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Noncovalent bonding of penicillin and cefazolin to dacron
Noncovalent Bonding of Penicillin and Cefazolin to Dacron Richard A. Harvey, PhD, New Brunswick, New Jersey John V. Tesariero, PhD, Newark, New Jersey Ralph S. Gmco, MD, FACS, New Brunswick, New Jersey
Our laboratory has described the bonding of anionic antibiotics to polytetrafluoroethylene grafts treated with cationic surfactants [l-7]. Although polytetrafluoroethylene has become the most popular prosthetic material used to bypass peripheral arterial occlusions and for vascular access, dacron remains the prosthesis of choice in the larger vessels in the chest and abdomen. This study was designed to evaluate the feasibility of bonding penicillin or cefazolin to dacron grafts treated with the surfactants, tridodecylmethylammonium chloride (TD&IAC) or benzalkonium chloride. Material and Methods Benzylpenicillin-14C was obtained from the Amersham Corporation, cefazolin14C was made available by Merck, Sharp and Dohme (West Point, PA), TDMAC-14C was synthesii by Amersham Corporation (Arlington Heights, IL), unlabeled surfactant was obtained from Polyscience (Warrington, PA), knitted dacron prostheses, 6 mm in diameter, were obtained from USC1 Surgical Products (Billerica, MA), and benzalkonium chloride was a product of Matheson, Coleman, and Bell (Norwood, OH). Radiochemicalassays: Binding of surfactant. Dacron grafts were submerged at room temperature for 30 minutes in a 5 percent solution of TDMAC in 95 percent ethanol or 5 percent benzalkonium chloride in water. TDMAC-14C used in radiolabeling experiments contained 0.5 pCi/ml. After incubation, the dacron was drained, dried for at least 1 hour at room temperature, and washed in five changes of water using a vortex stirrer. The specimens were then autoclaved at 255°F and 18 pounds per square inch for 20 minutes. Binding of radiolabeled antibiotic. After cooling, the control and surfactant-treated grafts were incubated for 1 hour at room temperature with penicillin-14C or cefazolin-14C, 10 mg/ml containing 0.25 rCi ml. The dacron was washed in five changes of sterile water as just de-
Vohmm 147. Fobuaty
1994
scribed. Radioactivity associated with the dacron and in local tissue was determined by liquid scintillation counting as described previously [2]. Bioassay: A modified agar diffusion technique was utilized in all of the studies and has been described elsewhere (21. The purpose of the bioassay was to demonstrate that bound antibiotic can give rise to biologically active molecules capable of inhibiting the growth of bacteria. Briefly, dacron grafts (6 mm internal diameter) were cut into 0.5 cm segments and embedded in brain-heart infusion medium containing 3 X 106 Staphylococcus aureus bacteria per milliliter of agar. In each assay, a control segment, a surfactant treated segment, a penicillin or cefazolm soaked segment, and a surfactant and penicillin or surfactant and cefazolin treated segment were utilized. Five assays were performed for each combination of antibiotic and surfactant. Eighteen to 24 hours after incubation at 37’C, zones of inhibition around the dacron sections were measured and compared with zones of inhibition achieved with antibiotic agar well diffusion standards to calculate the approximate concentration of antibiotic present on the dacron. The concentrations of penicillin and cefazolin measured in the bioassay were then compared with the concentrations of penicillin and cefazolin measured by liquid scintillation counting before and after completion of the bioassay. The Staph. aureus used in these assays has been described in a previous report 121.The minimum inhibitory concentration of penicillin is 0.015 pg/ml, of cefazolin 0.15 pglml, and of benzalkonium chloride 0.93 &ml. TDMAC has no antibacterial activity against this bacterium. A checkerboard susceptibility study was performed to evaluate the interaction between antibiotic and surfactant and this demonstrated a minimum inhibitory concentration of 0.015 and 0.932 fig/ml for penicillin and benzalkonium, respectively and 0.1 and 0.46 pg/ml for cefazolin and benzalkonium, respectively. The activity of penicillin and cefazolin was unchanged by TDMAC. In viva studies Sprague-Dawley rata that weighed 250 to 275 g were housed in individual cages. At operation, a 1 cm incision was made in the medial aspect of the thigh, and a pouch was created by blunt dissection in the adductor muscle. Segments of dacron were bound with 5 percent TDMAC in ethanol or 5 percent benzalkonium chloride in ethanol. Control and surfactant treated segments were then soaked in penicillin14C (10 mg/ml) or cefazolin-14C (10 mg/ml) for 30 minutes. Thirty-five segments in each group were then placed in the muscular
205
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Penklllk-WC . C9luolk-14C
1
WIWII
Figure 1. Binding of tridodecyimethyiammontum chloride- MC ( lDMAC- 14C), peniciiiin- 14C, and cefazoiin- 14C as a function of the concentration of surfactant or antibiotic used in the incubatkm. Dacron grafts were incubated with TDMAC- 14C or unia&+drmwACEolswedbyraaklabeled bkWl4C) h “AWaial and hWhoctiT&-&? 14C; 0 indicates peniciiiin- 14C; 0 indicates cefazoiin- 14C.
pouch and harvested at 1,3,6,12,24, and 72 hours and at
10 days after implantation. Five animals were sacrificed at each time interval. The concentration of antibiotic remaining on the dacron was measured by liquid scintillation counting. Five milligrams of adjacent thigh muscle was also harvested, and the antibiotic concentration was determined. In 75 rats, TDMAC-bonded, benzalkonium-bonded, or untreated dacron grafts were implanted. At completion of implantation in tlie muscle pouch, 10 mg of penicillin-14C or cefazolin-14C in 1 ml of water was instilled directly over the graft and the wound closed as just described. Five rats in each group were sacrificed at 1,3,6,12, and 24 hours. The conceritration of antibiotic in dacron was then determined by liquid scintillation counting. In the last group of 75 rats, surfactant-bound dacron grafts and control dacron grafts were placed in the muscle pouch. After wound closure, 10 mg of penicillin-14C or cefholin-14C in 1 ml of water was given by tail vein injection. Five rats in each group were sacrificed at 1,3,6,12, and 24 hours and the grafts harvested. The concentration of penicillin-14C and cefazolin-14C on the dacron was then determined by liquid scintillation counting. Results Dacron grafts treated with TDMAC-14C and washed exhaustively with water retained significant quantities of the cationic surfactant (Figure 1). Over the concentration range tested, the amount of TDMAC bound to the graft increased with the concentration of surfactant used in the incubation. A concentration of 5 percent TDMAC (50 mg/ml) was used as the standard in all studies reported. Exposure of TDMAC-treated prostheses to various concentrations of penicillin-14C or cefazolin-14C resulted in the binding of therapeutic amounts of antibiotic (Figure 1). The binding of penicillin-14C or cefazolin-14C reached a maximum at high levels of free antibiotic which indicated saturation of a limited number of binding sites. Untreated grafts 206
UhlDH”
Figure 2. ouanfnatkm of bound penicillin and cefazoiin &tetmined by mkrobWogk assay and by liquid scintiiiation counting before and after bioassay. q indicates antibiotic oniy; n indicates benzaikonium plus antWotic; 0 indicates trldodecyimethyiammonium chloride pius antibiotic.
binded l,OOO-fold less antibiotic (less than 4 pg/cm) than the TDMAC-treated prostheses. Since the retention of radioactivity on the TDMAC-treated dacron grafts may represent binding of an inactivated form of the molecule, quantitation of binding by a biologic assay was performed. The treated grafts showed an antimicrobial activity that corresponded to antibiotic levels similar to those determined by direct radiochemical assay (Figure 2). Thus, bound radioactivity reflects the adsorption of biologically active antibiotic. The determination of penicillin-14C or cefazolin-14C retained after the bioassays showed that part of the antibiotic dissociates from the surface of the graft during the assay (Figure 2), thus explaining the antibacterial activity of the prosthesis. It is clear that the retention of penicillin or cefazolin on the TDMAC-treated dacron surface depends on the chemical nature of the bathing solution; that is, the antibiotic does not dissociate when washed with water but is partially released when exposed to the complex broth employed in the microbiologic assay. In an effort to simulate in vivo elution, conditions, grafts treated first with surfactants TDMAC or benzalkonium chloride and then radiolabeled antibiotic were placed in surgically prepared pouches in the adductor muscle of rats. At various intervals, the dacron grafts were removed and counted to determine retained antibiotic. Figure 3 shows that the initial binding as well as the retention of penicillin14C was greater with the TDMAC-treated grafts compared with dacron incubated with benzalkonium chloride (p <0.02). With either surfactant, the binding and retention of antibiotic is greater than that observed with untreated grafts (p <0.02). Similar data were obtained with cefazolin-14C (Figure 3), although dissociation from the TDMAC surface appeared to be somewhat more rapid than with penicillin-14C. To determine whether the binding of antibiotic to The American Journal of Surgery
Noncovalent
Bonding
Cefazolin
> Penicillin >
0
24
48
72
Hours after implantation
Ffgwe 4. l’fewe kvefs of penk#Un-14C and cduoUn- 14C affer pkcemenf ofgra& h rai rmmcfapouch. 0 amI n h&ate unifwi@d~AandAhdkaie~~~~~
0 and* IndkategratYstmatedwffh~ chkrfde. @en symbofsare cefazoffn- 14C and cksed sydk am panfcfflbt- 14C. F&u-a 3. R-ion offwumfpenkfflh- 14C and cefazoffn-14C to grafb afbr @ammen! h rat musck pouch. El and W hdkafe
u~gnlk;A~Afndkaie~rafis~adwlfhbenzalkonkrm (8K); 0 ad 0 fndkate pft8 tmated wfth trfdmhcylv CMorkk (7DMAC). Open symfwfs art3 cefazoffn- 14C and c&bed syn&ok are penkillh- 14C.
prostheses results in therapeutic levels of the drug in the local environment, tissue samples adjacent to the grafts (Figure 3) were counted for penicillin-14C or cefazolin-14C. Tissue levels of antibiotic derived from the TDMAC and penicillin-14C or the benzalkonium chloride and penicillin-14C grafts were significantly higher (p <0.03) than the untreated control levels during the first hour of implantation in the muscle pouch (Figure 4). After 1 hour, the tissue concentrations of antibiotic were equal to or in some instances less than the untreated control concentrations. In contrast, tissue adjacent to the TDMAC and cefazolin14C graft showed significantly higher (p <0.03) levels of antibiotic at all time intervals (Figure 4). The benzalkonium chloride and cefazolin graft was somewhat less effective in maintaining local tissue concentrations of antibiotic. Procedures involving first the preparation of surfactant and antibiotic treated grafts in the laboratory followed by implantation in a test animal have the advantage that binding and subsequent elution are clearly separate processes. However, in certain clinical situations it may be desirable to implant a surfactant treated graft and then irrigate the surgical field, including the graft, with an appropriate solution of antibiotic. To demonstrate the feasibility of local irrigation with antibiotic, grafts treated with TDMAC or benzalkonium chloride, as well as untreated grafts, were placed in muscle pouches. Before closing the wound, 10 mg of penicillin-14C or cefazolin-14C in 1 ml of water was directly irrigated over the graft, and the pouch was then closed. After variVohnna 147, Fobrwy 1984
ous intervals, the grafts were removed and analyzed for bound antibiotic. Figure 5 shows that the TDMAC-treated dacron grafts were highly efficient in binding cefazolin-14C. In contrast, the benzalkonium chloride treated grafts were essentially indistinguishable from the untreated control grafts. Virtually identical results were obtained with penicillin-14C (not presented herein). A second clinically relevant method for administering antibiotic is by intravenous injection. TDMAC- or benzalkonium chloride-treated grafts were placed in a muscle pouch, and the host rat was injected in the tail vein with 10 mg of penicillin-14C or cefazolin-14C in 1 ml of water. Grafts were removed at various intervals after injection and the amount of bound antibiotic was determined (Figure 6). As was seen with local irrigation, the TDMACtreated grafts were effective in sequestering systemic antibiotics. Binding by the benzalkonium chloride treated grafts was similar to that observed in the untreated control grafts. commsnts Dacron grafts adsorb the surfactant TDMAC-14C, and these treated prostheses can bind therapeutic amounts of penicillin-14C or cefazolin-14C. This observation is significant because of the clinical usefulness of dacron and because the data document the general validity of findings previously reported only for polytetrafluoroethylene. Penicillin-G and cefazolin are similar but chemically distinct, representing prototypes for the penicihin and cephalosporin families of antibiotics. Both contain a single negative charge suggesting an ionic interaction with the positive TDMAC molecule bound to the surface of the dacron graft. In contrast to these chemical similarities, the two antimicrobial agents show 207
Harvey et al
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Hours rttef Imphntdom 5. Retenttonof bound cefazolln-14C after local Irrigation with antlbktk and placement In rat muscle pouch. 0 lndlcates unhated gram A tndkates grafts treated wHhbenzalkonlum; 0 lndkates grafts treated w/th trhlork+cy/methykmmonlum chkrme.
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markedly different biologic properties. For example, penicillin compared with cefazolin shows a much shorter biologic half-life (30 minutes versus 185 minutes), reduced binding to serum proteins, and an increased susceptibility to the action of the P-lactamases [&IO]. The longer half-life of cefazolin in conjunction with its somewhat more rapid release from the dacron surface when placed in the rat
208
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Figure 6. Blndingof Intravenouslyadmlnkteredpenlclllln- 14C or cefazolln-14C to grafts In rat muscle pouch. I3 and H Indicate untreated grafts; A and A lndlcate grafts treated with benzalkonlum; 0 and 0 lndlcate graffs treafed wffh frklodecyfn?ethyf8mm&fmchkrlde.~s~an,cefarokh-14Catnfcbsed symbolsare f.wnklllk- 14C.
muscle pouch (Figure 3) may, in part, explain the higher tissue levels of cefazolin observed (Figure 4). The successful application of antibiotic bonding to dacron prostheses extends this technique to vascular grafting in the larger vessels of the chest and abdomen. Clinically, this will allow the technique to be applied to the repair of abdominal aortic aneurysms in which infection is less common but a far more devastating complication. These sites are also theoretically advantageous because one of the drawbacks of cationic binding systems is their potential predisposition to thrombosis. The diameter and pressure characteristics of the aorta make this complication improbable. These studies are also noteworthy in that they si-
The American Journal of Surgery
multaneously evaluate antibiotic bonding with both a penicillin and a cephalosporin. The similarity of bonding supports the biochemical hypotheses of the technique. Nevertheless, the biologic properties of these antibiotics are quite different. The more rapid elution of cefazolin from the grafts which, in turn, produce higher local tissue concentrations of the drug, makes the cephalosporins an ideal group of antibiotics for this type of infection prophylaxis. Finally, the demonstration that dacron, like polytetrafluoroethylene, can bind antibiotics administered locally or parenterally, suggests a therapeutic model with broad clinical utility. Ultimately, however, the clinical use of cationic binding systems will depend on their ability to prevent graft infection and to have no ill effects on the biocompatability and durability of the prosthesis. Both of these parameters will be the subject of future investigation. Preliminary studies in the dog show no deleterious effects of antibiotic binding on the ultrastructure of these grafts after 6 weeks of implantation in the abdominal aorta (Figure 7). Summary Dacron grafts treated with the surfactant, benzalkonium chloride or TDMAC bind significant quantities of penicillin-14C or cefazolin-14C. The treated grafts showed strong antibacterial activity which indicated that bound radioactivity corresponds to the reversible adsorption of biologically active molecules. Bound penicillin-14C or cefazo-
Volume 147, February 1984
lin-14C slowly dissociates when the grafts are placed in a surgically prepared muscle pouch in the rat. This slow release of antibiotic produces therapeutic levels of antibiotic in the adjacent tissue. Binding can also be achieved by in situ irrigation of surfactant treated grafts with antibiotic or by injection of the antibiotic. References 1. Greco RS, HarveyRA, HenryR, RrahladA. Preventfonof waft infection by antibiotic bonding.Surg Forum 1980;31:2930. 2. HarveyRA, Creco RS. The non-covalentbondingof antibiotics to a polytetrafluoroethylene-benzalkonium graft. Ann Swg 1981;194:642-7. 3. HenryR, HarveyRA, GrecoRS. Antibioticbondingto vascutar prostfteses.J Thorac CardiovascSurg 1981;82:272-7. 4. HarveyRA,GrecoRS.Antibi&blndingtovascufarpm&eses. Fed Proc 1982;41:1556. 5. PrahladA. HarveyRA, GrecoRS. Dfffu&n of antibioticsfrom a polytetrafluoroetfrylene-benzalkonkrm surface. Am Surg 1981;47:515-8. 6. Greco RS, Harvey RA. The rote of antibioticbondingin the prevention of vascular prosthetic infectlons. Ann Surg 1982;195:16&71. 7. GrecoRS, HarveyRA, SmitowPL, TesorteroJV. Preventfonof vascular prostheticinfectfonby a benzafkonium-oxaclllin bondedpolytetrafluoroethylene graft. SurgQynecolDbstet 1982;155:28-32. 8. SakentineR, Huber SL. Cephafosporlns:an update. Am J Intraven Ther Clin Nutr 1982;9:19-25. 9. Neu HC. Clinicalpharmacoklneticsin preventiveantimicrobtal therapy.SouthMed J 1977;70:14-23. 10. Neu HC. The in vltroactivity,humsnphamacoloeyandcllnlcal effectivenessof new B-lactam antibiotics.Ann Rev Pharmacol Toxidol 1982;22:599-042.
209
Our laboratory has described the bonding of anionic antibiotics to polytetrafluoroethylene grafts treated with cationic surfactants [l-7]. Although polytetrafluoroethylene has become the most popular prosthetic material used to bypass peripheral arterial occlusions and for vascular access, dacron remains the prosthesis of choice in the larger vessels in the chest and abdomen. This study was designed to evaluate the feasibility of bonding penicillin or cefazolin to dacron grafts treated with the surfactants, tridodecylmethylammonium chloride (TD&IAC) or benzalkonium chloride. Material and Methods Benzylpenicillin-14C was obtained from the Amersham Corporation, cefazolin14C was made available by Merck, Sharp and Dohme (West Point, PA), TDMAC-14C was synthesii by Amersham Corporation (Arlington Heights, IL), unlabeled surfactant was obtained from Polyscience (Warrington, PA), knitted dacron prostheses, 6 mm in diameter, were obtained from USC1 Surgical Products (Billerica, MA), and benzalkonium chloride was a product of Matheson, Coleman, and Bell (Norwood, OH). Radiochemicalassays: Binding of surfactant. Dacron grafts were submerged at room temperature for 30 minutes in a 5 percent solution of TDMAC in 95 percent ethanol or 5 percent benzalkonium chloride in water. TDMAC-14C used in radiolabeling experiments contained 0.5 pCi/ml. After incubation, the dacron was drained, dried for at least 1 hour at room temperature, and washed in five changes of water using a vortex stirrer. The specimens were then autoclaved at 255°F and 18 pounds per square inch for 20 minutes. Binding of radiolabeled antibiotic. After cooling, the control and surfactant-treated grafts were incubated for 1 hour at room temperature with penicillin-14C or cefazolin-14C, 10 mg/ml containing 0.25 rCi ml. The dacron was washed in five changes of sterile water as just de-
Vohmm 147. Fobuaty
1994
scribed. Radioactivity associated with the dacron and in local tissue was determined by liquid scintillation counting as described previously [2]. Bioassay: A modified agar diffusion technique was utilized in all of the studies and has been described elsewhere (21. The purpose of the bioassay was to demonstrate that bound antibiotic can give rise to biologically active molecules capable of inhibiting the growth of bacteria. Briefly, dacron grafts (6 mm internal diameter) were cut into 0.5 cm segments and embedded in brain-heart infusion medium containing 3 X 106 Staphylococcus aureus bacteria per milliliter of agar. In each assay, a control segment, a surfactant treated segment, a penicillin or cefazolm soaked segment, and a surfactant and penicillin or surfactant and cefazolin treated segment were utilized. Five assays were performed for each combination of antibiotic and surfactant. Eighteen to 24 hours after incubation at 37’C, zones of inhibition around the dacron sections were measured and compared with zones of inhibition achieved with antibiotic agar well diffusion standards to calculate the approximate concentration of antibiotic present on the dacron. The concentrations of penicillin and cefazolin measured in the bioassay were then compared with the concentrations of penicillin and cefazolin measured by liquid scintillation counting before and after completion of the bioassay. The Staph. aureus used in these assays has been described in a previous report 121.The minimum inhibitory concentration of penicillin is 0.015 pg/ml, of cefazolin 0.15 pglml, and of benzalkonium chloride 0.93 &ml. TDMAC has no antibacterial activity against this bacterium. A checkerboard susceptibility study was performed to evaluate the interaction between antibiotic and surfactant and this demonstrated a minimum inhibitory concentration of 0.015 and 0.932 fig/ml for penicillin and benzalkonium, respectively and 0.1 and 0.46 pg/ml for cefazolin and benzalkonium, respectively. The activity of penicillin and cefazolin was unchanged by TDMAC. In viva studies Sprague-Dawley rata that weighed 250 to 275 g were housed in individual cages. At operation, a 1 cm incision was made in the medial aspect of the thigh, and a pouch was created by blunt dissection in the adductor muscle. Segments of dacron were bound with 5 percent TDMAC in ethanol or 5 percent benzalkonium chloride in ethanol. Control and surfactant treated segments were then soaked in penicillin14C (10 mg/ml) or cefazolin-14C (10 mg/ml) for 30 minutes. Thirty-five segments in each group were then placed in the muscular
205
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.
Penklllk-WC . C9luolk-14C
1
WIWII
Figure 1. Binding of tridodecyimethyiammontum chloride- MC ( lDMAC- 14C), peniciiiin- 14C, and cefazoiin- 14C as a function of the concentration of surfactant or antibiotic used in the incubatkm. Dacron grafts were incubated with TDMAC- 14C or unia&+drmwACEolswedbyraaklabeled bkWl4C) h “AWaial and hWhoctiT&-&? 14C; 0 indicates peniciiiin- 14C; 0 indicates cefazoiin- 14C.
pouch and harvested at 1,3,6,12,24, and 72 hours and at
10 days after implantation. Five animals were sacrificed at each time interval. The concentration of antibiotic remaining on the dacron was measured by liquid scintillation counting. Five milligrams of adjacent thigh muscle was also harvested, and the antibiotic concentration was determined. In 75 rats, TDMAC-bonded, benzalkonium-bonded, or untreated dacron grafts were implanted. At completion of implantation in tlie muscle pouch, 10 mg of penicillin-14C or cefazolin-14C in 1 ml of water was instilled directly over the graft and the wound closed as just described. Five rats in each group were sacrificed at 1,3,6,12, and 24 hours. The conceritration of antibiotic in dacron was then determined by liquid scintillation counting. In the last group of 75 rats, surfactant-bound dacron grafts and control dacron grafts were placed in the muscle pouch. After wound closure, 10 mg of penicillin-14C or cefholin-14C in 1 ml of water was given by tail vein injection. Five rats in each group were sacrificed at 1,3,6,12, and 24 hours and the grafts harvested. The concentration of penicillin-14C and cefazolin-14C on the dacron was then determined by liquid scintillation counting. Results Dacron grafts treated with TDMAC-14C and washed exhaustively with water retained significant quantities of the cationic surfactant (Figure 1). Over the concentration range tested, the amount of TDMAC bound to the graft increased with the concentration of surfactant used in the incubation. A concentration of 5 percent TDMAC (50 mg/ml) was used as the standard in all studies reported. Exposure of TDMAC-treated prostheses to various concentrations of penicillin-14C or cefazolin-14C resulted in the binding of therapeutic amounts of antibiotic (Figure 1). The binding of penicillin-14C or cefazolin-14C reached a maximum at high levels of free antibiotic which indicated saturation of a limited number of binding sites. Untreated grafts 206
UhlDH”
Figure 2. ouanfnatkm of bound penicillin and cefazoiin &tetmined by mkrobWogk assay and by liquid scintiiiation counting before and after bioassay. q indicates antibiotic oniy; n indicates benzaikonium plus antWotic; 0 indicates trldodecyimethyiammonium chloride pius antibiotic.
binded l,OOO-fold less antibiotic (less than 4 pg/cm) than the TDMAC-treated prostheses. Since the retention of radioactivity on the TDMAC-treated dacron grafts may represent binding of an inactivated form of the molecule, quantitation of binding by a biologic assay was performed. The treated grafts showed an antimicrobial activity that corresponded to antibiotic levels similar to those determined by direct radiochemical assay (Figure 2). Thus, bound radioactivity reflects the adsorption of biologically active antibiotic. The determination of penicillin-14C or cefazolin-14C retained after the bioassays showed that part of the antibiotic dissociates from the surface of the graft during the assay (Figure 2), thus explaining the antibacterial activity of the prosthesis. It is clear that the retention of penicillin or cefazolin on the TDMAC-treated dacron surface depends on the chemical nature of the bathing solution; that is, the antibiotic does not dissociate when washed with water but is partially released when exposed to the complex broth employed in the microbiologic assay. In an effort to simulate in vivo elution, conditions, grafts treated first with surfactants TDMAC or benzalkonium chloride and then radiolabeled antibiotic were placed in surgically prepared pouches in the adductor muscle of rats. At various intervals, the dacron grafts were removed and counted to determine retained antibiotic. Figure 3 shows that the initial binding as well as the retention of penicillin14C was greater with the TDMAC-treated grafts compared with dacron incubated with benzalkonium chloride (p <0.02). With either surfactant, the binding and retention of antibiotic is greater than that observed with untreated grafts (p <0.02). Similar data were obtained with cefazolin-14C (Figure 3), although dissociation from the TDMAC surface appeared to be somewhat more rapid than with penicillin-14C. To determine whether the binding of antibiotic to The American Journal of Surgery
Noncovalent
Bonding
Cefazolin
> Penicillin >
0
24
48
72
Hours after implantation
Ffgwe 4. l’fewe kvefs of penk#Un-14C and cduoUn- 14C affer pkcemenf ofgra& h rai rmmcfapouch. 0 amI n h&ate unifwi@d~AandAhdkaie~~~~~
0 and* IndkategratYstmatedwffh~ chkrfde. @en symbofsare cefazoffn- 14C and cksed sydk am panfcfflbt- 14C. F&u-a 3. R-ion offwumfpenkfflh- 14C and cefazoffn-14C to grafb afbr @ammen! h rat musck pouch. El and W hdkafe
u~gnlk;A~Afndkaie~rafis~adwlfhbenzalkonkrm (8K); 0 ad 0 fndkate pft8 tmated wfth trfdmhcylv CMorkk (7DMAC). Open symfwfs art3 cefazoffn- 14C and c&bed syn&ok are penkillh- 14C.
prostheses results in therapeutic levels of the drug in the local environment, tissue samples adjacent to the grafts (Figure 3) were counted for penicillin-14C or cefazolin-14C. Tissue levels of antibiotic derived from the TDMAC and penicillin-14C or the benzalkonium chloride and penicillin-14C grafts were significantly higher (p <0.03) than the untreated control levels during the first hour of implantation in the muscle pouch (Figure 4). After 1 hour, the tissue concentrations of antibiotic were equal to or in some instances less than the untreated control concentrations. In contrast, tissue adjacent to the TDMAC and cefazolin14C graft showed significantly higher (p <0.03) levels of antibiotic at all time intervals (Figure 4). The benzalkonium chloride and cefazolin graft was somewhat less effective in maintaining local tissue concentrations of antibiotic. Procedures involving first the preparation of surfactant and antibiotic treated grafts in the laboratory followed by implantation in a test animal have the advantage that binding and subsequent elution are clearly separate processes. However, in certain clinical situations it may be desirable to implant a surfactant treated graft and then irrigate the surgical field, including the graft, with an appropriate solution of antibiotic. To demonstrate the feasibility of local irrigation with antibiotic, grafts treated with TDMAC or benzalkonium chloride, as well as untreated grafts, were placed in muscle pouches. Before closing the wound, 10 mg of penicillin-14C or cefazolin-14C in 1 ml of water was directly irrigated over the graft, and the pouch was then closed. After variVohnna 147, Fobrwy 1984
ous intervals, the grafts were removed and analyzed for bound antibiotic. Figure 5 shows that the TDMAC-treated dacron grafts were highly efficient in binding cefazolin-14C. In contrast, the benzalkonium chloride treated grafts were essentially indistinguishable from the untreated control grafts. Virtually identical results were obtained with penicillin-14C (not presented herein). A second clinically relevant method for administering antibiotic is by intravenous injection. TDMAC- or benzalkonium chloride-treated grafts were placed in a muscle pouch, and the host rat was injected in the tail vein with 10 mg of penicillin-14C or cefazolin-14C in 1 ml of water. Grafts were removed at various intervals after injection and the amount of bound antibiotic was determined (Figure 6). As was seen with local irrigation, the TDMACtreated grafts were effective in sequestering systemic antibiotics. Binding by the benzalkonium chloride treated grafts was similar to that observed in the untreated control grafts. commsnts Dacron grafts adsorb the surfactant TDMAC-14C, and these treated prostheses can bind therapeutic amounts of penicillin-14C or cefazolin-14C. This observation is significant because of the clinical usefulness of dacron and because the data document the general validity of findings previously reported only for polytetrafluoroethylene. Penicillin-G and cefazolin are similar but chemically distinct, representing prototypes for the penicihin and cephalosporin families of antibiotics. Both contain a single negative charge suggesting an ionic interaction with the positive TDMAC molecule bound to the surface of the dacron graft. In contrast to these chemical similarities, the two antimicrobial agents show 207
Harvey et al
1
4
1000
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2
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1
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. . . . . . . ““.O
0
24
Hours rttef Imphntdom 5. Retenttonof bound cefazolln-14C after local Irrigation with antlbktk and placement In rat muscle pouch. 0 lndlcates unhated gram A tndkates grafts treated wHhbenzalkonlum; 0 lndkates grafts treated w/th trhlork+cy/methykmmonlum chkrme.
F&we
markedly different biologic properties. For example, penicillin compared with cefazolin shows a much shorter biologic half-life (30 minutes versus 185 minutes), reduced binding to serum proteins, and an increased susceptibility to the action of the P-lactamases [&IO]. The longer half-life of cefazolin in conjunction with its somewhat more rapid release from the dacron surface when placed in the rat
208
8
0
12 noun
a.
I-
0
4
alter
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8
12
injection
Figure 6. Blndingof Intravenouslyadmlnkteredpenlclllln- 14C or cefazolln-14C to grafts In rat muscle pouch. I3 and H Indicate untreated grafts; A and A lndlcate grafts treated with benzalkonlum; 0 and 0 lndlcate graffs treafed wffh frklodecyfn?ethyf8mm&fmchkrlde.~s~an,cefarokh-14Catnfcbsed symbolsare f.wnklllk- 14C.
muscle pouch (Figure 3) may, in part, explain the higher tissue levels of cefazolin observed (Figure 4). The successful application of antibiotic bonding to dacron prostheses extends this technique to vascular grafting in the larger vessels of the chest and abdomen. Clinically, this will allow the technique to be applied to the repair of abdominal aortic aneurysms in which infection is less common but a far more devastating complication. These sites are also theoretically advantageous because one of the drawbacks of cationic binding systems is their potential predisposition to thrombosis. The diameter and pressure characteristics of the aorta make this complication improbable. These studies are also noteworthy in that they si-
The American Journal of Surgery
multaneously evaluate antibiotic bonding with both a penicillin and a cephalosporin. The similarity of bonding supports the biochemical hypotheses of the technique. Nevertheless, the biologic properties of these antibiotics are quite different. The more rapid elution of cefazolin from the grafts which, in turn, produce higher local tissue concentrations of the drug, makes the cephalosporins an ideal group of antibiotics for this type of infection prophylaxis. Finally, the demonstration that dacron, like polytetrafluoroethylene, can bind antibiotics administered locally or parenterally, suggests a therapeutic model with broad clinical utility. Ultimately, however, the clinical use of cationic binding systems will depend on their ability to prevent graft infection and to have no ill effects on the biocompatability and durability of the prosthesis. Both of these parameters will be the subject of future investigation. Preliminary studies in the dog show no deleterious effects of antibiotic binding on the ultrastructure of these grafts after 6 weeks of implantation in the abdominal aorta (Figure 7). Summary Dacron grafts treated with the surfactant, benzalkonium chloride or TDMAC bind significant quantities of penicillin-14C or cefazolin-14C. The treated grafts showed strong antibacterial activity which indicated that bound radioactivity corresponds to the reversible adsorption of biologically active molecules. Bound penicillin-14C or cefazo-
Volume 147, February 1984
lin-14C slowly dissociates when the grafts are placed in a surgically prepared muscle pouch in the rat. This slow release of antibiotic produces therapeutic levels of antibiotic in the adjacent tissue. Binding can also be achieved by in situ irrigation of surfactant treated grafts with antibiotic or by injection of the antibiotic. References 1. Greco RS, HarveyRA, HenryR, RrahladA. Preventfonof waft infection by antibiotic bonding.Surg Forum 1980;31:2930. 2. HarveyRA, Creco RS. The non-covalentbondingof antibiotics to a polytetrafluoroethylene-benzalkonium graft. Ann Swg 1981;194:642-7. 3. HenryR, HarveyRA, GrecoRS. Antibioticbondingto vascutar prostfteses.J Thorac CardiovascSurg 1981;82:272-7. 4. HarveyRA,GrecoRS.Antibi&blndingtovascufarpm&eses. Fed Proc 1982;41:1556. 5. PrahladA. HarveyRA, GrecoRS. Dfffu&n of antibioticsfrom a polytetrafluoroetfrylene-benzalkonkrm surface. Am Surg 1981;47:515-8. 6. Greco RS, Harvey RA. The rote of antibioticbondingin the prevention of vascular prosthetic infectlons. Ann Surg 1982;195:16&71. 7. GrecoRS, HarveyRA, SmitowPL, TesorteroJV. Preventfonof vascular prostheticinfectfonby a benzafkonium-oxaclllin bondedpolytetrafluoroethylene graft. SurgQynecolDbstet 1982;155:28-32. 8. SakentineR, Huber SL. Cephafosporlns:an update. Am J Intraven Ther Clin Nutr 1982;9:19-25. 9. Neu HC. Clinicalpharmacoklneticsin preventiveantimicrobtal therapy.SouthMed J 1977;70:14-23. 10. Neu HC. The in vltroactivity,humsnphamacoloeyandcllnlcal effectivenessof new B-lactam antibiotics.Ann Rev Pharmacol Toxidol 1982;22:599-042.
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