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Antibiotic bonding to vascular prostheses
J THoRAc CARDIOVASC
SURG
82:272-277, 1981
Antibiotic bonding to vascular prostheses This study examines the efficacy of biochemically bonding an antibiotic (oxacillin) to a vascular graft using benzalkonium as a bonding agent. In both an in vitro bioassay and a subcutaneous pocket in the rats, the antibiotic-bonded graft demonstrated superior antibacterial activity when compared to the control graft and a graft simply soaked in the antibiotic. The possibility is raised that such a vascular prosthesis may be useful in eradicating graft infections.
Ricki Henry, M.D., Richard A. Harvey, Ph.D., and Ralph S. Greco, M.D., F.A.C.S., Piscataway, N. J.
Prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. Mortality rates vary from twenty-five to seventy-five percent depending upon the site of the infection and mode of therapy. 1 Presently, the accepted treatment of graft infections consists of removal of the graft, ligation of the host vessel, extra-anatomic bypass, and massive doses of systemic antibiotics. 2 Unfortunately, even this aggressive treatment is attended by excessive morbidity and mortality. This is due to the inability of antibiotics to penetrate the graft matrix that harbors the bacteria. Also, the clinical manifestations of infection become apparent after the infection is well established. Finally, thrombosis and suture-line disruption are not amenable to antibiotic therapy. The use of prophylactic antibiotics to prevent postoperative infections has many advocates. To date, there has been only one properly controlled study of this approach, and the results support the efficacy of this practice." However, the use of prophylactic antibiotics is not without complication and does not entirely eliminate graft infection. From the Department of Surgery, CMDNJ-Rutgers Medical School, Piscataway, N. J. Presented in part at the annual meeting of the Association for Academic Surgery in November, 1979. Supported by a grant from the General Research Support Fund of the College of Medicine and Dentistry of New Jersey, Rutgers Medical School. Received for publication Oct. 6, 1980. Accepted for publication Jan. 20, 1981. Address for reprints: Ralph S. Greco, M.D., Department of Surgery, CMDNJ-Rutgers Medical School, P.O. Box 101, Piscataway, N. J. 08854.
272
This study was initiated to evaluate an alternative approach to the prevention of graft infection by altering the prosthetic material itself by noncovalently bonding antibiotics to the graft surface and then evaluating its resistance to induced infection. The bonding of an antibiotic to the graft surface is an extension of the graphite-benzalkonium-heparin model developed by Whiffen and associates." They showed that graphite-coated plastic adsorbs heparin and becomes an antithrombogenic surface. Pretreatment of surfaces with benzalkonium chloride potentiated the heparin adsorption and antithrombogenic properties. The exact nature of this bond is not known, but it is postulated that the lipophilic end of the benzalkonium chloride is adsorbed to the graphite and the hydrophilic end is free to electrically bind the negatively charged heparin molecule." Previous studies in our laboratory have demonstrated that an analogous system to the graphite-benzalkonium-heparin model can be developed by replacing the heparin with a negatively charged antibiotic. 5
Methods Bacteria and sensitivity of surfactants and antibiotics. A stock culture of coagulase-positive Staphylococcus aureus was obtained from the Microbiology Department at CMDNJ-Rutgers Medical School. The original source of the bacterium was a wound culture. The minimum inhibitory concentration for oxacillin was 0.1 j.tg/ml. The minimum inhibitory concentration for benzalkonium chloride was 0.8 j.tg/ml. A checkerboard susceptibility study was performed to determine the effect of the combination of oxacillin and benzalkonium on the organism. This resulted in the minimum inhibitory concentration of 0.075 j.tg oxacillin and 0.2
0022-5223/81/080272+06$OO.60/0 © 1981 The C. V. Mosby Co.
Volume 82 Number 2
August, 1981
JLg benzalkonium, figures that suggest an additive effect but not a synergistic effect. Graft preparation. Segments of polytetrafluoroethylene* (PTFE) grafts, 0.5 cm long, 30 JL thick, and 6 mm in diameter, were prepared as follows: Group I, PTFE grafts alone; Group II, PTFE grafts soaked in 25% aqueous benzalkonium chloride for 30 minutes; Group lll, PTFE grafts soaked in IO mg oxacillin overnight at room temperature and air dried for 12 hours; Group IV, PTFE grafts soaked in benzalkonium chloride (as in Group II) and then soaked in IO mg oxacillin overnight at room temperature and air dried for 12 hours. In vitro studies. Bioassays were performed on these grafts in the following manner. Petri dishes were prepared with 35 cc of mannitol salt agar inoculated with I cc of an overnight culture of coagulase-positive Staphylococcus aureus (5 x 107 organisms). The inoculum of Staphylococcus aureus was calculated from a standard curve by the Kirby-Bauer technique. One graft from each group was embedded in the agar as it solidified. The plates were incubated at 37° C for 24 hours. The zones of inhibition of bacterial growth were measured and recorded. These bioassays were performed "acutely," that is, immediately following the various types of treatment, and at various intervals up to 90 days following preparation of the grafts. In the longitudinal studies, all grafts (Groups I to IV) were prepared and kept under dry, sterile conditions until the time of the bioassay. They were then embedded in the agar containing 5 x I Q7 freshly prepared Staphylococcus aureus. Finally, two groups of parallel evaluations were conducted to clarify the amount of antibiotic remaining on the grafts after soaking or bonding. In the first group of studies, a comparison was made between observed zones of inhibition in Groups I through IV and zones of inhibition caused by standard filter-paper discs impregnated with known concentrations of oxacillin. It was found that 10 JLg oxacillin discs cause zones of inhibition between 20 and 25 mm and I JLg oxacillin discs, zones of inhibition of 7.5 to 12.5 mm. In the second group of studies, an attempt was made to evaluate the concentration of surfactant and antibiotic present within the graft matrix after the preparation just described. This was necessary because the concentration utilized in the preparation of Groups II to IV was known but the amount remaining on the graft was *Gore-Tex, registered trademark of W. L. Gore & Associates, Inc., Elkton, Md.
Antibiotic bonding to vascular prostheses
273
Table I Surfactant treatment Benzalkonium Benzalkonium Sodium dodecyl sulfate Benzalkonium Benzalkonium Dodecyl trimethyl ammonium bromide
Antibiotic Oxacillin Carbenicillin Oxacillin Oxacillin 14C-labeled penicillin 14C-labeled penicillin
Concentration (IJ-g/cm graft)
4.0 10.0'
<0.01 4.0 10.3 15.9
Legend: The concentrations of oxacillin and carbenicillin were measured by comparison to filter paper discs impregnated with known concentrations of antibiotic in the microbiological assay. Concentrations of 14C-labeled penicillin were measured by liquid scintillation counting. All grafts were washed five times in sterile water after the bonding process was completed.
not. These studies were carried out by employing a variety of antibiotics and surfactants in the bioassay and by employing 3H-Iabeled benzalkonium and 14C_ labeled penicillin, measured by liquid scintillation counting. Since oxacillin was utilized in the graft preparation, the data utilizing penicillin are inferential and based on the following observations. Oxacillin and penicillin have different chemical structures, but both have a single net negative charge at physiological pH. This suggests that they behave in a similar fashion electrostatically. The Staphylococcus aureus used in these studies is sensitive to both penicillin and oxacillin. Both antibiotics produced zones of inhibition and minimum inhibitory concentrations of similar magnitude. Finally, the ionic nature of the bonding process was clarified by utilizing a variety of antibiotics and surfactants with complementary charges. Thus, when the cationic benzalkonium chloride was utilized with the singly negatively charged oxacillin and the doubly negatively charged carbenicillin, twice as much antibiotic was bound with carbenicillin. Similarly, when oxacillin was utilized with benzalkonium, more than 400 times as much antibiotic was bound as with the negatively charged surfactant sodium dodecyl sulfate. Also, when t4C-labeled penicillin was bound with benzalkonium and with the more cationic dodecyl trimethyl ammonium bromide, 50% more penicillin was bound with the latter. These data are summarized in Table I. Finally, the concentration of 14C-labeled penicillin expressed in the microbiological assay and that measured by liquid scintillation were compared, and it became clear that the former represents less than 20% of the latter (Table II). Thus, when 14C-Iabeled penicillin was used, grafts in Groups III and IV contained 6.04 ± 1.15 and 24.60 ±3.77JLg of antibi-
The Journal of
274 Henry, Harvey, Greco
Thoracic and Cardiovascular Surgery
PENICILLIN G
T1CARCILLIN DISODIUM
a
CEFOXITIN
®-u---r
COO-No·
+r-~~CH20CONH2
CONH +--'-...) : s saCHs
N I'CH
'0
Fig. 1. The chemical structures of anionic antibiotics demonstrating the single negative charge of penicillin, oxacillin, and cephalothin and the double negative charge of carbenicillin and ticarcillin.
Table II Penicillin bound (lLg/cm graft) Microbiological assay 14C-labeled penicillin bound (lLg/cm graft) Radiological assay Difference: Tightly bound (lLg/em graft)
1.5 10.3
8.8
Legend: The concentration of penicillin in the microbiological assay was measured by comparison to filter paper discs impregnated with a known concentration of penicillin. "C-labeled penicillin was measured by liquid scintillation counting. All grafts were washed five times in sterile water after the bonding process was completed.
otic, respectively. When 3H-labeled benzalkonium was used, grafts in Groups II and IV contained 20 ± 3.51J,g of benzalkonium. In vivo studies. The grafts were prepared in the aforementioned manner. One graft was placed in a subcutaneous pocket in the left groin of each animal. Twenty Sprague-Dawley rats were used in each of the four experimental groups. Half of the rats in each group were infected by local inoculation and half by intravenous infusion of 1 x 107 Staphylococcus aureus. After 2 weeks, the grafts were harvested and the wounds and grafts cultured. The number of bacteria in the graft matrix was evaluated by removing all identifiable fibrin and serum from the surfaces of the graft and incubating the graft in thioglycollate broth for 24 hours. The turbidity of the broth then was evaluated by measuring
optical density in a spectrophotometer and comparing the observation to the measurements obtained from known inocula of Staphylococcus aureus. A comparison of optical density and the actual number of bacteria in the broth were achieved by culturing 0.1 ml of the broth and performing colony counts. Results In vitro bioassay. A total of 85 grafts in each of the four groups were studied at 17 different time intervals up to 90 days following preparation of the grafts. Observations were performed every 2 to 3 days during the first month of the study, and a final observation, utilizing five grafts in each group, was done at 3 months. These results are shown graphically in Fig. 2. Zones of inhibition (in millimeters) in the antibiotic-soaked and the antibiotic-bonded groups are relatively constant during the first 30 days, averaging between 25 and 30 mm of inhibition. The gradual decrease in activity in the 90 day study appears constant in these two groups, but a pattern of greater activity in the PTFEbenzalkonium-oxacillin graft remains true throughout the duration of the study. The statistical evaluation of these differences was accomplished by a two-way analysis of variance with time as a repeated measure. The mean zones of inhibition of the antibiotic-soaked and antibiotic-bonded groups are not significantly different. However, both antibiotic-treated groups showed
Volume 82 Number2 August. 1981
Antibiotic bonding to vascular prostheses
35
0---0
27 5
Gore-Texuntreated
. - . Gore-Tex-Benzalkonium
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TIME (IN DAYS)
Fig. 2. In vitro bioassay results. A total of 85 grafts in each of the four groups was studied at 17 time intervals up to 90 days following graft preparation. Groups III and IV, the antibiotic-treated groups, show superior antibacterial activity as measured by zones of inhibition compared to other groups throughout the 90 day trial. p < 0.001 utilizing analysis of variance with time as a repeated measure.
Table III. Implantation of grafts in a rat subcutaneous pocket: Local infection with Staphylococcus aureus
Group Group Group Group
I II III IV
Wound culture (positive ltotal]
Graft culture* (positive/total)
Spectrophorometer readings of graft (mean)
4/10 2/10 3/10 0/10
10/10 9/10 10/10 2/10
1.15 0.59 1.37 0.03
No. of bacteria
I X 107 ± 4 x 10' ± 5 x 107 ± 2 x 10' ±
0.5 x 10" 5 x 10' 0.8 x 10" 1 x 10'
Legend: Group I, PTFE. Group II. PTFE-benzalkonium. Group 1lI. PTFE-oxacillin. Group IV, PTFE-benzalkonium-oxacillin.
•p
< 0.004 (Group IV versus Group I. II. or 1lI).
greater activity than the PTFE-benzalkonium group or the PTFE control group throughout the study, except for the final 90 day observation; at 90 days, the PTFEbenzalkonium grafts have slightly higher zones of inhibition than do PTFE-oxacillin grafts. The statistical analysis of the two antibiotic-treated groups (III and IV) compared to Groups I and II is statistically significant with time as a repeated measure (p < 0,001). Groups I and II had some antibacterial activity, but it was neither significant nor consistent with time. In vivo studies. The results of the rat subcutaneous study are summarized in Tables III and IV. In the group of animals inoculated locally, the wound cultures were sterile in all of Group IV grafts, whereas 40% of Group
I, 20% of Group II, and 30% of Group III grafts were infected. Infection was characterized by both the appearance of purulent material around the graft and within the graft lumen as well as colony counts. More importantly, only 20% of the PTFE-benzalkoniumoxacillin group had positive graft cultures both by optical density and by colony counts, compared with 100% of the control group, 90% of the PTFE-benzalkonium group, and 100% of the PTFE-antibiotic group. The difference between Group IV and all other groups was significant at p < 0.004 according to Fisher's exact test. The relationship between the results of wound cultures, graft cultures, measurement of bacteria in the graft by optical density, and number of bacteria in the
276
The Journal of Thoracic and Cardiovascular Surgery
Henry, Harvey, Greco
Table IV. Intravenous infection with Staphylococcus aureus
Group Group Group Group
I II III IV
Wound culture (positive [total}
Graft culture* (positive/total)
Spectrophotometer readings of graft (mean)
4/10 3/10 2/10 0/10
9/10 4/10 3/10 2/10
0.56 0.07 0.18 0.09
No. of bacteria
4 3 I 3
X X X X
10· 10' 1(j2 10'
± ± ± ±
5 2 3 2
X X X X
10' 10' 10' 10'
Legend: Group I, PTFE. Group II, PTFE-benzalkonium. Group III, PTFE-oxacillin. Group IV, PTFE-benzalkonium-oxacillin. • p < 0.001 (Groups II, III, and IV versus Group I).
graft are shown in columns 2,3,4, and 5, respectively, in Table IV. In the animals inoculated by intravenous injection of Staphylococcus aureus , the wound cultures were again sterile in all of the PTFE-benzalkonium-antibiotic group, whereas 40% of control grafts, 30% of PTFEbenzalkonium grafts, and 20% of PTFE antibioticsoaked grafts were infected. This pattern is similar to that found in the locally inoculated animals. In the graft cultures, 20% of the PTFE-benzalkonium-oxacillin group were infected as compared to 30% of the PTFEoxacillin group, 40% of the PTFE-benzalkonium group, and 90% of the control group. All treatment groups were statistically significant as compared to the control group (p < 0.001). Again, the comparison between optical density and colony counts is summarized in Table IV.
Discussion The susceptibility of vascular prostheses to infection is well documented." Despite the demonstration that implanted grafts may become infected by bacteremic challenge, the most common source of infection is contamination at the time of operation. The graft matrix, a foreign body with no intrinsic blood supply, is the likeliest site for bacterial invasion. Although the use of prophylactic systemic antibiotics has recently been supported in a randomized controlled trial, their use does not completely eradicate graft infections, and systemic antibiotics are not without complication. In 1978, our laboratory demonstrated that oxacillin, a negatively charged antibiotic, could be substituted for heparin in the graphite-benzalkonium model. Also, it is now clear that a rather large group of antibiotics, including the penicillins and their derivatives as well as the cephalosporins and cefoxitin, contain a net negative charge at physiological pH, so that they are amenable to the bonding process (Fig. 1). Thus it is possible that noncovalently bonding antibiotics to vascular grafts can be an effective means of placing small concentrations of antibiotic in the graft matrix for a variable period of
time. This procedure becomes a reasonable avenue of research in attempts at complete eradication of prosthetic infections. The in vitro studies show the superiority of the PTFE-benzalkonium-oxacillin surface in inhibiting the growth of Staphylococcus aureus under conditions of the bioassay and the persistence of this activity for a period of 3 months following graft preparation. Though there is some variability in the frequent assays done during the first month of the in vitro bioassay, the general pattern and statistical analysis corroborate that both antibiotic groups have superior activity to the PTFEbenzalkonium and PTFE control groups. The 90 day observations show persistent activity for the PTFEbenzalkonium-oxacillin group when compared to all other groups, including the PTFE-oxacillin graft. The one observation at 90 days among five grafts shows greater activity by the PTFE-benzalkonium group than the PTFE-oxacillin group. Since that is a single observation and is not statistically significant, it is not clear whether benzalkonium alone is superior to oxacillin alone as the duration of the study increases. The activity of the PTFE control is more difficult to explain. When one takes into account that the zones of inhibition include the 6 mm graft, they are very small and may be explained by the electronegative charge of the surface. Finally, comparison of the observed zones of inhibition to those achieved with standard oxacillin discs is presented only to provide a reference point to allow comparison to a known concentration of oxacillin. This technique was employed because the concentration of antibiotic utilized in the preparation of the bonded grafts and soaked grafts is known. However, the amount remaining in the graft matrix is only measured indirectly in the microbiological assay and, undoubtedly, represents far less than the concentration employed initially. This is clearly borne out by use of 14C-labeled penicillin. These studies do not clarify whether the antibacterial activity of the bonded grafts is a function of a biochemical interaction or the additive effect of two antibacte-
Volume82 Number 2 August, 1981
rial compounds. On the other hand, the data obtained with radioisotope-labeled benzalkonium and '4C_la_ beled penicillin, though inferential, show definite noncovalent bonding between antibiotic and surfactant. The demonstration that the cationic surfactant, benzalkonium, binds twice as much of the doubly negatively charged carbenicillin as the singly charged oxacillin in the microbiological assay supports a noncovalent bonding process. Similarly, the binding of 400 times as much oxacillin by the cationic surfactant, benzalkonium, as the anionic surfactant, sodium dodecyl sulfate, reinforces the noncovalent nature of the interaction. With 14C-labeled penicillin, it is likewise clear that microbiological assay measures loosely bound antibiotic representing less than 20% of the total amount of 14C-labeled penicillin bound per centimeter of graft measured by liquid scintillation. The remainder is less tightly bound, a point which may explain the greater activity found with the bonded grafts over time. Also, the grafts utilized in these studies, with the exception of those summarized in Tables II and III, were air dried and not exhaustively washed with water. Therefore, they contained a far higher concentration of the dissociable oxacillin. Thus, although oxacillin and penicillin are antibiotics with different antibacterial activities, they are electrostatically identical, and the substitution of one for the other in these studies appears valid. The studies using the rat subcutaneous pocket provide in vivo support consistent with the results in the in vitro bioassay. The most significant data are seen in the locally inoculated animals, in which wound and graft cultures of the PTFE-benzalkonium-oxacillin group show a pattern of superior antibacterial activity when compared to the control group, the PTFE-benzalkonium group, and most importantly the PTFEoxacillin group. The activity of the bonded grafts in this nonvascular model is supported both by measurement of optical density of the graft itself and by colony counts of the thioglycollate broth in which the graft was placed. In the intravenously inoculated group, the results of the wound cultures are similar to those seen in
Antibiotic bonding to vascular prostheses
27 7
the locally infected animals. The results of the graft cultures are different only in that all three treatment groups are statistically significant compared to the control group. This may well be a result of the lower inoculum reaching the graft when the bacteria are injected intravenously. These studies support the premise that appropriately charged antibiotics can be bonded to vascular prostheses by means of a surfactant with a complementary biochemical charge. Most importantly, they show that a PTFE-benzalkonium-antibiotic surface can be produced without the need for graphite, which has been associated in the past with serious operational difficulties. The PTFE grafts treated with both a surfactant and an antibiotic show superior inhibition of bacterial growth in an in vitro bioassay up to 90 days after graft preparation. This antibacterial activity appears to be present in a nonvascular subcutaneous pocket for at least 2 weeks. Though the effect of treatment on pseudointima formation has yet to be determined, it is possible that the use of a PTFE-benzalkonium-oxacillin surface, with or without perioperative parenteral antibiotics, will be a useful adjunct to attempts at complete eradication of vascular prosthetic infection. REFERENCES Christensen J, Eklof B: Synthetic arterial grafts. II. Infection complications. Scand J Thorac Cardiovasc Surg 11:43-50, 1977
2 Beebe HG: Complications of Vascular Surgery, Philadelphia, 1973, J. B. Lippincott Company 3 Kaiser AB, Clayson KR, Mulherin JL, Roach AC, Allen TR, Edwards WH, Dale WA: Antibiotic prophylaxis in vascular surgery. Ann Surg 188:283-289, 1978 4 Whiffen JD, Dutton R, Young WP, Gott VL: Heparin application to graphite-coated intravascular prostheses. Surgery 56:404-412, 1964 5 Jagpal R, Greco RS: Studies on a graphite-benzalkoniumoxacillin surface. Am Surg 45:774-779, 1979 6 Goldstone J, Moore WS: Infection in vascular prostheses. Clinical manifestations and surgical management. Am J Surg 128:225-233, 1974
SURG
82:272-277, 1981
Antibiotic bonding to vascular prostheses This study examines the efficacy of biochemically bonding an antibiotic (oxacillin) to a vascular graft using benzalkonium as a bonding agent. In both an in vitro bioassay and a subcutaneous pocket in the rats, the antibiotic-bonded graft demonstrated superior antibacterial activity when compared to the control graft and a graft simply soaked in the antibiotic. The possibility is raised that such a vascular prosthesis may be useful in eradicating graft infections.
Ricki Henry, M.D., Richard A. Harvey, Ph.D., and Ralph S. Greco, M.D., F.A.C.S., Piscataway, N. J.
Prosthetic infection following reconstructive vascular operations is an infrequent but often fatal complication which generally persists until the graft is removed. Mortality rates vary from twenty-five to seventy-five percent depending upon the site of the infection and mode of therapy. 1 Presently, the accepted treatment of graft infections consists of removal of the graft, ligation of the host vessel, extra-anatomic bypass, and massive doses of systemic antibiotics. 2 Unfortunately, even this aggressive treatment is attended by excessive morbidity and mortality. This is due to the inability of antibiotics to penetrate the graft matrix that harbors the bacteria. Also, the clinical manifestations of infection become apparent after the infection is well established. Finally, thrombosis and suture-line disruption are not amenable to antibiotic therapy. The use of prophylactic antibiotics to prevent postoperative infections has many advocates. To date, there has been only one properly controlled study of this approach, and the results support the efficacy of this practice." However, the use of prophylactic antibiotics is not without complication and does not entirely eliminate graft infection. From the Department of Surgery, CMDNJ-Rutgers Medical School, Piscataway, N. J. Presented in part at the annual meeting of the Association for Academic Surgery in November, 1979. Supported by a grant from the General Research Support Fund of the College of Medicine and Dentistry of New Jersey, Rutgers Medical School. Received for publication Oct. 6, 1980. Accepted for publication Jan. 20, 1981. Address for reprints: Ralph S. Greco, M.D., Department of Surgery, CMDNJ-Rutgers Medical School, P.O. Box 101, Piscataway, N. J. 08854.
272
This study was initiated to evaluate an alternative approach to the prevention of graft infection by altering the prosthetic material itself by noncovalently bonding antibiotics to the graft surface and then evaluating its resistance to induced infection. The bonding of an antibiotic to the graft surface is an extension of the graphite-benzalkonium-heparin model developed by Whiffen and associates." They showed that graphite-coated plastic adsorbs heparin and becomes an antithrombogenic surface. Pretreatment of surfaces with benzalkonium chloride potentiated the heparin adsorption and antithrombogenic properties. The exact nature of this bond is not known, but it is postulated that the lipophilic end of the benzalkonium chloride is adsorbed to the graphite and the hydrophilic end is free to electrically bind the negatively charged heparin molecule." Previous studies in our laboratory have demonstrated that an analogous system to the graphite-benzalkonium-heparin model can be developed by replacing the heparin with a negatively charged antibiotic. 5
Methods Bacteria and sensitivity of surfactants and antibiotics. A stock culture of coagulase-positive Staphylococcus aureus was obtained from the Microbiology Department at CMDNJ-Rutgers Medical School. The original source of the bacterium was a wound culture. The minimum inhibitory concentration for oxacillin was 0.1 j.tg/ml. The minimum inhibitory concentration for benzalkonium chloride was 0.8 j.tg/ml. A checkerboard susceptibility study was performed to determine the effect of the combination of oxacillin and benzalkonium on the organism. This resulted in the minimum inhibitory concentration of 0.075 j.tg oxacillin and 0.2
0022-5223/81/080272+06$OO.60/0 © 1981 The C. V. Mosby Co.
Volume 82 Number 2
August, 1981
JLg benzalkonium, figures that suggest an additive effect but not a synergistic effect. Graft preparation. Segments of polytetrafluoroethylene* (PTFE) grafts, 0.5 cm long, 30 JL thick, and 6 mm in diameter, were prepared as follows: Group I, PTFE grafts alone; Group II, PTFE grafts soaked in 25% aqueous benzalkonium chloride for 30 minutes; Group lll, PTFE grafts soaked in IO mg oxacillin overnight at room temperature and air dried for 12 hours; Group IV, PTFE grafts soaked in benzalkonium chloride (as in Group II) and then soaked in IO mg oxacillin overnight at room temperature and air dried for 12 hours. In vitro studies. Bioassays were performed on these grafts in the following manner. Petri dishes were prepared with 35 cc of mannitol salt agar inoculated with I cc of an overnight culture of coagulase-positive Staphylococcus aureus (5 x 107 organisms). The inoculum of Staphylococcus aureus was calculated from a standard curve by the Kirby-Bauer technique. One graft from each group was embedded in the agar as it solidified. The plates were incubated at 37° C for 24 hours. The zones of inhibition of bacterial growth were measured and recorded. These bioassays were performed "acutely," that is, immediately following the various types of treatment, and at various intervals up to 90 days following preparation of the grafts. In the longitudinal studies, all grafts (Groups I to IV) were prepared and kept under dry, sterile conditions until the time of the bioassay. They were then embedded in the agar containing 5 x I Q7 freshly prepared Staphylococcus aureus. Finally, two groups of parallel evaluations were conducted to clarify the amount of antibiotic remaining on the grafts after soaking or bonding. In the first group of studies, a comparison was made between observed zones of inhibition in Groups I through IV and zones of inhibition caused by standard filter-paper discs impregnated with known concentrations of oxacillin. It was found that 10 JLg oxacillin discs cause zones of inhibition between 20 and 25 mm and I JLg oxacillin discs, zones of inhibition of 7.5 to 12.5 mm. In the second group of studies, an attempt was made to evaluate the concentration of surfactant and antibiotic present within the graft matrix after the preparation just described. This was necessary because the concentration utilized in the preparation of Groups II to IV was known but the amount remaining on the graft was *Gore-Tex, registered trademark of W. L. Gore & Associates, Inc., Elkton, Md.
Antibiotic bonding to vascular prostheses
273
Table I Surfactant treatment Benzalkonium Benzalkonium Sodium dodecyl sulfate Benzalkonium Benzalkonium Dodecyl trimethyl ammonium bromide
Antibiotic Oxacillin Carbenicillin Oxacillin Oxacillin 14C-labeled penicillin 14C-labeled penicillin
Concentration (IJ-g/cm graft)
4.0 10.0'
<0.01 4.0 10.3 15.9
Legend: The concentrations of oxacillin and carbenicillin were measured by comparison to filter paper discs impregnated with known concentrations of antibiotic in the microbiological assay. Concentrations of 14C-labeled penicillin were measured by liquid scintillation counting. All grafts were washed five times in sterile water after the bonding process was completed.
not. These studies were carried out by employing a variety of antibiotics and surfactants in the bioassay and by employing 3H-Iabeled benzalkonium and 14C_ labeled penicillin, measured by liquid scintillation counting. Since oxacillin was utilized in the graft preparation, the data utilizing penicillin are inferential and based on the following observations. Oxacillin and penicillin have different chemical structures, but both have a single net negative charge at physiological pH. This suggests that they behave in a similar fashion electrostatically. The Staphylococcus aureus used in these studies is sensitive to both penicillin and oxacillin. Both antibiotics produced zones of inhibition and minimum inhibitory concentrations of similar magnitude. Finally, the ionic nature of the bonding process was clarified by utilizing a variety of antibiotics and surfactants with complementary charges. Thus, when the cationic benzalkonium chloride was utilized with the singly negatively charged oxacillin and the doubly negatively charged carbenicillin, twice as much antibiotic was bound with carbenicillin. Similarly, when oxacillin was utilized with benzalkonium, more than 400 times as much antibiotic was bound as with the negatively charged surfactant sodium dodecyl sulfate. Also, when t4C-labeled penicillin was bound with benzalkonium and with the more cationic dodecyl trimethyl ammonium bromide, 50% more penicillin was bound with the latter. These data are summarized in Table I. Finally, the concentration of 14C-labeled penicillin expressed in the microbiological assay and that measured by liquid scintillation were compared, and it became clear that the former represents less than 20% of the latter (Table II). Thus, when 14C-Iabeled penicillin was used, grafts in Groups III and IV contained 6.04 ± 1.15 and 24.60 ±3.77JLg of antibi-
The Journal of
274 Henry, Harvey, Greco
Thoracic and Cardiovascular Surgery
PENICILLIN G
T1CARCILLIN DISODIUM
a
CEFOXITIN
®-u---r
COO-No·
+r-~~CH20CONH2
CONH +--'-...) : s saCHs
N I'CH
'0
Fig. 1. The chemical structures of anionic antibiotics demonstrating the single negative charge of penicillin, oxacillin, and cephalothin and the double negative charge of carbenicillin and ticarcillin.
Table II Penicillin bound (lLg/cm graft) Microbiological assay 14C-labeled penicillin bound (lLg/cm graft) Radiological assay Difference: Tightly bound (lLg/em graft)
1.5 10.3
8.8
Legend: The concentration of penicillin in the microbiological assay was measured by comparison to filter paper discs impregnated with a known concentration of penicillin. "C-labeled penicillin was measured by liquid scintillation counting. All grafts were washed five times in sterile water after the bonding process was completed.
otic, respectively. When 3H-labeled benzalkonium was used, grafts in Groups II and IV contained 20 ± 3.51J,g of benzalkonium. In vivo studies. The grafts were prepared in the aforementioned manner. One graft was placed in a subcutaneous pocket in the left groin of each animal. Twenty Sprague-Dawley rats were used in each of the four experimental groups. Half of the rats in each group were infected by local inoculation and half by intravenous infusion of 1 x 107 Staphylococcus aureus. After 2 weeks, the grafts were harvested and the wounds and grafts cultured. The number of bacteria in the graft matrix was evaluated by removing all identifiable fibrin and serum from the surfaces of the graft and incubating the graft in thioglycollate broth for 24 hours. The turbidity of the broth then was evaluated by measuring
optical density in a spectrophotometer and comparing the observation to the measurements obtained from known inocula of Staphylococcus aureus. A comparison of optical density and the actual number of bacteria in the broth were achieved by culturing 0.1 ml of the broth and performing colony counts. Results In vitro bioassay. A total of 85 grafts in each of the four groups were studied at 17 different time intervals up to 90 days following preparation of the grafts. Observations were performed every 2 to 3 days during the first month of the study, and a final observation, utilizing five grafts in each group, was done at 3 months. These results are shown graphically in Fig. 2. Zones of inhibition (in millimeters) in the antibiotic-soaked and the antibiotic-bonded groups are relatively constant during the first 30 days, averaging between 25 and 30 mm of inhibition. The gradual decrease in activity in the 90 day study appears constant in these two groups, but a pattern of greater activity in the PTFEbenzalkonium-oxacillin graft remains true throughout the duration of the study. The statistical evaluation of these differences was accomplished by a two-way analysis of variance with time as a repeated measure. The mean zones of inhibition of the antibiotic-soaked and antibiotic-bonded groups are not significantly different. However, both antibiotic-treated groups showed
Volume 82 Number2 August. 1981
Antibiotic bonding to vascular prostheses
35
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Fig. 2. In vitro bioassay results. A total of 85 grafts in each of the four groups was studied at 17 time intervals up to 90 days following graft preparation. Groups III and IV, the antibiotic-treated groups, show superior antibacterial activity as measured by zones of inhibition compared to other groups throughout the 90 day trial. p < 0.001 utilizing analysis of variance with time as a repeated measure.
Table III. Implantation of grafts in a rat subcutaneous pocket: Local infection with Staphylococcus aureus
Group Group Group Group
I II III IV
Wound culture (positive ltotal]
Graft culture* (positive/total)
Spectrophorometer readings of graft (mean)
4/10 2/10 3/10 0/10
10/10 9/10 10/10 2/10
1.15 0.59 1.37 0.03
No. of bacteria
I X 107 ± 4 x 10' ± 5 x 107 ± 2 x 10' ±
0.5 x 10" 5 x 10' 0.8 x 10" 1 x 10'
Legend: Group I, PTFE. Group II. PTFE-benzalkonium. Group 1lI. PTFE-oxacillin. Group IV, PTFE-benzalkonium-oxacillin.
•p
< 0.004 (Group IV versus Group I. II. or 1lI).
greater activity than the PTFE-benzalkonium group or the PTFE control group throughout the study, except for the final 90 day observation; at 90 days, the PTFEbenzalkonium grafts have slightly higher zones of inhibition than do PTFE-oxacillin grafts. The statistical analysis of the two antibiotic-treated groups (III and IV) compared to Groups I and II is statistically significant with time as a repeated measure (p < 0,001). Groups I and II had some antibacterial activity, but it was neither significant nor consistent with time. In vivo studies. The results of the rat subcutaneous study are summarized in Tables III and IV. In the group of animals inoculated locally, the wound cultures were sterile in all of Group IV grafts, whereas 40% of Group
I, 20% of Group II, and 30% of Group III grafts were infected. Infection was characterized by both the appearance of purulent material around the graft and within the graft lumen as well as colony counts. More importantly, only 20% of the PTFE-benzalkoniumoxacillin group had positive graft cultures both by optical density and by colony counts, compared with 100% of the control group, 90% of the PTFE-benzalkonium group, and 100% of the PTFE-antibiotic group. The difference between Group IV and all other groups was significant at p < 0.004 according to Fisher's exact test. The relationship between the results of wound cultures, graft cultures, measurement of bacteria in the graft by optical density, and number of bacteria in the
276
The Journal of Thoracic and Cardiovascular Surgery
Henry, Harvey, Greco
Table IV. Intravenous infection with Staphylococcus aureus
Group Group Group Group
I II III IV
Wound culture (positive [total}
Graft culture* (positive/total)
Spectrophotometer readings of graft (mean)
4/10 3/10 2/10 0/10
9/10 4/10 3/10 2/10
0.56 0.07 0.18 0.09
No. of bacteria
4 3 I 3
X X X X
10· 10' 1(j2 10'
± ± ± ±
5 2 3 2
X X X X
10' 10' 10' 10'
Legend: Group I, PTFE. Group II, PTFE-benzalkonium. Group III, PTFE-oxacillin. Group IV, PTFE-benzalkonium-oxacillin. • p < 0.001 (Groups II, III, and IV versus Group I).
graft are shown in columns 2,3,4, and 5, respectively, in Table IV. In the animals inoculated by intravenous injection of Staphylococcus aureus , the wound cultures were again sterile in all of the PTFE-benzalkonium-antibiotic group, whereas 40% of control grafts, 30% of PTFEbenzalkonium grafts, and 20% of PTFE antibioticsoaked grafts were infected. This pattern is similar to that found in the locally inoculated animals. In the graft cultures, 20% of the PTFE-benzalkonium-oxacillin group were infected as compared to 30% of the PTFEoxacillin group, 40% of the PTFE-benzalkonium group, and 90% of the control group. All treatment groups were statistically significant as compared to the control group (p < 0.001). Again, the comparison between optical density and colony counts is summarized in Table IV.
Discussion The susceptibility of vascular prostheses to infection is well documented." Despite the demonstration that implanted grafts may become infected by bacteremic challenge, the most common source of infection is contamination at the time of operation. The graft matrix, a foreign body with no intrinsic blood supply, is the likeliest site for bacterial invasion. Although the use of prophylactic systemic antibiotics has recently been supported in a randomized controlled trial, their use does not completely eradicate graft infections, and systemic antibiotics are not without complication. In 1978, our laboratory demonstrated that oxacillin, a negatively charged antibiotic, could be substituted for heparin in the graphite-benzalkonium model. Also, it is now clear that a rather large group of antibiotics, including the penicillins and their derivatives as well as the cephalosporins and cefoxitin, contain a net negative charge at physiological pH, so that they are amenable to the bonding process (Fig. 1). Thus it is possible that noncovalently bonding antibiotics to vascular grafts can be an effective means of placing small concentrations of antibiotic in the graft matrix for a variable period of
time. This procedure becomes a reasonable avenue of research in attempts at complete eradication of prosthetic infections. The in vitro studies show the superiority of the PTFE-benzalkonium-oxacillin surface in inhibiting the growth of Staphylococcus aureus under conditions of the bioassay and the persistence of this activity for a period of 3 months following graft preparation. Though there is some variability in the frequent assays done during the first month of the in vitro bioassay, the general pattern and statistical analysis corroborate that both antibiotic groups have superior activity to the PTFEbenzalkonium and PTFE control groups. The 90 day observations show persistent activity for the PTFEbenzalkonium-oxacillin group when compared to all other groups, including the PTFE-oxacillin graft. The one observation at 90 days among five grafts shows greater activity by the PTFE-benzalkonium group than the PTFE-oxacillin group. Since that is a single observation and is not statistically significant, it is not clear whether benzalkonium alone is superior to oxacillin alone as the duration of the study increases. The activity of the PTFE control is more difficult to explain. When one takes into account that the zones of inhibition include the 6 mm graft, they are very small and may be explained by the electronegative charge of the surface. Finally, comparison of the observed zones of inhibition to those achieved with standard oxacillin discs is presented only to provide a reference point to allow comparison to a known concentration of oxacillin. This technique was employed because the concentration of antibiotic utilized in the preparation of the bonded grafts and soaked grafts is known. However, the amount remaining in the graft matrix is only measured indirectly in the microbiological assay and, undoubtedly, represents far less than the concentration employed initially. This is clearly borne out by use of 14C-labeled penicillin. These studies do not clarify whether the antibacterial activity of the bonded grafts is a function of a biochemical interaction or the additive effect of two antibacte-
Volume82 Number 2 August, 1981
rial compounds. On the other hand, the data obtained with radioisotope-labeled benzalkonium and '4C_la_ beled penicillin, though inferential, show definite noncovalent bonding between antibiotic and surfactant. The demonstration that the cationic surfactant, benzalkonium, binds twice as much of the doubly negatively charged carbenicillin as the singly charged oxacillin in the microbiological assay supports a noncovalent bonding process. Similarly, the binding of 400 times as much oxacillin by the cationic surfactant, benzalkonium, as the anionic surfactant, sodium dodecyl sulfate, reinforces the noncovalent nature of the interaction. With 14C-labeled penicillin, it is likewise clear that microbiological assay measures loosely bound antibiotic representing less than 20% of the total amount of 14C-labeled penicillin bound per centimeter of graft measured by liquid scintillation. The remainder is less tightly bound, a point which may explain the greater activity found with the bonded grafts over time. Also, the grafts utilized in these studies, with the exception of those summarized in Tables II and III, were air dried and not exhaustively washed with water. Therefore, they contained a far higher concentration of the dissociable oxacillin. Thus, although oxacillin and penicillin are antibiotics with different antibacterial activities, they are electrostatically identical, and the substitution of one for the other in these studies appears valid. The studies using the rat subcutaneous pocket provide in vivo support consistent with the results in the in vitro bioassay. The most significant data are seen in the locally inoculated animals, in which wound and graft cultures of the PTFE-benzalkonium-oxacillin group show a pattern of superior antibacterial activity when compared to the control group, the PTFE-benzalkonium group, and most importantly the PTFEoxacillin group. The activity of the bonded grafts in this nonvascular model is supported both by measurement of optical density of the graft itself and by colony counts of the thioglycollate broth in which the graft was placed. In the intravenously inoculated group, the results of the wound cultures are similar to those seen in
Antibiotic bonding to vascular prostheses
27 7
the locally infected animals. The results of the graft cultures are different only in that all three treatment groups are statistically significant compared to the control group. This may well be a result of the lower inoculum reaching the graft when the bacteria are injected intravenously. These studies support the premise that appropriately charged antibiotics can be bonded to vascular prostheses by means of a surfactant with a complementary biochemical charge. Most importantly, they show that a PTFE-benzalkonium-antibiotic surface can be produced without the need for graphite, which has been associated in the past with serious operational difficulties. The PTFE grafts treated with both a surfactant and an antibiotic show superior inhibition of bacterial growth in an in vitro bioassay up to 90 days after graft preparation. This antibacterial activity appears to be present in a nonvascular subcutaneous pocket for at least 2 weeks. Though the effect of treatment on pseudointima formation has yet to be determined, it is possible that the use of a PTFE-benzalkonium-oxacillin surface, with or without perioperative parenteral antibiotics, will be a useful adjunct to attempts at complete eradication of vascular prosthetic infection. REFERENCES Christensen J, Eklof B: Synthetic arterial grafts. II. Infection complications. Scand J Thorac Cardiovasc Surg 11:43-50, 1977
2 Beebe HG: Complications of Vascular Surgery, Philadelphia, 1973, J. B. Lippincott Company 3 Kaiser AB, Clayson KR, Mulherin JL, Roach AC, Allen TR, Edwards WH, Dale WA: Antibiotic prophylaxis in vascular surgery. Ann Surg 188:283-289, 1978 4 Whiffen JD, Dutton R, Young WP, Gott VL: Heparin application to graphite-coated intravascular prostheses. Surgery 56:404-412, 1964 5 Jagpal R, Greco RS: Studies on a graphite-benzalkoniumoxacillin surface. Am Surg 45:774-779, 1979 6 Goldstone J, Moore WS: Infection in vascular prostheses. Clinical manifestations and surgical management. Am J Surg 128:225-233, 1974