JOURNAL

OF SURGICAL

RESEARCH

Prophylactic

52.101-105

(19%)

Antibiotics

Prevent Bacterial Biofilm Graft Infection

THOMASM. BERGAMINI,M.D.,l JAMESC.PEYTON, M.S., ANDWILLIAMG.CHEADLE, M.D. Department Presented

of Surgery, University at the Annual

of Louisville

Symposium

School of Medicine,

of the Association

and the Veterans Administration

of Veterans

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’ colony-forming unit&ma 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 1530 pg/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 < 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 < 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. 0 1992 Academic Press, Inc.

INTRODUCTION Graft infection is a dreaded, serious complication of vascular reconstructions that frequently results in loss of organ function, limb, and life. Refinements in vascular surgical techniques, prosthetic biomaterials, and prophylactic antibiotics have resulted in a significant reduction of early postoperative graft infections [l, 21. 1 To whom reprint

requests should be addressed. 101

Administration

Medical Center, Louisville,

Surgeons, Milwaukee,

Wisconsin,

Kentucky

40292

May 9-11, 1991

During the past two decades graft infection has shifted to the late postoperative period, occurring months to years after implantation [3,4]. The late-appearing graft infections are due to Staphylococcus epidermidis, which colonize and adhere to the prosthesis and grow dormant within a surface biofilm. The low virulence pathogens ultimately produce inflammation of the perigraft tissues that clinically present as either an anastomotic aneurysm, or as a graft-cutaneous sinus tract or per&aft abscess in patients without systemic signs of sepsis (fever, leukocytosis, bacteremia). The vascular surgery patient’s own endogenous flora is the most likely source of S. epidermidis that colonize the graft. Several tissues which come into direct contact with the graft at the time of implantation have been shown to harbor the low virulence bacteria [5-71. S. epidermidis has been recovered from the skin, subcutaneous fat, lymph nodes, and arterial wall of greater than one-third of patients undergoing vascular reconstruction, despite the use of aseptic vascular surgical technique and prophylactic antibiotics. Culturing S. epidermidis from the arterial wall at the time of the vascular reconstruction has been associated with a significant increase in the incidence of graft infection [8]. Minimizing the quantity of bacteria in the perigraft tissues during vascular surgical procedures with the judicious use of prophylactic antibiotics would most effectively prevent prostheses colonization and the late appearance of bacterial biofilm graft infection. The success of surgical prophylaxis in preventingpostoperative infection is dependent on the pharmacokinetits of antibiotic tissue penetrance [9]. Two prospective, randomized, double-blind clinical trials [lo, 111 have convincingly demonstrated that prophylactic antibiotics failed to prevent infection for procedures lasting longer than 3 hr, in which adequate antibiotic levels in the tissues were not maintained for the duration of the operation (the period of potential contamination) [9]. Pharmacokinetic studies of vascular surgery patients have demonstrated that adequate tissue antibiotic levels may not be maintained with current prophylactic regimens and have suggested that higher antibiotic doses at more frequent intervals may be needed [12, 131. This study investigates the effectiveness of standard and high dose 0022-4804/92 $1.50 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

102

JOURNAL

OF SURGICAL

RESEARCH:

prophylactic antibiotic regimens on the prevention of bacterial biofilm graft infection in a rat model. The level of antibiotic needed in the tissue at the time of graft implantation to significantly reduce the concentration of bacteria in the surface biofilm and prevent infection is determined. MATERIAL

AND

METHODS

Rut model. Adult female Sprague-Dawley rats (Harlan Sprague-Dawley, Indianapolis, IN), weighing between 200 to 250 g, were anesthetized with an intramuscular injection of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (15 mg/kg). The hair of the back was shaved and the skin cleansed with 10% povidone-iodine solution. One subcutaneous pocket was made on each side of the midline. Dacron grafts (1 cm’) colonized with slime-producing S. epiderhidis were implanted into the pockets and the incisions were closed primarily. Prior to implantation, the Dacron graft segments were colonized with S. epidermidis in tryptic soy broth for 18 hr at 35°C. After incubation, the graft segment had an adherent bacterial surface biofilm [ 141 containing 3.19 f 0.71 X lo7 colony-forming units/cm2 graft. The S. epidermidis study strain was a slime-producing clinical isolate. This study strain was chosen because it had intermediate sensitivity to cefazolin with a minimum inhibitory concentration (MIC) of 15-30 pg/ml and permitted the attainment of antibiotic tissue levels higher, equal to, and below the MIC of the bacteria for the duration of time between preoperative antibiotic administration and graft implantation. Seventy-four Dacron grafts were implanted at 0.5, 2, and 4 hr after antibiotic administration of intraperitoneal cefazolin. Sixteen control animals had grafts colonized with an adherent surface biofilm implanted without prior antibiotic administration. Cefazolin, a first-generation cephalosporin, was chosen as the prophylactic agent because it had minimal toxicity over a wide range of doses; it was a commonly used antibiotic for surgical prophylaxis in vascular surgical patients; and it achieved adequate tissue levels after a single dose with maintenance for 2 to 3 hr. The dosage of cefazolin was chosen because of the correlation between the tissue antibiotic levels achieved and the MIC of the study strain. Sixteen grafts were implanted at each of the three time intervals after the standard dose (15 or 30 mg/kg intraperitoneal), which was equivalent to a 1 to 2 g dose in man. The standard dose was chosen because the antibiotic tissue level peaked at levels equal to the MIC of the study strain and decreased below the MIC by 2 hr. Four grafts were implanted 2 hr after a second dose (redose) of the standard dose, which was given 2 hr after the first dose. Ten grafts were implanted at 0.5 hr and eight grafts were implanted at both 2 and 4 hr after the high dose (120 mg/kg intraperitoneal), which was chosen because anti-

VOL.

52, NO. 2, FEBRUARY

1992

biotic tissue levels peaked above the MIC within 1 hr of administration and decreased below the MIC after 4 hr. Tissue antibiotic levels were determined using a standard bioassay [15]. Specimens of subcutaneous tissue were taken at 0.5,1,2, and 4 hr after a single intraperitoneal dose of cefazolin for the standard and high doses. The subcutaneous tissues were homogenized and the tissue antibiotic levels were assayed on agar plates inoculated with Bacillus subtilis ATCC 6633 (American Type Culture Collection, Rockville, MD) spore suspension. The zone of inhibition was determined and calibrated with a standard curve. The study was approved by the University of Louisville and the Veterans Administration Medical Center Animal Care Committee. Graft explanation. The grafts were sterilely removed 1 week after implantation and the animals were sacrificed. The grafts were sonicated to disrupt the adherent surface biofilm and cultured on agar and broth media. The concentration of bacteria in the surface biofilm culture was obtained by culturing the sonication effluent on agar media after serial dilutions and incubation at 35°C [16]. Broth culture of the sonicated graft and effluent was incubated at 35°C for recovering the study strain at low concentrations in the bacterial biofilm. Data analysis. Statistical comparisons between groups were made using analysis of variance (ANOVA) with Tukey-Kramer Honestly Significant Difference Test. RESULTS

Effectiveness of dosage based on duration of procedure. The concentration of S. epidermidis study strain in the surface biofilm of the explanted Dacron grafts correlated with the time of implantation as shown in Fig. 1. The bacteria concentration of the surface bio8

0 Standard Dose eZa High Dose

7 6t ‘;;kl \ z

5

5

3

4

2 1

0

control

1/2hr

2hr

4hr

redoss

FIG. 1. The concentration of Staphylococcus epidermidis in the surface biofilm of the graft 1 week after implantation was significantly decreased when compared with controls for grafts implanted i hr after standard dose and i and 2 hr after high dose cefazolin. The asterisk indicates the significant decreases. The results were determined by quantitative agar culture of the graft surface biofilm after sonication and expressed as mean + standard error of the mean log CFIJ/cm’ explanted graft. CFU, colony-forming units.

BERGAMINI,

PEYTON,

AND

CHEADLE:

PREVENTION

TABLE Graft Biofilm

OF BIOFILM

Broth Culture

Standard High

16 10*

* P co.01 compared with graft implantation

Results

after preoperative

cefazolin 4 hr

2 hr

0.5 hr No. of grafts

No. of no growth

103

INFECTION

1

Time graft implanted

Antibiotic dose

GRAFT

No. of grafts

No. of grafts

No. of no growth

No. of no growth

3

16

0

16

0

6

8

3

8

0

at 2 and 4 hr for standard

film for grafts implanted 0.5 hr after a standard dose of cefazolin (1.2 + 0.39 log CFU/cm2 graft) was significantly decreased (P < 0.05) compared with controls (3.9 f 0.53 log CFU/cm2 graft). The bacteria biofilm concentration was not significantly decreased with the standard dose antibiotic for the longer procedures at 2 hr (2.0 f 0.43 log CFU/cm2 graft) and 4 hr (4.3 + 0.33 log CFU/ cm2 graft). The bacteria biofilm concentration achieved 2 hr after the redose (2.1 f 0.76 log CFU/cm’ graft) was similar to that achieved 2 hr after the standard dose. The bacteria concentration of the surface biofilm for grafts implanted 0.5 hr (0.3 f 0.21 log CFU/cm2 graft) and 2 hr (0.7 + 0.24 log CFU/cm2 graft) after high dose cefazolin was significantly decreased (P < 0.05) compared with controls. The bacteria biofilm concentration (2.8 + 0.15 log CFU/cm2 graft) was not significantly decreased with the high dose antibiotic for the 4-hr procedure. The result of no growth by mechanical disruption and culture in broth media was significantly achieved (P < 0.01) only for grafts implanted 0.5 hr after high dose cefazolin (Table 1). Determination of adequate tissue antibiotic levels. Pharmacokinetic studies of cefazolin yieldedpeak subcutaneous tissue levels within 1 hr of administration for each dose. Standard dose cefazolin achieved a peak tissue antibiotic level (20.9 +- 1.5 pg/g tissue) within the first hour, which was equivalent to the MIC of the study strain. The tissue antibiotic level subsequently decreased below the MIC of the study strain at 2 hr (12.8 f 1.8 pg/g tissue) and 4 hr (11.4 +- 3.0 pg/g tissue) after adminstration. The high dose achieved peak tissue antibiotic levels (37.1 k 4.3 pg/g tissue) within the first hour which were higher than the MIC of the study strain. The tissue antibiotic level subsequently decreased at 2 hr (23.5 + 2.0 pg/g tissue) and 4 hr (19.1 +- 2.1 pg/g tissue). The concentration of bacteria in the biofilm was significantly decreased only for grafts implanted in the presence of antibiotic subcutaneous tissue levels that were greater than or equal to the MIC of the study strain for both the standard dose (Fig. 2) and the high dose (Fig. 3). Grafts implanted with tissue antibiotic levels below the MIC of the study strain did not have a significant reduc-

dose and 4 hr for high dose.

tion in the bacteria biofilm concentration. No growth of the explanted graft in broth culture after sonication, a very sensitive culture technique for recovery of S. epidermidis, was achieved only when the tissue antibiotic levels were above the minimum inhibitory concentration of the study strain (Fig. 3). The effectiveness of prophylactic antibiotics on the prevention of graft infection was dependent on the use of a dosage regimen that maintained tissue antibiotic levels equal to or higher than the MIC of the colonizing bacteria during the time of prostheses implantation. DISCUSSION The effectiveness of prophylactic antibiotics on the prevention of graft infection is dependent on the maintenance of adequate tissue antibiotic levels for the duration of the vascular surgical procedure. Two clinical studies of prophylactic antibiotics have demonstrated that the wound infection rate after surgical prophylaxis was not decreased when bacterial contamination occurred

40

STANDARD DOSE

;

z '=r

35

.d

r 03 0

.5

1

2

3

T"

4

TIME (hours)

FIG. 2. The concentration of Staphylococcus epidermidis in the surface biofilm of the graft (open circle) was significantly decreased (*) after standard dose cefazolin only when the subcutaneous tissue antibiotic level (solid circle) was equivalent to the minimum inhibitory concentration of the study strain (shaded box). CFU, colonyforming units.

104

JOURNAL

OF SURGICAL

RESEARCH:

HIGH DOSE

TIME (hours)

FIG. 3. The concentration of Staphylococcus epidermidis in the surface biofilm of the graft (open circle) was significantly decreased (*) after high dose cefazolin with subcutaneous tissue levels (solid circle) higher than or equal to the minimum inhibitory concentration of study strain (shaded box). CFU, colony-forming units.

late in the procedure and in the presence of inadequate tissue levels. In a prospective, randomized, controlled double-blind trial on patients undergoing hysterectomy [lo], cefazolin did not significantly decrease the wound infection rate compared with the control group for operations lasting longer than 3.5 hr. Similarly, in another prospective, double-blind, randomized clinical trial of colorectal operations [ll], cefoxitin failed to significantly decrease the wound infection rate for procedures that lasted longer than 4 hr. Pharmacokinetic studies of the wound antibiotic tissue level of cefazolin and cefoxitin after a single intravenous dose has demonstrated that the antibiotic activity was less than 10 pg/ml after 3 hr and would not achieve adequate tissue activity to protect the wound against surgical infections for procedures longer than this time period [9]. The results of our study is the first to confirm that these principles of surgical prophylaxis on the wound infection rate applies to the prevention of vascular graft infection. The prophylactic antibiotic was not effective against the bacterial biofilm graft infection for prostheses implanted more than 2 hr after the standard dose and more than 4 hr after the high dose of cefazolin. Bacterial biofilm graft infections occur most commonly in patients following emergency surgery or multiple revascularizations. In a recent clinical series of 15 patients, the bacterial biofilm graft infection occurred following prostheses implantation for a ruptured abdominal aortic aneurysm in four patients and after multiple operative procedures on the involved graft segment in eight patients [3]. The suboptimum sterile technique of emergency operations and the increased incidence of S. epidermidis recovered from the arterial wall in patients undergoing multiple revascularizations [8] can predispose to prostheses colonization. The length of these more difficult procedures may result in failure of prophylactic antibiotics in preventing graft infection due to

VOL.

52, NO. 2, FEBRUARY

1992

inadequate maintenance antibiotic tissue levels for the duration of the procedure. Lalka et al. [12] have shown that the penetrance of prophylactic antibiotics into the arterial wall of patients undergoing elective vascular surgery was inadequate. The tissue antibiotic levels achieved with the preoperative dose of cefazolin or cefamandole were below the minimum inhibitory concentration of the bacteria recovered from the arterial wall. The conclusion of this paper, which showed that prophylactic antibiotics should be administered at higher doses and at more frequent intervals, was prefaced by the fact that the tissue antibiotic level required to prevent graft infection was not known. The results of this animal model suggest that perigraft tissue levels equal to or greater than the MIC of the bacteria are adequate to prevent graft infection. The effectiveness of the antibiotic at a given tissue concentration is dependent on the sensitivity of the bacteria. In the experimental design of this rat model of bacterial biofilm graft infection, a study strain with intermediate sensitivity to cefazolin was chosen to demonstrate the effectiveness of tissue levels higher, equal to, and lower than the MIC. 5’. epidermidis recovered from the perigraft tissues of patients undergoing vascular surgery have been sensitive to the first- and second-generation cephalosporins with low MICs [7,12]. Pharmacokinetic studies of standard dose cefazolin (1 g intravenously) used for prophylaxis maintain wound tissue levels above 10 pg/ml for 3 hr and would be effective against sensitive S. epidermidis [9]. Operations lasting longer than approximately 3 hr would need supplemental antibiotics to maintain adequate prophylaxis. Several methods of antibiotic administration can be used to maintain adequate tissue levels for long surgical procedures. Redosing the antibiotic at 2- to 3-hr intervals during the operation, the use of an antibiotic with a longer half-life, or the application of topical antibiotics during the procedure, are equally effective in preventing infection as long as the tissue level is maintained above the MIC of the potentially contaminating bacteria [ 1,9]. The use of well-vascularized muscle flaps for coverage of the prostheses may achieve higher perigraft antibiotic tissue levels compared with subcutaneous tissues. This principle has been applied to the local treatment of graft infections but still needs further investigation. Another potential method of maintaining adequate perigraft tissue levels is the development of an antibiotic bonded graft. Clearly, the prevention of graft infection is dependent on many factors. First, bacterial biofilm infection of various biomaterials is believed to be dependent on the immune status of the host [4, I?‘]. S. epidermidis is a well-recognized pathogen of the immunocompromised patient, involving central venous lines, arteriovenous shunts, and peritoneal dialysis catheters. Defects in both the humoral and cellular defense mechanisms have been recognized in patients with peritonitis from perito-

BERGAMINI,

PEYTON,

AND

CHEADLE:

PREVENTION

neal dialysis catheters [ 171. The immunosuppressive effects of aortic surgery may predispose to the survival and growth of the low virulent S. epidermidis in a bacterial biofilm adherent to the prosthetic surface [18]. The improved effectiveness of higher antibiotic doses at repeated intervals over standard doses in preventing S. aureus infection has been shown in rats after the immunosuppressive effects of hemorrhagic shock [19]. Second, the incidence of infection directly correlates with the quantity of bacteria that contaminate the prostheses. Blood components that can coat the prosthetic surface, including fibronectin, fibrinogen, and vitronectin, can increase the quantitative adherence of S. epidermidis to biomaterials [20]. Patients undergoing multiple vascular procedures have an increased incidence of bacterial colonization of the arterial wall and repeated exposure of the graft to potential sources of contamination. The results of antibiotic prophylaxis may not be similar with increased bacterial adherence to the graft or in the immunosuppressed host. The addition of these factors to the effectiveness of antibiotic tissue levels in the prevention of bacterial biofilm graft infection in this model need further investigation. In summary, prophylactic antibiotics can be effective in the prevention of bacterial biofilm graft infection. The effectiveness of prophylactic antibiotics is dependent on the use of a dosage regimen that maintains adequate tissue antibiotic activity during the time of prosthesis implantation. S. epidermidis is an ubiquitous organism recovered from many perigraft tissues, including skin, arterial wall, subcutaneous tissue, and lymph nodes. The majority of bacteria recovered from vascular surgical patients are sensitive to the commonly used prophylactic antibiotics. Prophylactic antibiotics can effectively prevent bacterial biofilm graft infection in the presence of tissue levels equal to or greater than the MIC of the contaminating bacteria. Vascular procedures lasting longer than 3 hr should have redosing of the first- or second-generation cephalosporins or use of an antibiotic with a longer half-life to maintain adequate tissue antibiotic levels for the duration of the operation.

GRAFT

INFECTION

105

and Towne, J. B. In situ replacement of vascular prostheses infected by bacterial biofilms. J. Vast. Surg. 13: 575, 1991. 4. Bergamini, T. M. Vascular prostheses infection caused by bacterial biofilms. Semin. Vascular Surg. 3: 101, 1990. 5. Wakefield, T. W., Pierson, C. L., Schaberg, D. R., Messina, L. M., Lindenauer, S. M., Greenfield, L. J., Zelenock, G. B., and Stanley, J. C. Artery, periarterial adipose tissue, and blood microbiology during vascular reconstructive surgery: Perioperative and early postoperative observations. J. Vast. Surg. 11: 624, 1990. 6. Bunt, T. J., and Mohr, J. D. Incidence of positive inguinal lymph node cultures during peripheral revascularization. Am. Surg. 50:

522,1984. 7. Levy, M. F., Schmitt,

8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

REFERENCES 18. Pitt, H. A., Postier, R. G., MacGowan, W. A. L., Frank, L. W., Surmak, A. J., Sitzman, J. V., and Bouchier-Hayes, D. Prophylactic antibiotics in vascular surgery. Topical, systemic, or both? Ann. Surg. 192:356,1980. Hasselgren, P.-O., Ivarsson, L., Risberg, B., and Seeman, T. Effects of prophylactic antibiotics in vascular surgery. A prospective, randomized, double-blind study. Ann. Surg. 200: 86, 1984. Bandyk, D. F., Bergamini, T. M., Kinney, E. V., Seabrook, G. R.,

OF BIOFILM

19.

20.

D. D., Edmiston, C. E., Bandyk, D. F., Krepel, C. J., Seabrook, G. R., andTowne, J. B. Sequential analysis of staphylococcal colonization of body surfaces of patients undergoing vascular surgery. J. Clin. Microbial. 28: 664, 1990. Durham, J. R., Malone, J. M., and Bernhard, V. M. The impact of multiple operations on the importance of arterial wall cultures. J. Vast. Surg. 5: 160, 1987. Bergamini, T. M., and Polk, H. C., Jr. Pharmacodynamics of antibiotic penetration of tissue and surgical prophylaxis. Surg. Gynecol. Obstet. 168: 283, 1989. Shapiro, A., Munoz, A., Tager, I., Schoenbaum, S., and Polk, B. Risk factors for infection at the operative site after abdominal or vaginal hysterectomy. N. Engl. J. Med. 307: 1661, 1982. Kaiser, A. B., Herrington, J. L., Jr., Jacobs, J. K., Mulherin, J. D., Jr., Roach, A. C., and Sawyers, J. L. Cefoxitin versus erythromycin, neomycin, and cefazolin in colorectal operations. Ann. Surg. 198: 525, 1983. Lalka, S. G., Malone, J. M., Fisher, D. F., Jr., Bernhard, V. M., Sullivan, D., Stoeckelmann, D., and Bergstrom, R. F. Efficacy of prophylactic antibiotics in vascular surgery: An arterial wall microbiologic and pharmacologic perspective. J. Vosc. Surg. 5: 501, 1989. Guglielmo, B. J., Salazar, T. A., Rodondi, L. C., Carver, M., Goldstone, J., and Stoney, R. J. Alteredpharmacokinetics of antibiotics during vascular surgery. Am. J. Surg. 157: 410, 1989. Bergamini, T. M., Bandyk, D. F., Govostis, D., Kaebnick, H. W., and Towne, J. B. Infection of vascular prostheses caused by bacterial biofilms. J. VCLSC.Surg. 1: 21, 1988. Pitkin, D. H., Sachs, C., Zajac, I., and Actor, P. Distribution of sodium cefazolin in serum, muscle, bone marrow, and bone of normal rabbits. Antimicrob. Agents Chemother. 11: 760, 1977. Bergamini, T. M., Bandyk, D. F., Govostis, D., Vetsch, R., and Towne, J. B. Identification of Staphylococcus epidermidis vascular graft infections: a comparison of culture techniques. J. Vast. Surg. 9: 665, 1989. Lamperi, S., and Carozzi, S. Immunological defenses in CAPD. Blood Purif. 7: 126, 1989. Keane, R. M., Munster, A. M., Birmingham, W., Winchurch, R. A., Godacz, T. R., and Ernest, C. B. Suppression of lymphocyte function after aortic reconstruction. Use of nonimmunosuppressive anesthesia. Arch. Surg. 117: 1133, 1982. Livingston, D. H., Shumate, C. R., Polk, H. C., Jr., and Malangoni, M. A. More is better. Antibiotic management after hemorrhagic shock. Ann. Surg. 208: 451, 1988. Cheung, A. L., and Fischetti, V. A. The role of fibrinogen in mediating staphylococcal adherence to fibers. J. Surg. Res. 50: 150,199l.

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 tiss...
694KB Sizes 0 Downloads 0 Views