ANTiMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 1979, p. 776-780 0066-4804/79/12-0776/05$02.00/0
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Rosaramicin Versus Penicillin G in Experimental Pneumococcal Meningitis CHARLES M. NOLAN,* THOMAS P. MONSON, AND W. CLYDE ULMER, JR. Department of Medicine, University of Arkansas College of Medicine, Little Rock, Arkansas 72201 Received for publication 28 September 1979
Rosaramicin, a new macrolide antibiotic, was compared with penicillin G in the treatment of pneumococcal meningitis in rabbits. Animals were infected intracistemally with 104 colony-forming units of Streptococcus pneumoniae type III (rosaramicin minimal inhibitory/bactericidal concentrations, 0.25/0.5 ,ug/ml; penicillin G miniimal inhibitory/bactericidal concentrations, 0.03/0.06 pug/ml). Treatment was instituted 96 h later. Infusion of rosaramicin at 25 mg/kg per h intravenously for 8 h produced a peak cerebrospinal fluid (CSF) drug concentration of 1.54 pug/ml (range, 0.87-3.6 pug/ml). During this infusion the numbers of pneumococci in CSF decreased from 6.2 ± 0.5 to 3.36 ± 1.12 logio colony-forming units per ml. Penicillin G, infused at 30 mg/kg per h for 8 h, reached a similar concentration in CSF but caused a greater reduction (P < 0.01) in CSF bacteria, from 6.4 ± 0.36 to 1.3 ± 0.67 logio colony-forming units per ml. Penicillin G, at 100 mg/kg per day intramuscularly for 5 days, cured 7 of 10 rabbits with pneumococcal meningitis. A higher dose, 300 mg/kg per day for 5 days, was no more efficacious: 11 of 14 rabbits were cured. Rosaramicin at 100 mg/kg per day intramuscularly for 5 days cured only 5 of 15 rabbits with meningitis, but a higher dosage regimen of that drug (250 mg/kg per day intramuscularly) produced acute, fuhminant enterocecitis and death within 48 h in seven of eight rabbits. No cytotoxin was detected in the feces of one rabbit with acute enterocecitis. Thus the efficacy of rosaramicin in experimental pneumococcal meningitis, measured by bacterial clearance from CSF and by treatment outcome, was less than that of penicillin G. In addition, high-dose parenteral rosaramicin caused acute, fulminant enterocecitis in a high proportion of treated rabbits. Antimicrobial therapy of patients with pneumococcal meningitis who are allergic to penicillin is satisfactory but could be improved. The cephalosporis are effective against Streptococcus pneunoniae but they penetrate poorly into cerebrospinal fluid (CSF) (12). Vancomycin penetrates the meninges satisfactorily (T. Murray, L. Strausbaugh, and M. Sande, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 16th, Chicago, Ill., abstr. no. 246A, 1976), but it is virtually untested in patients with meningitis owing to a reputation for toxicity (5). Chloramphenicol is the current drug of choice for pneumococcal meningitis in patients allergic to penicillin, but it is less than ideal because of its propensity to suppress bone marrow hematopoietic function (15) and also because of its bacteriostatic mode of action on susceptible organisms. Rosaramicin, a new macrolide antibiotic, is a structural analog of erythromycin (20). This agent is active in vitro against S. pneumoniae and the other species of bacteria most often
associated with bacterial meningitis (6, 13). Rosaramicin should penetrate well into the central nervous system because, like chloramphenicol, it is highly soluble in lipid solvents (20). In addition, rosaramicin possesses a basic pK. and should thus avoid active transport out of the central nervous system by the pump that removes organic acids such as the penicillins and cephalosporins (17). On the other hand, this basic drug might suffer attenuation of its antimicrobial activity, which is primarily bacteriostatic, in the acidic milieu of the CSF in bacterial
meningitis.
We considered that the contradictory points concerning the potential usefulness of rosaramicin for bacterial meningitis deserved specific study. Consequently, several aspects of therapy with rosaramicin were compared with those of penicillin G in an experimental model of pneumococcal meningitis. Drug concentrations in CSF were measured, and the rates of clearance of bacteria from CSF were compared during parenteral administration of the antibiotics. An776
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tibacterial potency of the two drugs in CSF was also assessed by outcome of a 5-day course of therapy. MATERIALS AND MErHODS All studies were performed using laboratory standard rosaramicin sodium phosphate (provided by George Miller, Schering Corp., Bloomfield, N.J.) and penicillin G potassium (Eli Lilly & Co., Indianapolis, Ind.). The minimal inhibitory concentrations (MICs) of rosaramicin and penicillin G for the strain of S. pneumoniae type Ill used in these experiments were determined in Mueller-Hinton broth supplemented (vol/vol) with a 5% sheep erythrocyte lysate (21). Minimal bactericidal concentrations (MBCs) were subsequently measured by streaking on blood agar plates broth from MIC tubes showing no visible growth after overnight culture. Rosarmicin MICs and MBCs were performed in broth buffered at the following pH values: 6.8, 7.1, 7.4, 7.7, and 8.0. Serum and CSF concentrations of rosaramicin and penicillin G were performed by a cup-plate assay using Bacillus subtilis as the indicator organism (3). Serum standards were prepared in pooled rabbit serum; CSF standards were prepared in a 0.01 M phosphate buffer after initial experiments that showed equivalency among this buffer, pooled control CSF, and pooled infected CSF from which bacteria were removed by filtration through a 0.22-nm filter (Gelman Instrument Co., Ann Arbor, Mich.). In preliminary experiments, parallel assays of standard rosaramicin solutions in phosphate buffer were done with agar and standards buffered at pH values of 7.1, 7.4, and 8.0. In previous experiments using this model of infection (11), meningitis was produced in 2-kg New Zealand white rabbits of either sex by intravenous (i.v.) inoculation of 5 x 106 colony-forming units of S. pneumoniae type III. Approximately 75% of rabbits become infected, manifested by CSF cultures that are positive for pneumococci 24 h after i.v. inoculation. Infected rabbits develop progressive neurological dysfunction and characteristic CSF findings of a polymorphonuclear leukocyte pheocytosis, decreased glucose, and elevated protein concentrations. If untreated, infected animals invariably die within 6 days of infection. In preliminary experiments it was determined that progressive meningitis of the precise character noted above, including clinical signs, CSF abnormalities, and uniform mortality, could be produced by inoculation of 104 colony-forming units of pneumococci into the cisterna magna. In addition, over 90% of rabbits induced by this route become infected. Consequently, the latter more consistent means of producing experimental pneumococcal meningitis was used throughout this study. All studies of therapy with rosaramicin or penicillin G were performed in rabbits that had been infected 96 h previously. This time was chosen because the signs of infection, including neurological dysfunction, were well established, and because the concentration of bacteria in CSF was approximately 106 colony-forming units per ml, a number similar to that found at the time of diagnosis in humans with pneumococcal meningitis (7).
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Eleven rabbits with meningitis were anesthetized with i.v. sodium pentobarbital. A marginal ear vein was cannulated, and rosaramicin was infused i.v. (Constant Infusion Pump, model 922, Imed Corp., San Diego, Calif.) in a dose of 25 mg/kg per h for 8 h. An intramuscular (i.m.) loading dose of 25 mg/kg was given at the beginning of the infusion. CSF was obtained for enumeration of bacteria (colony-forming units per milliliter) by the pour-plate method by puncture of the cisterna magna with a 25-gauge needle immediately before the infusion. CSF was similrly obtained for bacterial enumeration and rosaraniicin assay at 4 and 8 h. Blood was also obtained at 4 and 8 h for rosaramicin assay. Serum and CSF samples were maintained at -20°C and assayed for rosaramicin within 1 week. Preliminary experiments showed that the rosaramicin and penicillin G were stable in serum and CSF for that period of time. Eight infected rabbits were infused i.v. with penicillin G, 30 mg/kg per h for 8 h, after a similar i.m. loading dose. CSF was obtained before the infusion and at 4 and 8 h for enumeration of bacteria and penicillin G assay. Blood was also obtained from four of the rabbits for assay of penicillin G at 4 and 8 h. Other rabbits infected for 96 h received treatment with rosaramicin or penicillin G for 5 days. One group of 12 rabbits received rosaramicin at 50 mg/kg i.m. every 12 h; another group of eight received rosaramicin at 125 mg/kg i.m. every 12 h. Ten rabbits received penicillin G at 50 mg/kg i.m. every 12 h; 14 others received penicillin G at 150 mg/kg i.m. every 12 h. Ten rabbits served as untreated controls. On all treated rabbits, enumeration of bacteria in CSF was done before treatment, on days 2 and 4 of antibiotic therapy, and on day 2 after completion of therapy on those that survived. From three rabbits in each treatment group, serum specimens were obtained for antibiotic assay 1 h after the third i.m. injection. CSF drug concentrations were not monitored in these experiments. Necropsy was performed immediately after death on a rabbit with pneumococcal meningitis that had received three injections of rosaramicin at 125 mg/kg i.m. every 12 h. A section of the cecum was removed, fixed immediately in 10% Formalin, and prepared for light microscopy by staining with hematoxylin and eosin. Approximately 1 g of feces was aspirated aseptically into a syringe from the sectioned cecum and was frozen immediately at -20°C. One week later this specimen of feces was diluted in 10 ml of 0.15 M NaCl solution, mixed thoroughly on a Vortex apparatus, and centrifuged at 5,000 x g for 30 min. The supernatant was filtered through a 0.22-nm filter (Gelman), and six serial 10-fold dilutions were prepared in Eagle miniimal essential medium (GIBCO, Laboratories, Grand Island, N.Y.). Each dilution was inoculated in triplicate onto monolayers of hamster lung fibroblast cells (strain V-79) prepared in polystyrene microtiter plates. The cell monolayers were examined after 4, 24, and 96 h for cytopathic effect. Serial 10-fold dilutions of a sterile filtrate of 1-week broth culture of Clostridium difficile (CDC strain A-180, kindly supplied by V. R. Dowell, Jr., Center for Disease Control, Atlanta, Ga.) were included in this experiment as a cytotoxicity control. A rabbit with meningitis that had received
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three injections of rosaramicin, 50 mg/kg i.m. every 12 h, and an untreated rabbit were sacrificed by rapid esnguination. Cecal biopsies were obtained for microscopic examination, and specimens of feces were prepared as described and tested for cytotoxicity for hamster lung fibrobl4sts. RESULTS
The MIC of rosaramicin for the pneumococcal strain used in the present studies was 0.25 jig/ml over a pH range of 6.8 to 8.0; likewise, the MBC over that pH range was uniformly 0.5 jg/ml. The MIC of penicillin G for the experimental pneumococci, determined only at pH 7.3, was 0.03 aLg/ml; its MBC was 0.06 ag/ml. In the cup-plate assays, rosaramicin was more inhibitory for the indicator organism in expenments in which the agar was buffered at pH 8.0 than in those with the agar buffered at pH 7.1. However, with the agar buffered at pH 8.0, zone sizes for rosaramicin added to the wells at pH values of 7.1 and 7.4 were not different from those obtained with the drug added to the wells at pH 8.0 at rosaramicin concentrations of 0.5 ag/ml and above. Consequently, the assays for CSF rosaramicin were performed with the agar prepared at pH 8.0 and standards prepared at pH 7.3, approximately that of control CSF. During the infusion of 25 mg/kg per h, the concentrations of rosaramicin in CSF were approximately 15% of those in serum (Table 1). The mean 4-h CSF rosaramicin concentration was twice the MBC for the infecting organism; the 8-h mean exceeded the MBC by threefold. Mean CSF levels of penicillin G during its infusion were imilar to those of rosaramicin even though the serum concentrations were considerably higher. During i.v. infusion, rosaramicin produced less
CSF bactericidal activity for the infecting pneumococcus than penicillin G did (Fig. 1). Although the mean concentration of bacteria in CSF was reduced almost 1,000-fold by rosaram-
icin, no rosaramicin-treated rabbit had a sterile CSF culture at the end of the 8-h infusion. Seven of eight rabbits infused with penicillin G had sterile CSF cultures at a 10' pour-plate dilution after 8 h of infusion. Results of treatment of rabbits with pneumococcal meningitis with two dosage regimens each of rosaramicin and penicillin G are shown in Table 2. Penicillin G, administered at 150 mg/ kg every 12 h, produced serum levels equivalent to those obtained in humans with bacterial meningitis (9) and yielded a high cure rate. The smaller dose of penicillin G was also efficacious. Among animals receiving rosaramicin at 50 mg/ kg every 12 h, quantitative cultures of CSF taken
Hours of antibiotic infusion FIG. 1. Clearance of bacteria from CSF of rabbits with pneumococcal meningitis during infusions of rosaramicin at 25 mg/kg per h (0) and penicillin G at 30 mg/kg per h (0). Each point represents mean standard error of the mean. The 8-h mean values are significantly different (P < 0.01, Student's t test). ±
TABLE 1. Concentrations of rosaramicin and penicilin G in serum and CSF of rabbits with pneumococcal Antibiotic
(dosage)
Rosaramicin (25 mg/kg per h)
meningitis during intravenous infusion" Concn (ug/ml) in: Timeof
infusion
(h) 4
8
Penicillin G (30 mg/kg per h)
Serum 7.9 (3.7-14.0)" 9.6 (6.6-14.5)
CSF
CSF
1.19 (0.5-2.1) 1.54 (0.87-3.6)
34.5 (28.9-40.2) 1.96 (0.94-2.50) 29.9 (23.2-36.7) 1.01 (0.34-1.95) a Eleven rabbits received rosaramicin; eight received penicillin G. b Mean CSF drug concentration/mean serum drug concentration x 100. c Range of individual values. 4
8
penetrationh (%) 15 16 6 3
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TABLE 2. Outcome of a 5-day course of treatment of rabbits with pneumococcal meningitis" Antibiotic regimen
Serum concn (jug/mn)
No. of rabbits
Treated
Survived
Rosaramicin 5 (33%)d 15 2.7 (2.2-3.5)c 100 mg/kg per dayb 1 (13%) 5.2 (4.6-6.3) 8 250 mg/kg per day Penicillin G 7 (70%) 10 5.5 (4.4-6.8) 100 mg/kg per day 19.8 (14.3-24.2) 14 11 (79%) 300 mg/kg per day 0 10 None aTreatment was initiated in rabbits infected 96 h previously. b Daily dosage was administered in two equal i.m. injections every 12 h. 'Mean (range) serum levels obtained on three rabbits 1 h after the third i.m. injection of antibiotic. d Percentage of those treated that survived.
after 48 h of therapy from those who survived contained fewer bacteria than cultures from those who ultimately died; the surviving group had a mean concentration of bacteria in CSF of 1.25 ± 0.5 logio colony-forming units per ml, whereas the group that died had 2.58 ± 0.69 logio colony-forming units per ml (P < 0.05, Student's t test). The higher dosage regimen of rosaramicin was associated with a higher fatality rate than the lower dose (P < 0.05, Fisher's exact test). All rabbits that died while receiving rosaramicin at 125 mg/kg every 12 h developed profuse diarrhea and prostration within 24 h of initiation of therapy; all deaths in that group occurred within 48 h of initiation of treatment. Three rabbits receiving the lower regimen of rosaramicin also died during the first 48 h of treatment, but only one of those developed diarrhea. Likewise, none of the four control rabbits that died during that period had diarrhea. The cecum of the necropsied rabbit that received rosaramicin i.m. at 125 mg/kg every 12 h was grossly distended with semiformed stool; the colon contained no feces. Microscopic examination of the cecum showed diffuse mucosal necrosis with total disruption of the normal villous architecture. The mucosa and submucosa were infiltrated by polymorphonuclear leukocytes. No pseudomembranous patches of exudate were present on the mucosal surface, however. The gross appearance and microscopic appearance ofthe cecum of the rabbit that received the lower dosage of rosaramicin were indistinguishable from those of the normal rabbit. Filtrates of fecal specimens from the ceca of the two rosaramicin-treated rabbits and the normal animal showed no cytotoxicity for hamster lung fibroblasts. By contrast, the C. difficile culture produced a cytopathic effect upon the cell monolayers at dilutions of 1:200,000 and above.
DISCUSSION In this experimental model of pneumococcal meningitis, penetration of rosaramicin from blood to CSF during intravenous infusion was equal to or greater than that achieved clinically or experimentally for penicillin G (9), ampicillin (19), gentamicin (18), and several cephalosporins (12). Nevertheless, the CSF bactericidal activity of rosaramicin toward the infecting pneumococcus was less than that of penicillin G (Fig. 1). Even though CSF concentrations of the two drugs were similar, the penicillin concentration in CSF exceeded by more than 30-fold its MBC for the infecting organism, whereas the CSF level of rosaramicin was only threefold higher than its MBC. Sande et al. have shown that the CSF bactericidal activities of two other antibiotics, chloramphenicol and gentamicin, continue to increase as their CSF concentrations are raised progressively above their MICs for the infecting pathogen (18; W. M. Scheld, R. S. Brown, Jr., D. D. Fletcher, and M. A. Sande, Clin. Res. 27:355a, 1979). Thus, it would probably be necessary to achieve CSF concentrations of rosaramicin higher than those obtained in the i.v. infusion studies to enhance the CSF bactericidal activity of this agent against pneumococci. This would ordinarily be done by increasing the systemic dosage of the antimicrobial agent. However, in the present study rosaramicin was infused at 25 mg/kg per h, a rate equivalent to a 13-g daily dose in humans. Disproportionately low serum levels of rosaramicin have been noted in several animal species during i.v. infusion (G. Miller, personal communication). This phenomenon has been attributed to the lipid solubility of the drug, a property that is associated with rapid distribution from plasma to tissues. Thus, if the CSF concentration of a drug and its efficacy in bacterial meningitis are determined in part by
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attainment of a high concentration of the drug in serum, then the extreme affinity of rosaramicin for lipid solvents works to its disadvantage. On the other hand, rosaramicin's lipid solubility could permit significant penetration from blood directly into brain tissue (2, 14). However, this possibility must remain speculative because brain concentrations of rosaramicin were not measured in the present study. Only 33% of rabbits receiving rosaramicin at 50 mg/kg i.m. every 12 h survived a 5-day course of therapy. Rabbits cured by this regimen of rosaramicin showed a larger reduction in the colony counts of pneumococci in CSF during the first 48 h of therapy than that noted in animals that died during therapy. It was thus considered that a larger dose of rosaramicin would be likely to enhance survival by producing higher CSF drug concentrations and a more consistent bactericidal effect. However, rosaramicin at a dose of 125 mg/kg i.m. every 12 h was associated with an even higher fatality rate. The appearance of rabbits that died while receiving the higher dose of rosaramicin resembled that previously described for rodents with acute antibiotic-induced enterocecitis (10, 16). Furthermore, the gross and microscopic changes that have been associated with acute enterocecitis in laboratory animals were present in the cecum of one affected rabbit at necropsy. However, no cytotoxin could be detected in an extract of feces from the cecum of that rabbit, and no attempt was made to culture its feces for C. difficik, the bacterium that has recently been linked firmly with antibiotic-associated colitis in humans (1) and its counterpart in experimental animals (10). It is of interest that the administration of large doses of erythromycin by mouth to guinea pigs also produced gut necrosis (4) even though this antimicrobial agent and rosaramicin both possess excellent inhibitory activity in vitro against C. difficile (8). Further work will thus be required to ascertain the cause of acute, fuhminant enterocecitis produced in rabbits by the administation of high-dose parenteral rosaraxmcin. In conclusion, this study in an animal model uncovered no unique advantages for rosaramicin in the treatment of pneumococcal meningitis. Further investigation in this area appears unwarranted. ACKNOWLEDGMENTS We acknowledge the excellent secretarial assistance of Inelle Reynolds. This study was supported in part by a grant-in-aid from Schering Codrporation, Bloomfield, N.J.
LITERATURE CrE 1. Bartlett, J. G., T. W. Chang, ML Gurwith, S. L Gorbach, and A. B. Onderdonk. 1978. Antibiotic-associated pseudomembranous colitis due to toxin-producing
ANTIMICROB. AGENTS CHEMOTHER. clostridia. N. Engl. J. Med. 298:531-534. 2. Beam, T. R., Jr., and J. C. Allen. 1979. Assessment of antibiotic efficacy in acute bacterial meningitis. Clin. Pharm. Ther. 25:199-203. 3. Bennett, J. V., J. L Brochie, E. J. Benner, and W. M. M. Kirby. 1966. Simplified, accurate method for antibiotic assay of clinical specimens. Appl. Microbiol. 14: 170-177. 4. Boyd, E. M., and M. A. Price-Jones. 1960. The comparative acute oral toxicity of spiramycin adipate in 5. 6. 7.
8.
9.
10.
mice, rats, guinea pigs, and rabbits. Antibiot. Chemother. 10:273-284. Cook, F. V., and W. E. Farrar. 1978. Vancomycin revisited. Ann. Intern. Med. 88:813-818. Crowe, C. C., and W. E. Sanders, Jr. 1974. Rosamicin: evaluation in vitro and comparison with erythromycin. Antimicrob. Agents Chemother. 5:272-275. Feldman, W. E., Jr. 1977. Relation of concentrations of bacteria and bacterial antigen in cerebrospinal fluid to prognosis in patients with bacterial meningitis. N. Engl. J. Med. 296:433-435. George, W. L, B. D. Kirby, V. L Sutter, and S. M. Finegold. 1979. Antimicrobial susceptibility of Clostridium difficile, p. 267-271. In D. Schlessinger (ed.), Microbiology-1979. American Society for Microbiology, Washington, D.C. Hieber, J. P., and J. D. Nelson. 1977. A pharmacologic evaluation of penicillin in children with purulent meningitis. N. Engl. J. Med. 297:410-413. Lusk, R. IL, R. Fekety, J. Silva, R. A. Browne, D. EL Ringler, and G. D. Abrams. 1978. Clindamycin-induced enterocolitis in hamsters. J. Infect. Dis. 137:464-
475. 11. O'Donoghue, J. M., A. E. Schweid, and H. N. Beaty. 1974. Experimental pneumococcal meningitis. 1. A rabbit model. Proc. Soc. Exp. Biol. Med. 146:571-574. 12. Sande, M. A., R. J. Sheretz, 0. Zak, and L J. Strausbaugh. 1978. Cephalosporin antibiotics in therapy of experimental Streptococcus pneumoniae and Haemophilus influenzae meningitis in rabbits. J. Infect. Dis. 137:S161-S168. 13. Sanders, C. C., and W. E. Sanders, Jr. 1977. In vitro activity of rosaramicin against Neisseria and Haemophilus, including penicillinase-producing strains. Antimicrob. Agents Chemother. 12:293-294. 14. Schanker, L S. 1966. Passage of drugs into and out of the central nervous system, p. 1044-1050. Antimicrob. Agents Chemother. 1965. 15. Scott, J. L, S. M. Finegold, G. A. Belkin, and J. S. Lawrence. 1965. A controlled double-blind study of the hematologic toxicity of chloramphenicol. N. Engl. J. Med. 272:1137-1142. 16. Small, J. D. 1968. Fatal enterocolitis in hamsters given
lincomycin hydrochloride. Lab. Anim. Care 18:411-420. 17. Spector, R., and A. V. Lorenzo. 1974. The effects of salicylate and probenecid on the cerebrospinal fluid transport of penicillin, amino salicylic acid, and iodide. J. Pharmacol. Exp. Ther. 188:55-65. 18. Strausbaugh, L J., and M. A. Sande. 1978. Factors influencing the therapy of experimental Proteus mirabibis meningitis in rabbits. J. Infect. Dis. 137:251-260. 19. Taber, L H., M. D. Yow, and F. G. Neiberg. 1967. The penetration of broad-spectrum antibiotics into the cerebrospinal fluid. Ann. N. Y. Acad. Sci. 145:473-481. 20. Wagman, G. A., J. A. Waitz, J. Marquez, A. Murawski, E. M. Oden, R. T. Testa, and M. J. Weinstein. 1972. A new micromonospora-produced macrolide antibiotic, rosamicin. J. Antibiot. Tokyo 25:641646. 21. Washington, J. A., and A. L Barry. 1974. Dilution test procedures, p. 410-417. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
Vol. 16, No. 6
Rosaramicin Versus Penicillin G in Experimental Pneumococcal Meningitis CHARLES M. NOLAN,* THOMAS P. MONSON, AND W. CLYDE ULMER, JR. Department of Medicine, University of Arkansas College of Medicine, Little Rock, Arkansas 72201 Received for publication 28 September 1979
Rosaramicin, a new macrolide antibiotic, was compared with penicillin G in the treatment of pneumococcal meningitis in rabbits. Animals were infected intracistemally with 104 colony-forming units of Streptococcus pneumoniae type III (rosaramicin minimal inhibitory/bactericidal concentrations, 0.25/0.5 ,ug/ml; penicillin G miniimal inhibitory/bactericidal concentrations, 0.03/0.06 pug/ml). Treatment was instituted 96 h later. Infusion of rosaramicin at 25 mg/kg per h intravenously for 8 h produced a peak cerebrospinal fluid (CSF) drug concentration of 1.54 pug/ml (range, 0.87-3.6 pug/ml). During this infusion the numbers of pneumococci in CSF decreased from 6.2 ± 0.5 to 3.36 ± 1.12 logio colony-forming units per ml. Penicillin G, infused at 30 mg/kg per h for 8 h, reached a similar concentration in CSF but caused a greater reduction (P < 0.01) in CSF bacteria, from 6.4 ± 0.36 to 1.3 ± 0.67 logio colony-forming units per ml. Penicillin G, at 100 mg/kg per day intramuscularly for 5 days, cured 7 of 10 rabbits with pneumococcal meningitis. A higher dose, 300 mg/kg per day for 5 days, was no more efficacious: 11 of 14 rabbits were cured. Rosaramicin at 100 mg/kg per day intramuscularly for 5 days cured only 5 of 15 rabbits with meningitis, but a higher dosage regimen of that drug (250 mg/kg per day intramuscularly) produced acute, fuhminant enterocecitis and death within 48 h in seven of eight rabbits. No cytotoxin was detected in the feces of one rabbit with acute enterocecitis. Thus the efficacy of rosaramicin in experimental pneumococcal meningitis, measured by bacterial clearance from CSF and by treatment outcome, was less than that of penicillin G. In addition, high-dose parenteral rosaramicin caused acute, fulminant enterocecitis in a high proportion of treated rabbits. Antimicrobial therapy of patients with pneumococcal meningitis who are allergic to penicillin is satisfactory but could be improved. The cephalosporis are effective against Streptococcus pneunoniae but they penetrate poorly into cerebrospinal fluid (CSF) (12). Vancomycin penetrates the meninges satisfactorily (T. Murray, L. Strausbaugh, and M. Sande, Program Abstr. Intersci. Conf. Antimicrob. Agents Chemother. 16th, Chicago, Ill., abstr. no. 246A, 1976), but it is virtually untested in patients with meningitis owing to a reputation for toxicity (5). Chloramphenicol is the current drug of choice for pneumococcal meningitis in patients allergic to penicillin, but it is less than ideal because of its propensity to suppress bone marrow hematopoietic function (15) and also because of its bacteriostatic mode of action on susceptible organisms. Rosaramicin, a new macrolide antibiotic, is a structural analog of erythromycin (20). This agent is active in vitro against S. pneumoniae and the other species of bacteria most often
associated with bacterial meningitis (6, 13). Rosaramicin should penetrate well into the central nervous system because, like chloramphenicol, it is highly soluble in lipid solvents (20). In addition, rosaramicin possesses a basic pK. and should thus avoid active transport out of the central nervous system by the pump that removes organic acids such as the penicillins and cephalosporins (17). On the other hand, this basic drug might suffer attenuation of its antimicrobial activity, which is primarily bacteriostatic, in the acidic milieu of the CSF in bacterial
meningitis.
We considered that the contradictory points concerning the potential usefulness of rosaramicin for bacterial meningitis deserved specific study. Consequently, several aspects of therapy with rosaramicin were compared with those of penicillin G in an experimental model of pneumococcal meningitis. Drug concentrations in CSF were measured, and the rates of clearance of bacteria from CSF were compared during parenteral administration of the antibiotics. An776
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tibacterial potency of the two drugs in CSF was also assessed by outcome of a 5-day course of therapy. MATERIALS AND MErHODS All studies were performed using laboratory standard rosaramicin sodium phosphate (provided by George Miller, Schering Corp., Bloomfield, N.J.) and penicillin G potassium (Eli Lilly & Co., Indianapolis, Ind.). The minimal inhibitory concentrations (MICs) of rosaramicin and penicillin G for the strain of S. pneumoniae type Ill used in these experiments were determined in Mueller-Hinton broth supplemented (vol/vol) with a 5% sheep erythrocyte lysate (21). Minimal bactericidal concentrations (MBCs) were subsequently measured by streaking on blood agar plates broth from MIC tubes showing no visible growth after overnight culture. Rosarmicin MICs and MBCs were performed in broth buffered at the following pH values: 6.8, 7.1, 7.4, 7.7, and 8.0. Serum and CSF concentrations of rosaramicin and penicillin G were performed by a cup-plate assay using Bacillus subtilis as the indicator organism (3). Serum standards were prepared in pooled rabbit serum; CSF standards were prepared in a 0.01 M phosphate buffer after initial experiments that showed equivalency among this buffer, pooled control CSF, and pooled infected CSF from which bacteria were removed by filtration through a 0.22-nm filter (Gelman Instrument Co., Ann Arbor, Mich.). In preliminary experiments, parallel assays of standard rosaramicin solutions in phosphate buffer were done with agar and standards buffered at pH values of 7.1, 7.4, and 8.0. In previous experiments using this model of infection (11), meningitis was produced in 2-kg New Zealand white rabbits of either sex by intravenous (i.v.) inoculation of 5 x 106 colony-forming units of S. pneumoniae type III. Approximately 75% of rabbits become infected, manifested by CSF cultures that are positive for pneumococci 24 h after i.v. inoculation. Infected rabbits develop progressive neurological dysfunction and characteristic CSF findings of a polymorphonuclear leukocyte pheocytosis, decreased glucose, and elevated protein concentrations. If untreated, infected animals invariably die within 6 days of infection. In preliminary experiments it was determined that progressive meningitis of the precise character noted above, including clinical signs, CSF abnormalities, and uniform mortality, could be produced by inoculation of 104 colony-forming units of pneumococci into the cisterna magna. In addition, over 90% of rabbits induced by this route become infected. Consequently, the latter more consistent means of producing experimental pneumococcal meningitis was used throughout this study. All studies of therapy with rosaramicin or penicillin G were performed in rabbits that had been infected 96 h previously. This time was chosen because the signs of infection, including neurological dysfunction, were well established, and because the concentration of bacteria in CSF was approximately 106 colony-forming units per ml, a number similar to that found at the time of diagnosis in humans with pneumococcal meningitis (7).
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Eleven rabbits with meningitis were anesthetized with i.v. sodium pentobarbital. A marginal ear vein was cannulated, and rosaramicin was infused i.v. (Constant Infusion Pump, model 922, Imed Corp., San Diego, Calif.) in a dose of 25 mg/kg per h for 8 h. An intramuscular (i.m.) loading dose of 25 mg/kg was given at the beginning of the infusion. CSF was obtained for enumeration of bacteria (colony-forming units per milliliter) by the pour-plate method by puncture of the cisterna magna with a 25-gauge needle immediately before the infusion. CSF was similrly obtained for bacterial enumeration and rosaraniicin assay at 4 and 8 h. Blood was also obtained at 4 and 8 h for rosaramicin assay. Serum and CSF samples were maintained at -20°C and assayed for rosaramicin within 1 week. Preliminary experiments showed that the rosaramicin and penicillin G were stable in serum and CSF for that period of time. Eight infected rabbits were infused i.v. with penicillin G, 30 mg/kg per h for 8 h, after a similar i.m. loading dose. CSF was obtained before the infusion and at 4 and 8 h for enumeration of bacteria and penicillin G assay. Blood was also obtained from four of the rabbits for assay of penicillin G at 4 and 8 h. Other rabbits infected for 96 h received treatment with rosaramicin or penicillin G for 5 days. One group of 12 rabbits received rosaramicin at 50 mg/kg i.m. every 12 h; another group of eight received rosaramicin at 125 mg/kg i.m. every 12 h. Ten rabbits received penicillin G at 50 mg/kg i.m. every 12 h; 14 others received penicillin G at 150 mg/kg i.m. every 12 h. Ten rabbits served as untreated controls. On all treated rabbits, enumeration of bacteria in CSF was done before treatment, on days 2 and 4 of antibiotic therapy, and on day 2 after completion of therapy on those that survived. From three rabbits in each treatment group, serum specimens were obtained for antibiotic assay 1 h after the third i.m. injection. CSF drug concentrations were not monitored in these experiments. Necropsy was performed immediately after death on a rabbit with pneumococcal meningitis that had received three injections of rosaramicin at 125 mg/kg i.m. every 12 h. A section of the cecum was removed, fixed immediately in 10% Formalin, and prepared for light microscopy by staining with hematoxylin and eosin. Approximately 1 g of feces was aspirated aseptically into a syringe from the sectioned cecum and was frozen immediately at -20°C. One week later this specimen of feces was diluted in 10 ml of 0.15 M NaCl solution, mixed thoroughly on a Vortex apparatus, and centrifuged at 5,000 x g for 30 min. The supernatant was filtered through a 0.22-nm filter (Gelman), and six serial 10-fold dilutions were prepared in Eagle miniimal essential medium (GIBCO, Laboratories, Grand Island, N.Y.). Each dilution was inoculated in triplicate onto monolayers of hamster lung fibroblast cells (strain V-79) prepared in polystyrene microtiter plates. The cell monolayers were examined after 4, 24, and 96 h for cytopathic effect. Serial 10-fold dilutions of a sterile filtrate of 1-week broth culture of Clostridium difficile (CDC strain A-180, kindly supplied by V. R. Dowell, Jr., Center for Disease Control, Atlanta, Ga.) were included in this experiment as a cytotoxicity control. A rabbit with meningitis that had received
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three injections of rosaramicin, 50 mg/kg i.m. every 12 h, and an untreated rabbit were sacrificed by rapid esnguination. Cecal biopsies were obtained for microscopic examination, and specimens of feces were prepared as described and tested for cytotoxicity for hamster lung fibrobl4sts. RESULTS
The MIC of rosaramicin for the pneumococcal strain used in the present studies was 0.25 jig/ml over a pH range of 6.8 to 8.0; likewise, the MBC over that pH range was uniformly 0.5 jg/ml. The MIC of penicillin G for the experimental pneumococci, determined only at pH 7.3, was 0.03 aLg/ml; its MBC was 0.06 ag/ml. In the cup-plate assays, rosaramicin was more inhibitory for the indicator organism in expenments in which the agar was buffered at pH 8.0 than in those with the agar buffered at pH 7.1. However, with the agar buffered at pH 8.0, zone sizes for rosaramicin added to the wells at pH values of 7.1 and 7.4 were not different from those obtained with the drug added to the wells at pH 8.0 at rosaramicin concentrations of 0.5 ag/ml and above. Consequently, the assays for CSF rosaramicin were performed with the agar prepared at pH 8.0 and standards prepared at pH 7.3, approximately that of control CSF. During the infusion of 25 mg/kg per h, the concentrations of rosaramicin in CSF were approximately 15% of those in serum (Table 1). The mean 4-h CSF rosaramicin concentration was twice the MBC for the infecting organism; the 8-h mean exceeded the MBC by threefold. Mean CSF levels of penicillin G during its infusion were imilar to those of rosaramicin even though the serum concentrations were considerably higher. During i.v. infusion, rosaramicin produced less
CSF bactericidal activity for the infecting pneumococcus than penicillin G did (Fig. 1). Although the mean concentration of bacteria in CSF was reduced almost 1,000-fold by rosaram-
icin, no rosaramicin-treated rabbit had a sterile CSF culture at the end of the 8-h infusion. Seven of eight rabbits infused with penicillin G had sterile CSF cultures at a 10' pour-plate dilution after 8 h of infusion. Results of treatment of rabbits with pneumococcal meningitis with two dosage regimens each of rosaramicin and penicillin G are shown in Table 2. Penicillin G, administered at 150 mg/ kg every 12 h, produced serum levels equivalent to those obtained in humans with bacterial meningitis (9) and yielded a high cure rate. The smaller dose of penicillin G was also efficacious. Among animals receiving rosaramicin at 50 mg/ kg every 12 h, quantitative cultures of CSF taken
Hours of antibiotic infusion FIG. 1. Clearance of bacteria from CSF of rabbits with pneumococcal meningitis during infusions of rosaramicin at 25 mg/kg per h (0) and penicillin G at 30 mg/kg per h (0). Each point represents mean standard error of the mean. The 8-h mean values are significantly different (P < 0.01, Student's t test). ±
TABLE 1. Concentrations of rosaramicin and penicilin G in serum and CSF of rabbits with pneumococcal Antibiotic
(dosage)
Rosaramicin (25 mg/kg per h)
meningitis during intravenous infusion" Concn (ug/ml) in: Timeof
infusion
(h) 4
8
Penicillin G (30 mg/kg per h)
Serum 7.9 (3.7-14.0)" 9.6 (6.6-14.5)
CSF
CSF
1.19 (0.5-2.1) 1.54 (0.87-3.6)
34.5 (28.9-40.2) 1.96 (0.94-2.50) 29.9 (23.2-36.7) 1.01 (0.34-1.95) a Eleven rabbits received rosaramicin; eight received penicillin G. b Mean CSF drug concentration/mean serum drug concentration x 100. c Range of individual values. 4
8
penetrationh (%) 15 16 6 3
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TABLE 2. Outcome of a 5-day course of treatment of rabbits with pneumococcal meningitis" Antibiotic regimen
Serum concn (jug/mn)
No. of rabbits
Treated
Survived
Rosaramicin 5 (33%)d 15 2.7 (2.2-3.5)c 100 mg/kg per dayb 1 (13%) 5.2 (4.6-6.3) 8 250 mg/kg per day Penicillin G 7 (70%) 10 5.5 (4.4-6.8) 100 mg/kg per day 19.8 (14.3-24.2) 14 11 (79%) 300 mg/kg per day 0 10 None aTreatment was initiated in rabbits infected 96 h previously. b Daily dosage was administered in two equal i.m. injections every 12 h. 'Mean (range) serum levels obtained on three rabbits 1 h after the third i.m. injection of antibiotic. d Percentage of those treated that survived.
after 48 h of therapy from those who survived contained fewer bacteria than cultures from those who ultimately died; the surviving group had a mean concentration of bacteria in CSF of 1.25 ± 0.5 logio colony-forming units per ml, whereas the group that died had 2.58 ± 0.69 logio colony-forming units per ml (P < 0.05, Student's t test). The higher dosage regimen of rosaramicin was associated with a higher fatality rate than the lower dose (P < 0.05, Fisher's exact test). All rabbits that died while receiving rosaramicin at 125 mg/kg every 12 h developed profuse diarrhea and prostration within 24 h of initiation of therapy; all deaths in that group occurred within 48 h of initiation of treatment. Three rabbits receiving the lower regimen of rosaramicin also died during the first 48 h of treatment, but only one of those developed diarrhea. Likewise, none of the four control rabbits that died during that period had diarrhea. The cecum of the necropsied rabbit that received rosaramicin i.m. at 125 mg/kg every 12 h was grossly distended with semiformed stool; the colon contained no feces. Microscopic examination of the cecum showed diffuse mucosal necrosis with total disruption of the normal villous architecture. The mucosa and submucosa were infiltrated by polymorphonuclear leukocytes. No pseudomembranous patches of exudate were present on the mucosal surface, however. The gross appearance and microscopic appearance ofthe cecum of the rabbit that received the lower dosage of rosaramicin were indistinguishable from those of the normal rabbit. Filtrates of fecal specimens from the ceca of the two rosaramicin-treated rabbits and the normal animal showed no cytotoxicity for hamster lung fibroblasts. By contrast, the C. difficile culture produced a cytopathic effect upon the cell monolayers at dilutions of 1:200,000 and above.
DISCUSSION In this experimental model of pneumococcal meningitis, penetration of rosaramicin from blood to CSF during intravenous infusion was equal to or greater than that achieved clinically or experimentally for penicillin G (9), ampicillin (19), gentamicin (18), and several cephalosporins (12). Nevertheless, the CSF bactericidal activity of rosaramicin toward the infecting pneumococcus was less than that of penicillin G (Fig. 1). Even though CSF concentrations of the two drugs were similar, the penicillin concentration in CSF exceeded by more than 30-fold its MBC for the infecting organism, whereas the CSF level of rosaramicin was only threefold higher than its MBC. Sande et al. have shown that the CSF bactericidal activities of two other antibiotics, chloramphenicol and gentamicin, continue to increase as their CSF concentrations are raised progressively above their MICs for the infecting pathogen (18; W. M. Scheld, R. S. Brown, Jr., D. D. Fletcher, and M. A. Sande, Clin. Res. 27:355a, 1979). Thus, it would probably be necessary to achieve CSF concentrations of rosaramicin higher than those obtained in the i.v. infusion studies to enhance the CSF bactericidal activity of this agent against pneumococci. This would ordinarily be done by increasing the systemic dosage of the antimicrobial agent. However, in the present study rosaramicin was infused at 25 mg/kg per h, a rate equivalent to a 13-g daily dose in humans. Disproportionately low serum levels of rosaramicin have been noted in several animal species during i.v. infusion (G. Miller, personal communication). This phenomenon has been attributed to the lipid solubility of the drug, a property that is associated with rapid distribution from plasma to tissues. Thus, if the CSF concentration of a drug and its efficacy in bacterial meningitis are determined in part by
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attainment of a high concentration of the drug in serum, then the extreme affinity of rosaramicin for lipid solvents works to its disadvantage. On the other hand, rosaramicin's lipid solubility could permit significant penetration from blood directly into brain tissue (2, 14). However, this possibility must remain speculative because brain concentrations of rosaramicin were not measured in the present study. Only 33% of rabbits receiving rosaramicin at 50 mg/kg i.m. every 12 h survived a 5-day course of therapy. Rabbits cured by this regimen of rosaramicin showed a larger reduction in the colony counts of pneumococci in CSF during the first 48 h of therapy than that noted in animals that died during therapy. It was thus considered that a larger dose of rosaramicin would be likely to enhance survival by producing higher CSF drug concentrations and a more consistent bactericidal effect. However, rosaramicin at a dose of 125 mg/kg i.m. every 12 h was associated with an even higher fatality rate. The appearance of rabbits that died while receiving the higher dose of rosaramicin resembled that previously described for rodents with acute antibiotic-induced enterocecitis (10, 16). Furthermore, the gross and microscopic changes that have been associated with acute enterocecitis in laboratory animals were present in the cecum of one affected rabbit at necropsy. However, no cytotoxin could be detected in an extract of feces from the cecum of that rabbit, and no attempt was made to culture its feces for C. difficik, the bacterium that has recently been linked firmly with antibiotic-associated colitis in humans (1) and its counterpart in experimental animals (10). It is of interest that the administration of large doses of erythromycin by mouth to guinea pigs also produced gut necrosis (4) even though this antimicrobial agent and rosaramicin both possess excellent inhibitory activity in vitro against C. difficile (8). Further work will thus be required to ascertain the cause of acute, fuhminant enterocecitis produced in rabbits by the administation of high-dose parenteral rosaraxmcin. In conclusion, this study in an animal model uncovered no unique advantages for rosaramicin in the treatment of pneumococcal meningitis. Further investigation in this area appears unwarranted. ACKNOWLEDGMENTS We acknowledge the excellent secretarial assistance of Inelle Reynolds. This study was supported in part by a grant-in-aid from Schering Codrporation, Bloomfield, N.J.
LITERATURE CrE 1. Bartlett, J. G., T. W. Chang, ML Gurwith, S. L Gorbach, and A. B. Onderdonk. 1978. Antibiotic-associated pseudomembranous colitis due to toxin-producing
ANTIMICROB. AGENTS CHEMOTHER. clostridia. N. Engl. J. Med. 298:531-534. 2. Beam, T. R., Jr., and J. C. Allen. 1979. Assessment of antibiotic efficacy in acute bacterial meningitis. Clin. Pharm. Ther. 25:199-203. 3. Bennett, J. V., J. L Brochie, E. J. Benner, and W. M. M. Kirby. 1966. Simplified, accurate method for antibiotic assay of clinical specimens. Appl. Microbiol. 14: 170-177. 4. Boyd, E. M., and M. A. Price-Jones. 1960. The comparative acute oral toxicity of spiramycin adipate in 5. 6. 7.
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mice, rats, guinea pigs, and rabbits. Antibiot. Chemother. 10:273-284. Cook, F. V., and W. E. Farrar. 1978. Vancomycin revisited. Ann. Intern. Med. 88:813-818. Crowe, C. C., and W. E. Sanders, Jr. 1974. Rosamicin: evaluation in vitro and comparison with erythromycin. Antimicrob. Agents Chemother. 5:272-275. Feldman, W. E., Jr. 1977. Relation of concentrations of bacteria and bacterial antigen in cerebrospinal fluid to prognosis in patients with bacterial meningitis. N. Engl. J. Med. 296:433-435. George, W. L, B. D. Kirby, V. L Sutter, and S. M. Finegold. 1979. Antimicrobial susceptibility of Clostridium difficile, p. 267-271. In D. Schlessinger (ed.), Microbiology-1979. American Society for Microbiology, Washington, D.C. Hieber, J. P., and J. D. Nelson. 1977. A pharmacologic evaluation of penicillin in children with purulent meningitis. N. Engl. J. Med. 297:410-413. Lusk, R. IL, R. Fekety, J. Silva, R. A. Browne, D. EL Ringler, and G. D. Abrams. 1978. Clindamycin-induced enterocolitis in hamsters. J. Infect. Dis. 137:464-
475. 11. O'Donoghue, J. M., A. E. Schweid, and H. N. Beaty. 1974. Experimental pneumococcal meningitis. 1. A rabbit model. Proc. Soc. Exp. Biol. Med. 146:571-574. 12. Sande, M. A., R. J. Sheretz, 0. Zak, and L J. Strausbaugh. 1978. Cephalosporin antibiotics in therapy of experimental Streptococcus pneumoniae and Haemophilus influenzae meningitis in rabbits. J. Infect. Dis. 137:S161-S168. 13. Sanders, C. C., and W. E. Sanders, Jr. 1977. In vitro activity of rosaramicin against Neisseria and Haemophilus, including penicillinase-producing strains. Antimicrob. Agents Chemother. 12:293-294. 14. Schanker, L S. 1966. Passage of drugs into and out of the central nervous system, p. 1044-1050. Antimicrob. Agents Chemother. 1965. 15. Scott, J. L, S. M. Finegold, G. A. Belkin, and J. S. Lawrence. 1965. A controlled double-blind study of the hematologic toxicity of chloramphenicol. N. Engl. J. Med. 272:1137-1142. 16. Small, J. D. 1968. Fatal enterocolitis in hamsters given
lincomycin hydrochloride. Lab. Anim. Care 18:411-420. 17. Spector, R., and A. V. Lorenzo. 1974. The effects of salicylate and probenecid on the cerebrospinal fluid transport of penicillin, amino salicylic acid, and iodide. J. Pharmacol. Exp. Ther. 188:55-65. 18. Strausbaugh, L J., and M. A. Sande. 1978. Factors influencing the therapy of experimental Proteus mirabibis meningitis in rabbits. J. Infect. Dis. 137:251-260. 19. Taber, L H., M. D. Yow, and F. G. Neiberg. 1967. The penetration of broad-spectrum antibiotics into the cerebrospinal fluid. Ann. N. Y. Acad. Sci. 145:473-481. 20. Wagman, G. A., J. A. Waitz, J. Marquez, A. Murawski, E. M. Oden, R. T. Testa, and M. J. Weinstein. 1972. A new micromonospora-produced macrolide antibiotic, rosamicin. J. Antibiot. Tokyo 25:641646. 21. Washington, J. A., and A. L Barry. 1974. Dilution test procedures, p. 410-417. In E. H. Lennette, E. H. Spaulding, and J. P. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C.
