling using technetium-99m. At 7 weeks grafts were excised and gamma activity was measured in proximal, middle, and distal segments. Luminal generation of TxB2 and 6-keto-PGF1 alpha from midportions of grafts was assayed. Indium-111 excess ratios at 6 weeks correlated with actual gamma activity of excised grafts (proximal r = 0.80, P less than 0.01; middle r = 0.73, P less than 0.05; distal r = 0.48, ns) but such a correlation did not exist for the technetium-99m subtraction technique (r = -0.05, -0.25, and 0.16, in the three segments, respectively, all ns). The ratio of graft to aortic TxB2 production revealed a positive correlation with graft gamma activity (r = 0.87, P less than 0.01), and the ratio of graft 6-keto-PGF1 alpha to TxB2 production also correlated with gamma counts (r = -0.64, P = 0.05). In this experimental setting technetium-99m subtraction analysis was an imprecise method of detecting graft platelet accumulation, whereas indium-111 excess ratios proved to be a more accurate method of quantitating vascular prosthetic thrombogenicity.     

Bacterial adherence to vascular prostheses: A determinant of graft infectivity

An in vitro model was developed to quantitatively measure bacterial adherence to the surface of prosthetic vascular graft material. Four strains of bacteria (Staphylococcus aureus, nonmucin-producing S. epidermidis [SP-2], mucin-producing S. epidermidis [RP-12], and Escherichia coli) were used to inoculate expanded polytetrafluoroethylene (ePTFE), woven Dacron, and velour knitted Dacron graft material. After graft specimens were incubated in a 10⁷ suspension of bacteria, they were washed to remove nonadherent organisms and ultrasonically oscillated to dislodge adherent organisms. Quantitative culture of the sonication effluent was used to calculate bacterial adherence, expressed as the number of colony-forming units found in each square centimeter of graft material per 10⁷ inoculum. All bacterial strains had a greater affinity to velour knitted Dacron graft than to ePTFE (p < 0.025). E. coli and S. aureus adhered to velour knitted Dacron in greater numbers than to woven Dacron (p < 0.04). The production of extracellular polysaccharide (mucin) by the RP-12 strain significantly increased adherence to both ePTFE and Dacron grafts compared with the other three bacterial strains tested (p < 0.04). Although E. coli was less adherent to ePTFE than nonmucin-producing staphylococcal strains (S. aureus and SP-2), no difference in adherence to knitted or woven Dacron graft material was demonstrated. The differential adherence of bacteria to prosthetic vascular grafts pays an important role in the pathogenesis of graft sepsis and determines relative graft infectivity. The in vitro model developed is well suited for further study of the mechanisms by which bacteria adhere to and colonize vascular grafts. (J VASC SURG 1986;3:732-40.)     

Evaluation of three techniques for documenting Staphylococcus epidermidis vascular prosthetic graft infections

Staphylococcus epidermidis (S. epidermidis) vascular prosthetic graft infections are notoriously hard to detect. Three different techniques of determining whether vascular prosthetic grafts were infected using a dog model were evaluated. Aortic angiograms were compared with nuclear magnetic resonance (NMR) imaging and systemic norepinephrine (NE) kinetics to determine if either newer technique would be more reliable than standard angiograms. Twelve dogs were randomized to control (n = 6) or infected groups (n = 6). All dogs had a 5 cm section of their infrarenal aorta replaced with knitted Dacron vascular prosthetic graft. The grafts in the infected group were contaminated by soaking them in a broth containing S. epidermidis. NE production and clearance rates were calculated for all animals after an infusion of 3H-NE using the steady-state radionuclide tracer methodology. One week following graft insertion, dogs were reanesthetized, and the 3H-NE infusion and measurements were repeated. Standard angiograms and NMR imaging were also performed. Once all tests were performed, the prosthetic grafts were removed for cultures. Comparisons between the initial and final norepinephrine measurements for each group were made using the nonparametric Wilcoxon two-sample test, while comparisons between the groups were made by chi square or the Student's t test. Angiogram results were similar for control and infected animals. Angiograms missed disruption of the proximal anastomosis found in three of the six infected dogs at graft removal. None of the six control animals, while five of the six infected animals, had localized areas of high signal intensity on NMR imaging (P less than 0.01) suggesting abscess formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of bacterial contamination on neointimal hyperplasia in vascular grafts

Neointimal hyperplasia (NH) is the most significant contributing factor to long-term vascular graft failure. Inflammation is known to be important in its development; however, the role of bacterial infection is unclear. We examined the effect of contamination with common organisms on the development of NH in expanded polytetrafluoroethylene grafts. Thirty adult pigs were randomized into one of four groups: no infection, contamination with Staphylococcus aureus, mucin-producing Staphylococcus epidermidis, or Pseudomonas aeruginosa. An expanded polytetrafluoroethylene graft (6 mm x 3 cm) was placed as a common iliac artery interposition graft and was inoculated with 1-2 x 10(8) of the selected organism before closure. Grafts were explanted 6 weeks postoperatively. Microbiologic, histological, and morphometric evaluations were performed. All grafts were patent at the time of euthanasia. The mean areas of NH were 5.45 mm(2) in sterile grafts, 8.36 mm(2) in S. aureus, 7.63 mm(2) in S. epidermidis, and 11.52 mm(2) in P. aeruginosa grafts. Comparison of means via analysis of variance showed that P. aeruginosa grafts had significantly higher formation of NH than sterile grafts (P = 0.025). NH production in infected grafts appears to be organism specific and is significantly higher with P. aeruginosa than common Gram-positive organisms. Increased NH from subclinical infection may be a significant factor contributing to late graft failures.     

Sonication is superior to scraping for retrieval of bacteria in biofilm on titanium and steel surfaces in vitro

Background and purpose Low-virulence implant infections are characterized by bacterial colonization of the implant with subsequent biofilm formation. In these cases, soft tissue biopsies often prove to be culture negative. Consequently, detachment of the causative adherent bacteria is crucial for correct microbiological diagnosis. Using an in vitro model, we compared 4 methods of biofilm sampling from metal surfaces.Methods Discs of titanium and steel were incubated in the presence of Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, and Propionibacterium acnes in Mueller Hinton broth. Non-adherent bacteria were removed by repeated rinsing of the discs. 10 parallels of each disc were subjected to 1 of 4 methods for bacterial recovery: (A) sonication of the discs, (B) scraping of the discs using surgical blades followed by streaking of the blades onto agar plates, (C) scraping of the discs followed by vortex mixing of the surgical blades, and (D) scraping of the discs followed by sonication of the surgical blades. Quantitative bacterial cultures were performed for each sampling method.Results With the exception of S. epidermidis on steel, sonication efficiently and reliably dislodged biofilm bacteria. The scraping methods employed did not detach bacteria embedded in biofilm.Interpretation Scraping of metal surfaces is not an adequate method for sampling of biofilm bacteria in vitro.