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Laboratory Animal Science Copyright 1999 by the American Association for Laboratory Animal Science Vol 49, No 3 June 1999 Model of Staphylococcus aureus Central Venous Catheter-Associated Infection in Rats Joseph S. Ulphani and Mark E. Rupp* Background and Purpose: Staphylococcus aureus is an important cause of intravascular catheter-associated bacteremia. We developed a rat central venous catheter (CVC)-associated infection model to study pathogenesis and treatment. Methods: A silastic lumen-within-lumen catheter and rodent-restraint jacket were designed. Subcutaneously tunneled catheters were inserted in the jugular vein of 20 male Sprague Dawley rats. Twelve rats (group 1) were inoculated with S. aureus via the CVC; three rats (group 2) were inoculated with S. aureus via the tail vein, five rats (group 3) served as uninfected controls; and three rats (group 4) were inoculated with S. aureus via the tail vein but did not undergo CVC insertion. Five to eight days after inoculation, animals were euthanized, CVCs were aseptically removed, and quantitative culture was done. Quantitative culture also was performed on blood, heart, liver, lungs, and kidneys. Results: Infection, characterized by bacteremia and metastatic disease, was observed in all rats inoculated via the CVC with as few as 100 colony-forming units (CFU) of S. aureus. Rats of group 2 were not as likely to develop CVC-associated infection, and none of the animals of groups 3 or 4 developed infection. Conclusions: This model of CVC-associated infection should prove suitable for studying pathogenesis and treatment of the condition. Each year about 20 million people admitted to U.S. hospitals undergo intravascular catheterization. About 50,000 to 120,000 develop nosocomial bacteremia (1, 2). Central venous catheter (CVC)-related infection is a common complication and has become a major medical problem. The mortality associated with nosocomial bacteremia approximates 25% (3). Staphylococci are the prevailing etiologic agents, accounting for 46.9% of episodes of primary nosocomial bacteremia (4). Although Staphylococcus aureus is less frequently encountered as an etiologic agent of catheter-related bacteremia than is S. epidermidis, it is a more virulent pathogen, resulting in greater morbidity and mortality (5, 6). Unfortunately, this problem is increasing at an alarming rate. According to the Centers for Disease Control’s National Nosocomial Infection Surveillance System, the rate of nosocomial bacteremia caused by S. aureus increased from 122% to 283% depending on the size of hospital studied during the 1980s (7). The rapid increase in staphylococcal bacteremia can largely be attributed to the extensive use of intravascular catheters. To study the pathogenesis and treatment of this condition, we developed a CVC-associated infection model. One important part of this model was construction of a novel Silastic lumen-within-lumen catheter, which was tunneled subcutaneously to simulate the experience with surgically implanted central venous catheters in humans. Equally important was the development of a special animal-restraint jacket, which contained the catheter and facilitated rapid and nonstressful serial blood collection. University of Nebraska Medical Center, Omaha, Nebraska *Address correspondence to: Dr. Mark E. Rupp, Department of Internal Medicine, Section of Infectious Diseases, 9854031 Nebraska Medical Center, Omaha, NE 68198-4031. Materials and Methods Bacteria: Staphylococcus aureus strain SA-160 used in our investigation was isolated from a patient with CVC-associated bacteremia. A single colony was incubated in 3 ml of trypticase-soy broth overnight at 37⬚C. The overnight subculture was centrifuged at 2,000 X g for 10 min, washed, and resuspended in phosphate buffered saline (PBS) (0.9% NaCl in 10 mM phosphate buffer, pH 7.4). Serial dilution was performed to obtain a concentration of 105 colony-forming units (CFU)/ml, which was confirmed by quantitative culture. One-tenth milliliter of the bacterial suspension was added to 0.3 ml of PBS to yield a volume that completely filled the catheter lumen. Animals: Male Sprague Dawley rats (Charles River, Wilmington, Mass.), weighing 400 to 500 g, were studied. The rats were acclimated for 7 days prior to surgery. They were housed in individual polypropylene shoe-box cages on cob bedding; cages and bedding were changed twice per week. Water and feed were given ad libitum. Feed consisted of standard laboratory rat diet (Harlan Inc., Indianapolis, Ind.). Animals were maintained under the following environmental conditions: lighting, 12-h on/off cycle; temperature, 22 to 25⬚C; air changes, minimum of 10/h; humidity, 50%. The rats were obtained from the supplier specified pathogen free and were maintained using a soiled bedding sentinel system. Catheter construction: A Silastic lumen-within-lumen catheter (U.S. patent no. 5762636) was constructed, using silicon rubber tubing (Specialty Manufacturing Inc., Saginaw, Mich.) of three diameters. A 1.27-cm-long, 20gauge blunt needle with a luerlock hub (Figure 1 [B]) was slid tip first into a 17.0-cm length of 0.064-cm-i.d. and 0.119- 283 Vol 49, No 3 Laboratory Animal Science June 1999 cm-o.d. tubing to form the inner cannula (Figure 1 [D]). The inner cannula was slid into a 10.0-cm-long tube of 0.15 cm i.d. and 0.195 cm o.d. (outer cannula), which encases the subcutaneous portion and the segment from the skin to the hub of the inner cannula (Figure 1 [E]). This portion of the catheter is subjected to maximal stress during catheter manipulation. The outer lumen simply serves to protect the inner cannula from stress caused either by handling or by the animal. A 1.52-cm-long heat-shrink tube (Cole-Parmer Instrument Co., Vernon Hills, Ill.) was used to secure the inner and outer cannulas to the needle (Figure 1 [C]). An injection adaptor (Medexinc, Hilliard, Ohio) was attached to the luerlock of the needle (Figure 1 [A]). A pair of collars with the same diameter as that of the outer cannula was glided over the projecting end of the inner cannula and was used for securing the catheter in position by affixing it to the surrounding tissue with a suture. A clamp was made by slitting a 0.076-cm-i.d. and 0.165-cm-o.d. silicon rubber tubing and mounting it as a marker on the inner cannula at a predetermined insertional length to prevent the collars from sliding off of the inner cannula (Figure 1 [H]). Catheters were sterilized before use by pressurized steam autoclave. Restraint jacket: An animal-restraint jacket (U.S. patent no. 5839393) was constructed, using an elongated sheet of stretchable material, elastic in only the longitudinal direction (Figure 2). Spaced from the opposing ends of the sheet, a pair of limb holes were formed. Velcro was mounted along the entire edge of the ends so that it could be fastened along the thorax of the animal. To receive the catheter, an aperture was formed midway between the ends of the sheet. Two elastic straps were attached adjacent to the aperture to hold the catheter. A cover flap wide enough to extend over the aperture and the straps was attached along one side of the sheet. Velcro was mounted on the edge of the other end of the flap to fasten it to the sheet. Placement of the CVC: All procedures and protocols incorporated in this study were conducted with the approval of the Institutional Animal Care and Use Committee. The surgical placement of CVCs was performed aseptically. Rats were anesthetized by intraperitoneal administration (90 to 10 mg/kg of body weight) of a ketamine-xylazine solution. The hair on an area over the scapula, cranial region of the thorax, and neck was shaved. The rats were restrained on an animal operating board, and their eyes were coated with boric acid ophthalmic ointment. The shaved area was cleansed with povidone-iodine solution and was draped. With the rat positioned on its left side, a 0.5-cm midline incision was made over the scapular region. The rat was turned on its back, and a small longitudinal incision was placed over the spot where the proximal part of the jugular, acromiodeltoid, and cephalic veins join to form the right external jugular vein. A trocar was passed subcutaneously from the ventral to the dorsal incision down the side of the neck. The catheter was advanced through the trocar to the ventral incision. The trocar was then slowly pulled out, leaving 4 to 5 cm of the catheter in a subcutaneous position. The right external jugular vein was exposed by carefully removing the connective tissue surrounding the vein. Two 3.0 silk sutures were placed around the vein ap- 284 Figure 1. Silastic lumen-within-lumen catheter: A, injection adapter (heparin lock); B, needle; C, heat-shrink tube; D, inner cannula; E, outer cannula; F, proximal collar; G, distal collar; H, clamp. Figure 2. Animal-restraint jacket: A, catheter; B, Velcro closure; C, limb holes; D, elastic straps; E, injection adapter (heparin lock). proximately 1.5 cm apart. The vein was ligated by tying the cephalad suture, and a small incision was made between the two sutures. A catheter filled with heparin solution (20 U of heparin/ml of 0.9% NaCl) was slowly inserted into the vein and was advanced into the sinus venosus. The lower suture was tied around the vein over the proximal collar, which prevented occlusion of the catheter. A stress loop was formed, the catheter was secured in place by tying a suture around the distal collar, and incisions were closed. After surgery, each animal was given buprenorphine hydrochloride (0.05 mg/kg, intravenously). Central venous catheter-associated infection: Fortyeight h after catheter placement, the 20 rats were assigned to three groups. Rats of group 1 (n = 12) were inoculated via the catheter with 100 l of an S. aureus suspension, ranging from 102 to 104 CFU. One rat received an inoculum of 102 CFU, three rats received an inoculum of 103 CFU, and eight rats received an inoculum of 104 CFU. The bacterial suspension was allowed to dwell within the catheter lumen for 15 min. The contents of the catheter were then flushed into the blood with 0.5 ml normal saline, and the catheter was filled with heparin solution. Central Venous Catheter-Associated Infection Model Rats of group 2 (n = 3) were inoculated via the tail vein and received an inoculum of 104 CFU. Group-3 (n = 5) rats (control) did not receive a bacterial inoculum. To assess whether the catheter had a potentiating role in bacteremia, an additional group (group 4, n = 3) of rats was studied. This group did not undergo any surgical procedure and was inoculated via the tail vein with an inoculum of 104 CFU of S. aureus. Five rats of group 1 were euthanized on day 5 by CO2 inhalation. The remaining seven rats of group 1 and rats of groups 2, 3, and 4 were sacrificed on day 8 after inoculation. The intravascular portion of the catheters along with the surrounding vein were removed aseptically; bacteria adhering to the catheter/tissue were removed by vortex washing in 5 ml of PBS; and a 0.1-ml aliquot of the bacterial suspension was taken for quantitative plate count. The heart, lungs, liver, and kidneys of each rat were removed, weighed, and homogenized with sterile disposable tissue grinders (Sage Products, Inc., Crystal Lake, Ill.) in 5 ml of PBS, then a 0.1-ml aliquot of the tissue suspension was taken for quantitative plate count. One milliliter of peripheral blood, obtained by puncture of the caudal vena cava at the time of euthanasia, also was collected for quantitative culture. Pulse-field gel electrophoresis: To ensure that staphylococci recovered from the rats was identical to the inoculum strain, the isolates were characterized by use of analysis of restriction fragment length polymorphisms with pulse-field gel electrophoresis (PFGE). Genomic DNA was prepared for PFGE by use of described methods (8). The following electrophoresis parameters were used: 6 V/cm; initial pulse time, 1 sec; final pulse time, 30 sec for 22 h at 14⬚C. Statistical analysis: Bacterial counts (CFU/ml or CFU/g) were log10 normalized, then were analyzed by use of the Kruskal-Wallis test with the Dunn’s test for multiple comparisons. Statistical analysis was performed, using Graphpad Prism version 2.0 (San Diego, Calif.). Results All rats of group 1 developed CVC-associated infection (Figure 3). Mean number of organisms recovered from the catheters at the time of euthanasia was 6.1 x 106 CFU/catheter. In contrast, mean number of organisms recovered from the catheters or rats of group 2 was 10 CFU/catheter, and no rats of groups 3 or 4 had bacteria recovered from the catheters (P < 0.01, for group 1 versus group 2, 3, or 4). Mean number of staphylococci recovered from the blood of rats in group 1 was 2.7 x 104 CFU/ml, compared with 7, 0, and 0 CFU/ml for groups 2, 3, and 4, respectively (P < 0.05 for group 1 versus group 2, 3, or 4) (Figure 3). All rats of group 1 had evidence of metastatic disease with large numbers of staphylococci recovered from the heart, lungs, liver, and kidneys (Figure 3). In contrast, there was minimal evidence of metastatic disease in group-2 rats (P < 0.01 for group 1 versus group 2, 3, or 4). Staphylococcus aureus was the only bacterial species recovered from the rats and, as indicated by PFGE (gel not shown), the isolates recovered were identical to the strain inoculated. Discussion The purpose of this study was to develop an effective, economical, invivo model to simulate human CVC-associated Figure 3. Staphylococcus aureus recovered from catheter (CFU/ catheter), blood (CFU/ml), and various organs (CFU/g). Group 1 (n = 12): catheterized and inoculated via the catheter, five euthanized day 5, seven euthanized day 8. Group 2 (n = 3): catheterized and inoculated via the tail vein, euthanized day 8. Group 3 (n = 5): catheterized, not inoculated (control), euthanized day 8. Group 4 (n = 3): not catheterized, inoculated via the tail vein, euthanized day 8. Bars represent mean value, and lines indicate SEM. infection. The pathogenesis of CVC-associated infection is complex and involves interactions between the microbe, the device, and the host. Therefore, in vivo models should be used to examine questions regarding pathogenesis and treatment. To better understand the rationale for this study, a brief description of previously used intravascular catheter-associated infection models is in order. For years, the rabbit model of infective endocarditis, first introduced by Garrison and Freedman (1970), modified by Durack and Beeson (1972), and later described in the rat by Santoro and Levison (1978), has been considered reliable to study catheter-associated infections (9–11). This model has proven useful for investigating the pathophysiology of infective endocarditis. However, some features of the endocarditis model constitute a substantial departure from catheter-associated infection in humans (12, 13). Most importantly, to make the animal susceptible to infection, trauma to cardiac valves is induced by moving the catheter backward and forward, and by keeping the tip of the catheter between the valve leaflets. This is pathophysiologically different from the condition that predisposes humans to catheter-associated infection. Also, a larger inoculum of bacteria was used to induce infection than that encountered in clinical practice in humans. In 1993, Paston et al. introduced a rat model of CVC-related sepsis (14). This model was useful in describing the dynamics of catheter-related sepsis. Our model differs from that of Paston et al. in several respects. The CVC in our model is subcutaneously tunneled, which more closely mimics the conditions in human surgically implanted catheters, such as the Hickman or Broviac types. Also, the catheters were kept patent, allowing collection of sequential blood samples for culture. Finally, the need for a tethering device was obviated by the use of a rodentrestraint jacket that protected the catheter yet allowed free animal mobility and ready access to the catheter. Dennis et al. described an intravascular catheter-associated infection model 285 Vol 49, No 3 Laboratory Animal Science June 1999 in the rabbit (15). Our model differs from that of Dennis et al. in that rats are used rather than rabbits. This introduces an economic advantage as rats are considerably less expensive than rabbits. In addition, the advantages of using the rodentrestraint jacket described herein have already been mentioned. It should be emphasized that the model developed in our laboratory simulates the human condition. We were successful in inducing CVC-associated infection with secondary bacteremia and metastatic disease with a range of inocula. Animals sacrificed as soon as day 5 after infection had a substantial degree of infection. As expected, progression of infection and metastatic disease was observed in animals sacrificed on day 8. Infection was present at all sites, which was the result of seeding of bacteria into the bloodstream from the infected CVC. The difference in the degree of infection seen in rats of groups 2 and 4 clearly indicates the importance of the indwelling vascular catheters in potentiation of the infection. This finding is in agreement with those of other studies (5, 16, 17). The route of inoculation also mimics that of the human condition. Migration of organisms down the external surface of the catheter appears to be important in the pathogenesis of infections involving short-term, nontunneled catheters (18, 19). In contrast, in long-term, subcutaneously tunneled catheters, the major route of inoculation appears to be via the catheter hubs and intraluminal migration (20–22). Our CVC-associated infection model has been fully developed and standardized to study staphylococcal disease. In this study, we have documented reproducible induction of CVC-associated infection with a range of inocula. The importance of the catheter and the route of inoculation was unambiguously documented in the tail vein inoculation experiments. In other studies, this model has been used to elucidate the importance of the polysaccharide intercellular adhesin/hemagglutinin (PIA/HA) of S. epidermidis and the autolysin of S. epidermidis in the pathogenesis of intravascular catheter-related infection and to study the efficacy of antistaphylococcal antibiotics (23–25). Future efforts will be directed at further study of the pathogenesis of staphylococcal intravascular catheter-associated infection. However, this model can be used for a broad range of applications, including the investigation of pharmacokinetics, pharmacodynamics, efficacy, and disposition of novel antimicrobial agents. The rodent intravascular catheter and the rodentrestraint jacket developed in our laboratory make the model efficient. Together these devices obviate elaborate preparation, equipment, and practical skills otherwise required for multiple blood sample collection studies. In conclusion, use of our in vivo model, which simulates human S. aureus CVC-associated infection, should prove suitable in elucidating the pathogenesis and treatment of this and other similar conditions. Acknowledgements We thank Firdous Ulphani for assistance with production of the rodent-restraint jacket. This work was supported in part by a Grant-in-Aid from the American Heart Association, (MER) no. 96006810. 286 References 1. Maki, A. G. 1992. Infection due to infusion therapy, p. 849–898. In J. V. Bennet and P. S. Brachman (ed.), Hospital infections. Little, Brown & Co., Boston, Mass. 2. Raad, I. I., and G. P. Bodey. 1992. Infectious complication of indwelling catheters. Clin. Infect. Dis. 15:197–208. 3. 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