Natural History of Aortic Valve Endocarditis in Rats

Transcription

1 INFECTION AND IMMUNITY, JUlY 192, p /2/7127-5$2./ Vol. 37, No. 1 Natural History of Aortic Valve Endocarditis in Rats ERIC HtRAIEF, MICHEL P. GLAUSER,* AND LAWRENCE R. FREEDMANt Division des Maladies Infectieuses, Dipartement de Me6decine, Centre Hospitalier Universitaire Vaudois, CH 111 Lausanne, Switzerland Received 21 December 191/Accepted 26 March 192 Sterile aortic vegetations were produced in rats by introducing a polyethylene catheter through the right carotid artery. The catheter was either left in place throughout the experiments or removed before bacterial challenge. Bacterial endocarditis was uniformly produced by intravenous injection of 17 colonyforming units of Staphylococcus aureus or Streptococcus intermedius, whether the catheter was left in place or removed. However, in rats with the catheter left in place, bacterial multiplication within the vegetations with both strains was accelerated, and mortality from Staphylococcus aureus infection was increased. Using 17 colony-forming units of serum-resistant Escherichia coli as a test microorganism, we found a marked difference in the production of endocarditis depending upon whether the catheter was left in place or removed before injection; only those animals infected with the catheter in place developed infection. From these experiments in rats, it was evident that the presence of a foreign body has a considerable influence on the ability of bacteria to grow within an intravascular vegetation. In addition, a striking difference in the virulence of the three strains studied was established; Staphylococcus aureus was the most, and E. coli the least, pathogenic. Studies of the natural history of intravascular infections (1, 2, 4, 9, 11, 16), of their treatment (15, 1, 19), and of their prophylaxis (6, 14, 21) have mainly been conducted on the rabbit. Since there is controversy concerning the relation of these studies to intravascular infection in humans (7,13, 17) and since experimental observations are not possible in humans, there is clearly a need to extend the rabbit studies to other animal species. The rat is a suitable experimental animal and offers several advantages, such as easy housing of a larger number of animals at less expense than the rabbit. Santoro and Levison have shown that the model of Streptococcus sanguis bacterial endocarditis utilized in rabbits is applicable to rats (2). The experiments reported here were undertaken to evaluate the severity of endocardial infection and its mortality, using different test microorganisms. Furthermore, the role of the catheter in the initiation and the evolution of bacterial endocarditis was studied. MATERIALS AND METHODS Microorganisms. Staphylococcus aureus (phage type 6/47/53/54/75/77/4/5) and Streptococcus intermedius strain MG () were isolated from patients with t Present address: Department of Internal Medicine, W. A. Wadsworth Medical Center, UCLA School of Medicine, Los Angeles, CA 973. bacterial endocarditis, and Escherichia coli was isolated from a septicemic patient with acute pyelonephritis (B. R. Yersin, M. P. Glauser, and L. R. Freedman, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 2th, New Orleans, La., abstr. no. 275, 19). The strains were maintained by stock cultures on blood agar plates and on MacConkey agar (Difco Laboratories, Detroit, Mich.), and by repeated passages through rats with endocarditis. Overnight cultures of the different stock cultures in brain heart infusion broth (Difco Laboratories) were diluted 1:1 in saline, and.5 ml of this suspension was used as the infecting inoculum. The density of the bacterial inoculum was determined in each experiment by serial 1- fold dilution on blood or MacConkey agar plates. Production of endocarditis. Female Wistar rats weighing 1 to 22 g were anesthetized by an intraperitoneal injection of 4 mg of pentobarbital sodium (Abbott Laboratories, Zurich, Switzerland) per kg. The right carotid artery was exposed by an anterior incision of the skin above the clavicles, and a loose silk ligature was passed around the artery. A sterile polyethylene catheter (Portex Ltd., Hythe, England; internal diameter,.4 mm; external diameter,. mm) filled with diluted heparin was introduced through the opened artery. It was then passed toward the aortic valve until pulsation and resistance indicated that the catheter had reached the aortic valve and the left ventricle. The catheter was then secured with the silk ligature and folded upon itself. Any excess catheter was cut off. The skin was closed over the catheter with two clips (Autoclips [9 mm] no and Autoclips Applyer; Clay Adams, Parsippany, N.J.). Direct mortality from the operation and anesthesia was around 3%. One to three days later, sterile fibrin platelet Downloaded from on June 15, 21 by guest 127

2 12 HERAIEF, GLAUSER, AND FREEDMAN vegetation had developed, and rats were infected by injecting a.5-ml inoculum containing 17 microorganisms per ml into a tail vein. In parallel experiments, the catheter was removed (usually 3 days after the operation), 3 min before bacterial challenge, under pentobarbital sodium anesthesia. All of the rats were similarly treated before bacterial challenge with either of the three microorganisms. Evaluation of the role of the catheter on the severity of endocarditis due to different microorganisms. Infection was established with different microorganisms, with the catheter left in place or removed, just before inoculation. Rats were sacrificed by ether anesthesia 1 or 3 days thereafter. One milliliter of blood was obtained for culture by puncture of the inferior vena cava. Vegetations, left kidney, and spleen were removed aseptically and were weighed and homogenized in sterile saline. To determine the number of bacteria per gram of tissue, we plated.1 ml of serial 1-fold dilutions in saline onto blood agar plates or MacConkey agar. One milliliter of blood was similarly plated. After overnight incubation for 1 to 24 h, the plates were read and the results were expressed as loglo colony-forming units per gram of tissue. This method permitted the detection of 11 colony-forming units per g of spleen or kidney and 12 colony-forming units per g of vegetation. Evaluation of the role of the catheter on the mortality from endocarditis due to different microorganisms. In a different series of experiments, rats were infected, with the catheter in place or removed, and observed daily or autopsied (or both) within 24 h of death to establish the presence of endocarditis as described above. To determine the role of the catheter alone in the mortality from this model, control rats were catheterized, with subsequent intravenous injection of sterile saline but not bacteria. The mortality from endocarditis or from the catheter alone in controls was recorded from the time of intravenous injection of bacteria or saline. Statistical evaluation. The number of bacteria in the vegetations was compared by using Student's unpaired t test. The chi-square test with Yates' correction was used to compare the differences between groups of animals. RESULTS The role of the catheter on mortality. Mortality in relation to the presence of the catheter, in the absence of infection, was observed within the first 3 days after the operation (Table 1). This mortality was apparently due to cardiac insufficiency and pulmonary edema, as suggested by autopsy findings. When the catheter was left in place, mortality from infectious endocarditis during the first 3 days after bacterial challenge was increased significantly only in those animals infected with Staphylococcus aureus. In contrast, infection with Streptococcus intermedius or E. coli did not significantly modify the mortality. The detrimental effect of a catheter retained during infection was obvious when observations INFECT. IMMUN. on mortality were made in animals from which the catheter had been removed. In rats with Staphylococcus aureus infection, the number of early deaths was significantly reduced after catheter removal compared with rats with the catheter still present. In addition, with E. coli infection, it was not possible to study mortality after removal of the catheter because E. coli endocarditis developed only in those animals with the catheter still in place. The role of the catheter on infection. The effect of the catheter observed in mortality studies was reflected bacteriologically in rats sacrificed 1 and 3 days after challenge with different microorganisms. For all three microorganisms tested, mean bacterial densities were significantly higher in the presence of the catheter after 24 h of infection (Fig. 1). With infection due to grampositive cocci, this difference was no longer observed after 3 days, suggesting that the catheter effect was most marked during the early phase of infection. With E. coli endocarditis, the role of the catheter on early infection was so critical that after removal of the catheter, only 3 of 12 rats had infected vegetations when examined 24 h after bacterial challenge and none of 11 rats had infections after 3 days. Thus, the catheter was necessary for inducing and maintaining endocardial infection with this serum-resistant E. coli. The virulence of the different strains. From the preceding results, it was obvious that the three bacteria used in these experiments had different capacities to induce severe infections in catheterized rats. Staphylococcus aureus endocarditis was accompanied by significant mortality early during infection, even in the absence of the catheter, but this was not the case for either Streptococcus intermedius or E. coli endocarditis (Table 1). Furthermore, increased virulence of Staphylococcus aureus in the rats was expected, considering the greater mean bacterial densities of infected vegetations 1 and 3 days after bacterial challenge (Fig. 1). E. coli was so weakly virulent that the presence of the catheter was necessary to induce infection of the vegetations. The difference between bacteria was also reflected in the results of blood cultures. All of the animals infected with Staphylococcus aureus and sacrificed 24 or 72 h after bacterial challenge were bacteremic whether the catheter was still in place or removed. In contrast, with Streptococcus intermedius endocarditis, when the catheter was in place, only 29% of the rats (7 of 24) were bacteremic when sacrificed at 24 h and 14% (1 of 7) were bacteremic at 72 h. This frequency was reduced to % (O of 6) and 7% (1 of 13), respectively, when the catheter was removed. Similar differences between Staphylococcus aureus and Streptococcus intermedius Downloaded from on June 15, 21 by guest

3 VOL. 37, 192 TABLE 1. ENDOCARDITIS IN RATS 129 The role of the catheter on mortality from endocarditis due to different microorganisms Time after Mortality (no. dead/total) after injection with: intravenous Control Staphylococcus aureus Streptococcus intermedius Escherichia coli injection (h) Catheter Catheter Catheter Catheter Catheter Catheter Catheter present present removed present removed present removed 24 2/25 (%) 13/47 (27%) 2/25 (%) 1/17 (5%) /12 (o) 2/16 (12%) 4 2/25 (%) 25/47a (53%) 4/25b (16%) 1/17 (5%) /12 (%7) 3/16 (1%) 72 4/25 (16%) 36/47a (76%) 7/25b (2%) 2/17 (11%) 1/12 (%) 3/16 (1%) a P <.5 by chi-square test for comparison of cumulated mortality from Staphylococcus aureus endocarditis with catheter in place compared with mortality in controls without infection. b p <.5 by chi-square for comparison of cumulated mortality from Staphylococcus aureus endocarditis with catheter in place compared with mortality after removal of the catheter. All other comparisons were not significant. infections were observed upon culture of the spleens and kidneys. DISCUSSION These studies confirm the ease with which it is possible to induce infective endocarditis of the aortic valve in rats (2). Thus, the rat is another animal species suitable for studies of infective endocarditis. Since recommendations concerning prophylactic antibiotic administration in humans have been influenced by experimental 11l I I 'I 'I 31 Log,, CFU /g S. aureus p1=.3 N.S? 6.1 * % *I* studies in the rabbit (6, 13, 14, 21) and since there is debate concerning the applicability of these studies to humans (7, 17), it will be of considerable interest to conduct experiments on the efficacy of prophylactic antibiotics in rats. It will also be of great interest to perform studies on the treatment of established endocardial infections in the rat. The experiments presented here were conducted to establish the natural history of endocarditis with three different microorganisms. They emphasize two important features in the S. sanguis p'=o.1 a N.Sa -_ i S oo L o E. LL Downloaded from on June 15, 21 by guest <21 Incidence of infection % h h h h p =.7 p =.1 24h 72h Time of sacrifice after bacterial challenge FIG. 1. The role of the catheter on bacterial densities in vegetations infected with different microorganisms. Each point represents one rat. The dash represents the mean bacterial densities of infected vegetations. Symbols: *, catheter left in place before bacterial challenge;, catheter removed before bacterial challenge. Superscript symbols: a, Student's unpaired t test for comparison of mean bacterial densities; b, chi-square test with Yates' correction for comparison of incidence of endocarditis. N.S., Not significant. The mean bacterial densities for Staphylococcus aureus and for Streptococcus intermedius endocarditis at 24 and 72 h, both with the catheter in place or removed, are significantly different at the P level of '.1. The same holds true for comparison of mean bacterial densities of Staphylococcus aureus and E. coli endocarditis after 24 h, with the catheter left in place. Other comparisons were not statistically different.

4 13 HIRAIEF, GLAUSER, AND FREEDMAN rat that had already been observed in rabbits: first, they show the detrimental effect of the catheter on the development of endocardial infection; second, they stress the differences in infections due to different bacteria. The deleterious effect of the catheter on the natural history of endocarditis has been amply documented (9, 1, 12; Yersin et al., 2th ICAAC, abstr. no. 275). It is not known in what way the catheter contributes to the maintenance of endocardial infection. Only one early study in rabbits has demonstrated a role for the catheter in the maintenance of infections in the left side of the heart (16), and it has been shown that removal of the catheter from animals with rightsided streptococcal endocarditis augments the speed of spontaneous sterilization of infections on that side of the heart (9). Furthermore, the presence of the catheter impairs the efficacy of antibiotic treatment of established endocardial infection (15). The present study is a clear demonstration of the importance of the catheter on infections in the left side of the heart in rats. The mean bacterial densities of vegetations containing Staphylococcus aureus and Streptococcus intermedius increased significantly more rapidly over 24 h in the presence of the catheter. Furthermore, the presence of the catheter in the left side of the heart was necessary to consistently induce E. coli endocarditis. In addition, it was recently demonstrated in the same model that the presence of the catheter reduces by 1,-fold the efficacy of prophylactic vancomycin in Streptococcus intermedius endocarditis (12). The mechanism(s) by which the catheter exercises these effects is unknown. The various virulence differences between the three bacteria studied have already been established in rabbits, and it has been shown that the inoculum size necessary to induce endocarditis depends on the test bacteria (1). The present studies confirm these results and extend these previous experiments to rats. With E. coli, they showed that, with an inoculum as high as 17 microorganisms per ml, this bacterium was consistently able to induce endocarditis only when the catheter was present. This lowered virulence occurred despite resistance of this E. coli strain to the spontaneous bactericidal activity of the serum (Yersin et al., 2th ICAAC, abstr. no. 275). Durack and Beeson have shown that serum-resistant E. coli microorganisms maintain endocardial infections of the left side of the heart in rabbits, whereas serum-sensitive strains are rapidly sterilized (3). With Streptococcus intermedius, our observations revealed a virulence in rats that was intermediate between E. coli and Staphylococcus aureus. Even with the catheter left in place, bacterial densities in animals with streptococcal infection were consistently lower INFECT. IMMUN. than those in animals with staphylococcal infection. It is not established whether this difference was due to differences in susceptibility to infection with these two microorganisms of the left side of the heart or whether it took longer for the streptococci to achieve maximal densities. With Staphylococcus aureus, the virulence was so great that even in the absence of the catheter infection by itself killed a significant number of rats. The present experiments confirm the feasibility and reproducibility of experimental endocarditis due to different microorganisms in rats. They establish some of the factors that influence its natural history and that are related both to the bacterial strain and to the presence of a foreign body. ACKNOWLEDGMENTS We thank S. Bovey for excellent secretarial assistance. LITERATURE CITED 1. Durack, D. T., and P. B. Beeson Experimental bacterial endocarditis I. Colonization of a sterile vegetation. Br. J. Exp. Pathol. 53: Durack, D. T., and P. B. Beeson Experimental bacterial endocarditis. II. Survival of bacteria in endocardial vegetations. Br. J. Exp. Pathol. 53: Durack, D. T., and P. B. Beeson Protective role of complement in experimental Escherichia coli endocarditis. Infect. Immun. 16: Durack, D. T., P. B. Beeson, and R. G. Petersdorf Experimental bacterial endocarditis. III. Production and progress of the disease in rabbits. Br. J. Exp. Pathol. 54: Durack, D. T., L. L. Pelietier, and R. G. Petersdorf II. Synergism between' penicillin and streptomycin against penicillin-sensitive streptococci. J. Clin. Invest. 53: Durack, D. T., and R. G. Petersdorf Chemotherapy of experimental streptococcal endocarditis. I. Comparison of commonly recommended prophylactic regimens. J. Clin. Invest. 52: Editorial Prophylaxis of bacterial endocarditis. Faith, hope and charitable interpretation. Lancet 1:519.. Facklam, R. R Physiological differentiation of viridans streptococci. J. Clin. Microbiol. 15: Francdoll, P. B., and L. R. Freedman Streptococcal infection of endocardial and other intravascular vegetations in rabbits. Natural history and effect of dexamethasone. Infect. Immun. 24: Freedman, L. R., and J. Valone, Jr Experimental infective endocarditis. Progr. Cardiovasc. Dis. 22: Garrison, P. K., and L. R. Freedman Experimental endocarditis I. Staphylococcal endocarditis in rabbits resulting from placement of a polyethylene catheter in the right side of the heart. Yale J. Biol. Med. 42: Heraef, E., M. P. Glauser, and L. R. Freedman Vancomycin prophylaxis of streptococcal endocarditis in rats, p In J. D. Nelson and C. Grassi (ed.), Current chemotherapy and infectious diseases, vol. 2. American Society for Microbiology, Washington, D.C. 13. Kaplan, E. L., B. F. Anthony, A. Bisno, et al Prevention of bacterial endocarditis. Circulation 56:139A- 143A. Downloaded from on June 15, 21 by guest

5 VOL. 37, Pefletler, L. L., D. T. Durack, and R. G. Petersdorf IV. Further observations on prophylaxis. J. Clin. Invest. 56: Pefetler, L. L., R. G. Petersdorf, and K. Nielson V. Effect of duration of infection and retained intracardiac catheter on response to treatment. J. Lab. Clin. Med. 7: Perlman, B., and L. R. Freedman Experimental endocarditis. II. A new method for the production of staphylococcal endocarditis of the aortic valve in rabbits. Yale J. Biol. Med. 44: ENDOCARDITIS IN RATS Petersdorf, R. G Antimicrobial prophylaxis of bacterial endocarditis. Am. J Med. 65: Sande, M. A., and K. B. Courtney Antimicrobial therapy of experimental endocarditis caused by Staphylococcus aureus. J. Infect. Dis. 131: Sande, M. A., and R. G. Irwing Penicillin-aminoglycoside synergy in experimental streptococcus viridans endocarditis. J. Infect. Dis. 129: Santoro, J., and M. E. Levison Experimental endocarditis in the rat. Infect. Immun. 19: Southwick, F. S., and D. T. Durack Chemotherapy of experimental streptococcal endocarditis. III. Failure of a bacteriostatic agent (tetracycline) in prophylaxis. J. Clin. Pathol. 27: Downloaded from on June 15, 21 by guest