Because bacteriostatic concentrations of vancomycin are effective in prophylaxis against endocarditis due to Streptococcus sanguis in rats, the efficacy of three other bacteriostatic antibiotics was investigated against three different streptococcal species that cause subacute endocarditis in humans: Streptococcus intermedius, S. sanguis, and Streptococcus mitior. Rats were challenged by intravenous injection of 2 × 105 colony-forming units of streptococci 24 hr after intracardiac insertion of a transaortic catheter and 30 min after intravenous injection of various doses of clindamycin, erythromycin, and doxycycline. Significant protection was achieved with all three antibiotics, but only clindamycin was fully effective against all three species at doses that simulated serum levels achievable in humans after oral administration. Endocarditis was prevented by antibiotic concentrations in serum far below minimal bactericidal concentrations for these streptococci. Furthermore, serum at the time of bacterial challenge was not bactericidal. Therefore, single doses of nonbactericidal antibiotics prevented endocarditis in rats by mechanisms other than bacterial killin

Francioli, P.

Glauser, M. P.

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THE JOURNAL OF INFECTIOUS DISEASES. VOL. 146, NO.6· DECEMBER 1982© 1982 by The University of Chicago. All rights reserved. 0022-1899/82/4606-0013$00.75Successful Prophylaxis Against Experimental Streptococcal Endocarditis withBacteriostatic AntibioticsM. P. Glauser and P. Francioli From the Division des Maladies Infectieuses, Departement deMedecine, Centre Hospitalier Universitaire Vaudois,Lausanne, SwitzerlandBecause bacteriostatic concentrations of vancomycin are effective in prophylaxisagainst endocarditis due to Streptococcus sanguis in rats, the efficacy of three otherbacteriostatic antibiotics was investigated against three different streptococcal speciesthat cause subacute endocarditis in humans: Streptococcus intermedius, S. sanguis, andStreptococcus mitior. Rats were challenged by intravenous injection of 2 x lOs colony-forming units of streptococci 24 hr after intracardiac insertion of a transaortic catheterand 30 min after intravenous injection of various doses of clindamycin, erythromycin,and doxycycline. Significant protection was achieved with all three antibiotics, but onlyclindamycin was fully effective against all three species at doses that simulated serumlevels achievable in humans after oral administration. Endocarditis was prevented byantibiotic concentrations in serum far below minimal bactericidal concentrations forthese streptococci. Furthermore, serum at the time of bacterial challenge was notbactericidal. Therefore, single doses of nonbactericidal antibiotics prevented endocar-ditis in rats by mechanisms other than bacterial killing.Recommendations for antibiotic prophylaxisagainst bacterial endocarditis require the adminis-tration of multiple high doses of bactericidal anti-biotics and parenteral administration for high-riskpatients [I]. These recommendations are basedlargely on studies in rabbits and have presented anumber of practical problems. Prolonged or par-enteral administration of prophylactic antibioticsis far more expensive and less acceptable to bothpatients and their dentists than a single oral dose.Poor compliance may jeopardize the effectivenessof regimens involving multiple or parenteral doses[2]. There are also important limitations in the ex-perimental data on which the recommendationsfor prophylaxis during dental procedures are based.The experimental studies involved only rabbitsand a single strain of Streptococcus sanguis [3, 4].Moreover, failure of certain antibiotic regimens inReceived for publication March 9, 1982, and in revised formJune 30, 1982.This work was presented in part at the 21st Interscience Con-ference on Antimicrobial Agents and Chemotherapy, held inChicago in November 1981, and at the Second InternationalSymposium on Infections in the Immunocompromised Host,held in Stirling, Scotland, in June 1982.We thank Dr. J. A. McCutchan for criticism and commentsand J. Kaelin for technical assistance.Please address requests for reprints to Dr. M. P. Glauser,Division des Maladies Infectieuses, Departernent de Medecine,Centre Hospitalier Universitaire Vaudois, 1011 Lausanne,Switzerland.806the rabbit model could be due to the number ofmicroorganisms used to induce infection, whichgreatly exceeded that found in humans after den-tal manipulation [2]. These problems as well aspossible failures with the presently recommendedregimens in humans [5] have prompted us to con-tinue experimental studies of the prevention ofbacterial endocarditis.Recent experiments with vancomycin in rats [6]and rabbits [7] introduced the possibility ofmechanisms other than bacterial killing operatingin the prevention of endocarditis. Therefore, thepresent study was undertaken to test three nonbac-tericidal antibiotics (erythromycin, clindamycin,and doxycycline), against three species of viridansstreptococci that are responsible for infective en-docarditis.Materials and MethodsMicroorganisms. Two previously describedspecies of a-hemolytic streptococci were used,Streptococcus intermedius (described elsewhere asS. sanguis [6]) and S. sanguis biotype II (providedby Dr. D. T. Durack, Duke University MedicalCenter, Durham, N.C. [3, 4]), as well as a strainof Streptococcus mitior that was also isolatedfrom a patient with bacterial endocarditis. MICsand MBCs of clindamycin (Upjohn Co., Kalama-zoo, Mich.), erythromycin (Eli Lilly and Co., In-Prophylaxis Against Endocarditis 807dianapolis), and doxycycline (Pfizer, Zurich) weredetermined by broth dilution tests, using Mueller-Hinton broth (Difco Laboratories, Detroit) and 5x lOs microorganisms as the inoculum. Specialcare was taken to avoid the carry-over of antibiot-ics [8]. The MBC was defined as the lowest con-centration of antibiotic that produced 99.9070 kill-ing after 24 hr of incubation.Antibiotic serum levels and serum bactericidalactivity. Serum levels of antibiotics were deter-mined in five rats at each time point by the agardiffusion technique [9]. The serum bactericidal ac-tivity of antibiotics against the three streptococciwas determined by standard methods [10] 30 minafter iv injection of 50 mg of clindamycin orerythromycin/kg of body weight or 10 mg of doxy-cycline/kg. The serum bactericidal activity was de-fined as the highest serum dilution that provided99.9% killing after 24 hr of incubation.Production and prophylaxis of endocarditis.Sterile vegetations were produced in female Wistarrats (weight, 180-200 g) by a modification of apreviously described method [6]. In brief, a poly-ethylene catheter (model no. PP 10; Portex, Hythe,Kent, England) was passed through the aorticvalve via the right carotid artery and secured witha silk ligature. Twenty-four hours after catheteri-zation, rats were injected in the tail vein with 0.5ml of various concentrations of antibiotics or0.85% NaC!. Thirty minutes later 0.5 ml of salinecontammg lOS cfu of bacteria, the minimal in-oculum needed to produce endocarditis in >80070of control rats, was injected iv. Rats were killed48-72 hr after bacterial challenge, and aorticvegetations were excised, weighed, homogenizedin 1 ml of 0.85% NaCI, serially diluted, andplated. Colony counts were done after 48 hr of in-cubation at 37 C. This method permitted the de-tection of 5 x 102 cfu/g of vegetation.Statistical analysis. The X2 test with Yates'scorrection and Student's t-test (unpaired) were usedfor statistical comparisons.ResultsAntibiotic sensitivity, antibiotic serum levels,and serum bactericidal activity. All three strep-tococcal species had very low MICs for the threeantibiotics tested (figure 1), but MBCs were large-ly above achievable serum levels (figure 2). At thetime of bacterial challenge, serum bactericidal ac-tivity against all three streptococcal species wasundetectable with any of the three antibiotics.Prophylaxis of endocarditis with clindamycin.Despite the lack of bacterial killing in vitro, clin-damycin at doses of 50 and 20 mg/kg achievedvery efficient prophylaxis against the three strep-tococci tested (figure 1). The 20 mg/kg dose pro-duced peak serum levels below the 4.5 Ilg/mlachieved in humans after an oral dose of 450 mg34CLiNDAMYCIN25 34 C26totLa~ t~ La* 10~C '0 C '0 C 10 mgikg*EJC 10 mg;kg*.rLlJ34C 20 1074.~-.C 50201086ERYTHROMYCIN DOXYCYCLINES. INTERMEDIUS S. SANGUIS S.MITIOR S.INTEAMEDtUS S.SANGUtS S. MITIORMIC:Q032 MIC:Q016 MIC:Q032 MIC-025 MIC 0.063 MIC 0032WBC,>128 MBC:>l28 MBC:128 MBC >126 MBC 16 MBC >1286.4 5,35.7 !li]]] ~ 159 6°1 ~_Ji] Lag lO~~g14 1.31.1 1. 2.4 to 1,4 12 1.6**~c 20 10 mgjkgC 20 10~ru74s. SANGUIS S. MITIORMIC:<QOO2 MIC;O.016MBC:>64 PJBC: 64S.INTERMEDIUSMIC:Q032MBC> 64L09-Jo~~W I~: 3.3 6.0 t~100~,~ 9215 :,a:" ~8ili 4Jl!5~ Ji!lo 01;:; **19 15C 50 20 10Figure 1. Incidence of endocarditis due to Streptococcus intermedius, Streptococcus sanguis, and Streptococcusmitiorafter prophylaxis with various doses of clindamycin, erythromycin, and doxycycline administered iv 30 min be-fore bacterial challenge. Numbers within the bars indicate the total number of rats in the groups; numbers beneath thebars indicate the doses (mg/kg of body weight) of the prophylactic antibiotics. Results for control rats (C) that wereinjected iv with 0.85070 NaCI are shown. Above the bars the log of the mean (± sn) bacterial density (MBD) recoveredfrom infected vegetation in each group are shown in boxes. The MICs and MBCs for the antibiotics are also indi-cated. Statistical comparisons are as follows: (*) indicates P< 0.01 in comparison with controls; (t) indicates P< 0.05in comparison with controls.808CLiNDAMYCIN ERYTHROMYCINGlauserand FrancioliDOXYCYCLINEl'9/ml'~I.1,I615141 ! lOmg/k9 •:r-j--------------!.----------------,,ILo 30· 2h 4h TIMEFigure 2. Serum levels of clindamycin, erythromycin, and doxycycline0.5,2, and 4 hr after single iv injections inrats. Dotted lines represent peak levels achieved in humans after oral doses of 450 mg of clindamycin, 500 mg oferythromycin, and 200 mg of doxycycline [11].[11] (figure 2). The 10 mg/kg dose was not uni-formly effective.Prophylaxis of endocarditis with erythromycin.Although the MICs and MBCs of erythromycinfor the three streptococcal species were similar tothose of clindamycin, erythromycin was less ef-ficient than clindamycin in preventing endocar-ditis. With the dose of 10 mg/kg, which producedpeak serum levels similar to the 0.5-2.0 IAg/mlachieved in humans after an oral dose of 500 mg[11] (figure 2), only endocarditis due to S. sanguisand S. mitior was significantly prevented. The in-cidence of infection with S. sanguis was "'30070.Prophylaxis of endocarditis with doxycycline.Although half of that in controls, the incidence ofendocarditis due to S. intermedius and S. sanguisin doxycycline-treated rats was still >40% with thedose of 10 mg/kg. This dose produced peak serumlevels similar to the 2.8 IAg/ml achieved in humansafter an oral dose of 200 mg [11] (figure 2).Quantitative cultures of infected vegetations.For all three streptococcal species, the number ofbacterialg of infected vegetations was similar incontrol rats to that in rats in which prophylaxisfailed (figure 1).DiscussionPrevious studies in rabbits that had been used toestablish current recommendations for the pro-phylaxis against bacterial endocarditis [1] sug-gested that bacterial killing is the mechanism re-sponsible for successfulprotection. This conclusionwas based on two observations. (1) There seemedto be a correlation between the efficacy of antibi-otics or combinations of synergistic antibiotics inkilling bacteria in vitro and the protection obtainedin vivo [3]. (2) Bacteriostatic antibiotics such astetracycline failed to protect from infection [12].These results served as a basis for the currentAmerican Heart Association recommendations ofhigh doses and prolonged administration of bacte-ricidal antibiotics for prophylaxis of endocarditisin humans [1]. However, these experiments in rab-bits involved extremely large bacterial inocula. Re-cent experiments from two different laboratoriesusing inocula that are 2 to 3 log smaller and asingle dose of vancomycin as the prophylactic an-tibiotic have shown that experimental endocarditiscan be prevented in the absence of bacterial killing[6, 7]. Even this smaller inoculum provides a strin-gent test of antibiotic prophylaxis because the re-sulting bacteremia is far greater than that ob-served in humans after oral procedures [2] andproduces 80%-100% endocarditis in animals. Therats [6], like high-risk human patients, have a per-manent intracardiac foreign body. Therefore, itwas of interest to use this model to reexamine theefficacy of single doses of orally absorbable, non-bactericidal antibiotics.Previous attempts to protect rabbits with clin-damycin at doses of 10mg/kg against high inocula(l08 cfu) of S. sanguis failed [4]. In the presentstudy, we found that this same dose produced sim-ilar peak concentrations in rats and often failed toprotect against smaller inocula (l05 cfu) of-threestreptococcal species. In contrast, excellent pro-tection was afforded by doubling the dose (20mg/kg). This protection was achieved by clin-damycin levels below those which follow a single450-mg oral dose in humans. Thus, peak serumlevels conveniently achievable in patients pro-Prophylaxis Against Endocarditistected rats from three streptococcal species whichcolonize the mouth and cause endocarditis inhumans. Furthermore, rats were protected at clin-damycin levels below the MBCs for these strep-tococci and in the absence of serum bactericidalactivity.Erythromycin protected rats from endocarditisdue to S. sanguis and S. mitior at doses resultingin serum levels achievable in humans after an oraldose of 500 mg. Against both S. intermedius in thepresent experiments and S. sanguis in rabbits [4],erythromycin was protective only at doses that re-sulted in serum levels far above those obtained inhumans after oral administration of 500 mg of thedrug [11]. Like tetracycline hydrochloride in rab-bits [12], doxycycline failed to provide sufficientprotection in rats against infection with two of thethree species tested. Thus, both erythromycin anddoxycycline protected rats against only somestreptococcal species at doses usually given orallyto patients. As with clindamycin, protection wasachieved in the absence of serum bactericidalactivity.Since sublethal concentrations of vancomycin inprevious studies [6, 7] and three bacteriostatic an-tibiotics in the present experiments provide protec-tion against endocarditis, mechanisms other thankilling must be involved. We investigated in twoways the possibility that bacteriostatic antibioticsmight have merely delayed the growth of bacteriain vegetations. (1) We compared the numbers ofstreptococci in vegetations from infected controlsand from animals infected in spite of prophylaxis.If antibiotics suppressed rather than prevented in-fection, rats in which prophylaxis failed shouldhave fewer organisms in their vegetations than un-treated control animals. In fact, we found thesame high numbers in both groups of rats (figure1). (2) We examined animals one week rather thanthree days after challenge (data not shown) andfound no increase in the percentage of animals inwhich prophylaxis failed. We conclude thatbacteriostatic antibiotics prevented, not just sup-pressed, infection - that is, prophylaxis was an all-or-none phenomenon.Another possible mechanism is suggested fromprevious experiments with vancomycin. In bothrats and rabbits, vancomycin appears to protectby inhibiting adherence of streptococci to sterilevegetations [6, 7]. This interpretation is supportedby demonstration in vitro that nonbactericidal809concentrations of vancomycin and penicillin pre-vent adherence of streptococci to platelet-fibrinclots [7, 13]. MICs of clindamycin have similarnegative effects on the adherence of Escherichiacoli [14] and alter surface structures of strepto-cocci that mediate adherence [15, 16]. Theseobservations raise the possibility that one mecha-nism by which clindamycin or other bacteriostaticantibiotics prevented endocarditis is interferencewith adherence.Regardless of the mechanism responsible forprotection, the present experiments suggest thatthe use of stringent antibiotic regimens aimed atkilling bacteria might not be required for adequateprevention of bacterial endocarditis in humans.ReferencesI. Kaplan, E. L., Anthony, B. F., Bisno, A., Durack, D.,Houser, H., Maillard, H. D., Sanford, 1., Shulman,S. T., Stillerrnan, M., Taranta, A., Wenger, N. Preven-tion of bacterial endocarditis. Circulation 56:139A-143A, 1977.2. Petersdorf', R. G. Antimicrobial prophylaxis of bacte-rial endocarditis: prudent caution or bacterial overkill?Am. 1. Med, 65:220-223, 1978.3. Durack, D. T., Petersdorf', R. G. Chemotherapy of exper-imental streptococcal endocarditis. I. Comparison ofcommonly recommended prophylactic regimens. 1.Clin. Invest. 52:592-598, 1973.4. Pelletier, L. L., lr., Durack, D. T., Petersdorf, R. G.Chemotherapy of experimental streptococcal endocar-ditis. IV. Further observations on prophylaxis. 1. Clin.Invest. 56:319-330, 1975.5. Durack, D. T., Kaplan, E. L., Bisno, A. L. Twenty-fivecases of apparent endocarditis prophylaxis failure:results of a national survey [abstract]. Clinical Research29:384A, 1981.6. Bernard, 1.-P., Francioli, P., Glauser, M. P. Vancomycinprophylaxis of experimental Streptococcus sanguis: in-hibition of bacterial adherence rather than bacterial kill-ing. 1. Clin. Invest. 68:1113-1116, 1981.7. Scheld, W. M., Zak, 0., Vosbeck, K., Sande, M. A. Bac-terial adhesion in the pathogenesis of infective endocar-ditis: effect of subinhibitory antibiotic concentrationson streptococcal adhesion in vitro and the developmentof endocarditis in rabbits. 1. Clin. Invest. 68:1381-1384,1981.8. Pearson, R. D., Steigbigel, R. T., Davis, H. T., Chapman,S. W. Method for reliable determination of minimallethal antibiotic concentrations. Antimicrob. AgentsChemother. 18:699-708, 1980.9. Sabath, L. D., Anhalt, 1. P. Assay of antimicrobics.In E. H. Lennette, A. Balows, W. 1. Hausler, lr., and1. P. Truant [ed.]. Manual of clinical microbiology. 3rded. American Society for Microbiology, Washington,D.C., 1980, p, 485-490.81010. Anhalt, J. P., Sabath, L. D., Barry, A. L. Special tests:bactericidal activity, activity of antimicrobics in com-bination, and detection of fJ-lactamase production. InE. H. Lennette, A. Balows, W. J. Hausler, Jr., andJ. P. Truant [ed.]. Manual of clinical microbiology. 3rded. American Society for Microbiology, Washington,D.C., 1980, p. 478-484.II. Braude, A. I. Pharmacology principles. W. B. Saunders,Philadelphia, 1976, p, 68.12. Southwick, F. S., Durack, D. T. Chemotherapy of exper-imental streptococcal endocarditis. III. Failure of a bacte-riostatic agent (tetracycline) in prophylaxis. J. Clin.Pathol. 27:261-264, 1974.13. Neuhaus, E., Lowy, F., Chang, D., Horne, D., Tomasz,A., Steigbigel, N. Penicillin tolerant S. viridans: effect ofpenicillin on host-bacterial interaction [abstract no. 356].Glauserand FrancioliIn Program and Abstracts of the 20th Interscience Con-ference on Antimicrobial Agents and Chemotherapy.American Society for Microbiology, Washington, D.C.,1980.14. Vosbeck, K., Handschin, H., Menge, E.-B., Zak, O.Effects of subminimal inhibitory concentrations of anti-biotics on adhesiveness of Escherichia coli in vitro. Rev.Infect. Dis. 1:845-851, 1979.15. Gemmell, C. G., Peterson, P. K., Schmeling, D., Kim, Y.,Mathews, J., Wannamaker, L., Quie, P. G. Potentiationof opsonization and phagocytosis of Streptococcuspyogenes following growth in the presence of clin-damycin. J. Clin. Invest. 67:1249-1256, 1981.16. Fox, E. N. M proteins of group A streptococci. Bac-teriological Reviews 38:57-86, 1974.

Successful Prophylaxis Against Experimental Streptococcal Endocarditis with Bacteriostatic Antibiotics

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