Penicillin prophylaxis against experimental endocarditis due to a strain of Streptococcus intermedtus isolated from a patient with endocarditis was studied in rats. The minimum bactericidal concentration of penicillin for this strain was more than 64 mg/l and was higher than the peak penicillin serum level obtained in rats 30 min after the iv injection of 60 mg/kg, and in man after an oral dose of 2 g of phenoxymethyl penicillin. Moreover timed kill curves performed in the presence of 64 mg/l of penicillin showed no decrease in the number of colony-forming units during the first 6 h of incubation and only a 95% decrease after 24 h. In addition, no bactericidal activity could be detected in the serum 30 min after penicillin injection, that is at the time of bacterial challenge. Using the minimum bacterial inoculum needed to produce endocarditis in 90% of control animals (ID90), penicillin successfully prevented endocarditis due to this strain. We conclude that penicillin may prevent streptococcal endocarditis by other mechanisms than bacterial killin

Francioli, P.

Glauser, M. P.

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Journal of Antimicrobial Chemotherapy (1985) 15, Suppl. A. 297-302Successful prophylaxis of experimental streptococcal endocarditis withsingle doses of sublethal concentrations of penicillinP. Francioli and M. P. GlauserDivision des Maladies Infectieuses, Departement de Medecine,Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, SwitzerlandPenicillin prophylaxis against experimental endocarditis due to a strain ofStreptococcus intermedus isolated from a patient with endocarditis was studied inrats. The minimum bactericidal concentration of penicillin for this strain was morethan 64 mg/1 and was higher than the peak penicillin serum level obtained in rats30 min after the IV injecUon of 60 mg/kg, and in man after an oral dose of 2 g ofphenoxymethyl penicillin. Moreover timed kill curves performed in the presence of64 mg/1 of penicillin showed no decrease in the number of colony-forming unitsduring the first 6 h of incubation and only a 95% decrease after 24 h. In addition, nobactericidal activity could be detected in the serum 30 min after penicillin injection,that is at the time of bacterial challenge. Using the minimum bactenal inoculumneeded to produce endocarditis in 90% of control animals (ID90), penicillinsuccessfully prevented endocarditis due to this strain. We conclude that penicillinmay prevent streptococcal endocarditis by other mechanisms than bactenal killing.IntroductionPenicillin is one of the most commonly recommended antibiotics for prophylaxis ofbacterial endocarditis in susceptible patients undergoing oral procedures. Studies in therabbit model of endocarditis have suggested that high and repeated doses ofbactericidal antibiotics, including penicillin, were necessary to achieve successfulprotection (Durack & Petersdorf, 1973; Pelletier, Durack & Petersdorf, 1975)Furthermore, antibiotics like clindamycin or tetracycline were ineffective, suggestingthat bacterial killing was the main mechanism by which antibiotics protected againstendocarditis. However the number of micro-organisms used to induce infection inthese experiments was very high and probably greatly exceeded that found in manafter dental manipulation.Recent experiments performed with lower inocula have shown that single doses ofvancomycin, amoxicillin and even "bacteriostatic" antibiotics such as clindamycin orerythromycin were effective in preventing endocarditis (Bernard, Francioli & Glauser,1981; Glauser & Francioli, 1982; Glauser et al, 1983a). These studies raised thepossibility of mechanisms other than bacterial killing operating to preventendocarditis, since protection was achieved in the absence of bactericidal activity of theantibiotics used for prophylaxis.Mechanisms of action other than bacterial killing are not only interesting but highlydesirable if one considers that up to 40 to 50% of Streptococcus viridans isolated either2970305-7453/85/15A297 + 06 S02.00/0 © 1985 The British Society for Antimicrobial Chemotherapy298 P. FraDcioli and M. P. Gliuserfrom the mouth flora (Dankert & Hess, 1982) or from patients with endocarditis(Glauser el al., 19836) were shown to be tolerant to penicillin or amoxicillin.The purpose of the present study was to test the efficacy of penicillin, the antibioticmost commonly used for endocarditis prophylaxis, against a strain of Str. viridanshighly resistant to its bactericidal activity.Material and methodsMicro-organismA previously described strain of Str. intermedius isolated from a patient with bacterialendocarditis was used (Glauser & Francioli, 1982). Minimal inhibitory concentration(MIC) of penicillin were determined by broth dilution tests, using Mueller-Hintonbroth (Difco Laboratories, Detroit, U.S.A.), and 5 x 105 cfu as the inocula. The minimalbactericidal concentration (MBC) were determined by subculturing on penicillinase-containing blood agar plates 01 ml of an undiluted sample. After incubation for 48 hthe number of colonies on each plate was counted, and the MBC was determined asthe lowest dilution of antibiotic that showed 99-9% killing.Killing curve with 64 mg/1 of penicillin were performed in trypticase soya broth(Difco Laboratories, Detroit, U.S.A.) using an inoculum of 106 cfu/ml of an overnightculture of Str. intermedius. This concentration was chosen because it was well aboveachievable serum levels obtainable in humans after a 2 g oral dose of phenoxymethylpenicillin.Penicillin serum levels and serum bactericidal activitySerum levels of sodium benzyl penicillin were determined in five rats by an agardiffusion technique (Sabath & Anhalt, 1980) at 30 min, 1 and 2 h after iv injection of60 mg/kg. This dosage was chosen because it produces peak serum levels in rats similarto those achieved in man after an oral dose of 2 g of phenoxymethyl penicillin. Theserum bactericidal activity of penicillin against Str. intermedius was determined bystandard methods (Anhalt, Sabath & Barry, 1980) 30 min after iv injection of 60 mg ofpenicillin/kg of body weight. The serum bactericidal activity was defined as the highestserum dilution that gave 99-9% killing after 24 h of incubation.Production and prophylaxis of endocarditisSterile vegetations were produced in femal Wistar rats (weight 180-200 g) by amodification of a previously described method. In brief, a polyethylene catheter (modelno. PP10; Portex, Hythe, Kent, England) was passed through the aortic valve via theright carotid artery and secured with a silk ligature. Twenty-four hours aftercatheterization, rats were injected in the tail vein with saline or 60 mg/kg of sodiumpenicillin. Thirty minutes later 0-5 ml of saline containing 105 cfu of bacteria, theminimal inoculum needed to produce endocarditis in more than 90% of control rats,was injected intravenously (Glauser et al., 1983a). Rats were killed 72 h after bacterialchallenge, and aortic vegetations were excised, weighed, homogenized in 1 ml of 0-85%NaCl, serially diluted, and plated. Colony counts were done after 48 h of incubation at37°C.Penicillin prophylaxis against endocarditis 29924 hTimeFigure 1. Rate of m-vilro lulling of Str intermedium by 64 mg of penicillin/1 a concentration 4 times higherthan the serum level in rats 30 min after the iv injection of 60 mg/kg of penicillin O. Control; • , benzylpenicillin 64 mg/1Statistical analysisThe x2 test with Yates's correction was used for statistical comparisons.ResultsSusceptibility of Str. intermedius to penicillinMinimal inhibitory and bactericidal concentrations for the Str. intermedius of penicillinwere 0032 and >64mg/1 respectively. Survival of Str. intermedius in 64mg/1 ofpenicillin showed no decrease in cfu during the first 6 h of incubation and only a90-99% killing after 24 and 48 h of incubation (Figure 1). Therefore, this Str.intermedius was tolerant to the bactericidal action of penicillin.Penicillin serum levels and serum bactericidal activity (Figure 2)Penicillin serum levels (±s.D.) in 5 rats 30 min, 1 h and 2 h after injection of 60 mg/kgiv were 16±1, 2 ± 0 1 and approximately 001 mg/1, respectively. There was nodetectable serum bactericidal activity 30 min after injection of penicillin, that is at thetime of injection of bacteria in the prophylaxis experiments.Prophylaxis of Str. intermedius endocarditis by intravenously administered penicillin(Figure 3)The incidence of Str. intermedius endocarditis was reduced from 95% (22/23 rats) incontrols to 20% (4/20) in rats given penicillin 30 min before bacterial challenge(P<\0-*).DiscussionEarlier studies in rabbits have shown that high and prolonged serum levels ofbactericidal antibiotics, including penicillin, were necessary for adequate prophylaxis300 P. Francioli and M. P. Glauser2 -2hFigure 2. Serum levels ( ± S D ) of sodium penicillin 30 mm, 1 h and 2 h after a single IV injection of60 mg/kg in rats The dotted line represents peak level achieved in humans after an oral dose of 2 g ofphenoxymethyl penicillinagainst Str. viridans endocarditis (Durack & Petersdorf, 1973).Recently, strains of viridans streptococci isolated from the oral flora of children withcardiac disease (Dankert & Hess, 1982) and from their blood after dental extractions(Holloway, Dankert & Hess, 1980) were often shown to be tolerant to penicillin. Inaddition, we found that 17 out of 25 strains of viridans streptococci isolated frompatients with endocarditis were tolerant to amoxicillin, 13 of them having MBCs100Infected vegetationsOl CDO Oo 40*-P < I0"422/234/20Saline 6 0 mg/kgcontrols benzyl penclBinFigure 3. Incidence of endocarditis due to Str. intermedius after prophylaxis with 60 mg/kg of sodiumpenicillin administered iv 30 min before bacterial challenge. Numbers within the bars indicate the number ofrats in each groupPenicillin prophylaxis against endocarditis 301greater than 32 mg/1, a concentration which is higher than the peak serum levelsachieved after a 3 g oral dose (Glauser et al., 1983ft).To assess whether the phenomenon of in-vitro tolerance holds any significance invivo, Hess, Dankert & Durack (1983) studied the ability of penicillin to preventexperimental endocarditis caused by tolerant and non-tolerant strains of Str. sanguis.They found that a single dose of penicillin was successful when the strain ofstreptococcus was highly sensitive to the bactericidal action of the antibiotic, but wasmuch less effective when the strains were tolerant and not killed in vitro byconcentrations of penicillin similar to those achieved in vivo. Moreover the efficacy ofpenicillin against tolerant strains were greatly increased by the addition ofstreptomycin which provided a better bactericidal activity. These results wereinterpreted as showing that killing was indeed a critical factor for efficacy ofprophylaxis and, because of the occurrence of tolerance among viridans streptococci,this raises concern about the efficacy of the single dose amoxicillin regimen recentlyrecommended by the Working Party of the British Society for AntimicrobialChemotherapy (1982).In contrast to the experiments mentioned above (Durack and Petersdorf, 1973;Hess, Dankert and Durack 1983), we and others recently found that single doses ofvancomycin (Bernard et al., 1981; Scheld et al., 1981), amoxicillin (Glauser et al., 1983a)and even bacteriostatic antibiotics (Glauser & Francioh, 1982) could afford excellentprotection against Str. viridans endocarditis in the absence of bacterial killing. Withthe present study, penicillin can be added to the list of antibiotic operating in theabsence of bacterial killing. Indeed, the Str. intermedius strain had a MBC forpenicillin much higher than the peak serum level of the antibiotic and therefore wasnot expected to be killed by the drug in vivo. This was also attested by the killingcurves and the absence of any detectable bactericidal activity in the serum at the timeof injection of the bacteria.The discrepancy between the present results and those of Durack & Petersdorf(1973) and Hess et al. (1983) regarding the requirement for penicillin to be bactericidalin order to prevent endocarditis might be explained by the differences in the size of theinocula used for bacterial challenge. Indeed, we have shown in rats that streptococcalendocarditis can be efficiently prevented by single doses of non-bactericidalconcentrations of amoxicillin, provided that, like in the present experiment, theinoculum size is not higher than the minimum inoculum able to induce endocarditis in90% of control animals (ID90). With higher inocula the efficacy of prophylaxis ofamoxicillin was abolished unless the strain of streptococcus used was highly sensitiveto the bactericidal action of the drug (Glauser et ai, 1983a). Therefore, a bactericidalactivity appears to be required for efficient prophylaxis only when infection is inducedby inocula 10 or 100 times higher than the ID9 0 . Durack & Petersdorf (1973) used aninoculum of 108 cfu and presented evidence that this inoculum was much higher thanthe ID9 0 (Pelletier, Durack & Petersdorf, 1975). Hess, Dankert & Durack (1983) usedan inoculum of 107 cfu, a size which is likely to be also above the ID9 0 . In contrast, thepresent experiments using penicillin as prophylactic antibiotic were earned out with aninoculum known to be close to the ID9 0 (Glauser et al., 1983a). These differences ininoculum size probably explain why, in the absence of killing, penicillin preventedendocarditis in the present experiment, while it did not in other studies. Even thoughthe inoculum we used was smaller, it provides a stringent test of antibiotic prophylaxisbecause the resulting bacteraemia is far greater than that observed in humans after302 P. FrancioU and M. P. Glauseroral procedure (Petersdorf, 1978) and produces 80-100% endocarditis in animals.The mechanisms by which antibiotics can prevent endocarditis in the absence ofkilling are not completely elucidated. The cell-wall active antibiotics such as penicillin,amoxicillin or vancomycin have been shown in vitro to interfere with streptococcaladhesiveness to platelets fibrin matrices mimiking cardiac vegetations. Moreover thereare in-vivo experimental suggestions that inhibition of adherence might be one of themechanisms of action of antibiotics involved in endocarditis prophylaxis. (Scheld etal., 1981; Lowy et al, 1983; Glauser et al, 1983a).Whatever the mechanism of protection, the present experiment demonstrates thatshort-lived serum levels of penicillin similar to those achieved in man after an oral doseof 2 gm of phenoxymethyl penicillin can efficiently prevent the development ofendocarditis, provided the bacterial inoculum size used for challenge is limited to theID9 0 .ReferencesAnhalt, J. P., Sabath, L. D. & Barry, A. L. (1980) Special tests: bactericidal activity, activity ofantimicrobics in combination, and detection of /Mactamase production. In Manual ofClinical Microbiology 3rd edn. (Lennette, E H., Blows, A., Hausler, W. J. Jr. & Truant,J. P. Eds), pp 478-84. American Society for Microbiology, Washington, D.C.Bernard, J. P., Francioli, P & Glauser, M. P. (1981). Vancomycin prophylaxis of experimentalstreptococcus sanguis endocarditis: inhibition of bacterial adherence rather than bacterialkilling. Journal of Clinical Investigation 68, 1113-5.Dankert, J. & Hess, J. (1982). Penicillin-sensitive streptococcal endocarditis. Lancet ii, 1219.Durack, D T & Petersdorf, R. G (1973). Chemotherapy of experimental streptococcalendocarditis. I. Comparison of commonly recommended prophylactic regimens Journal ofClinical Investigation 52, 592-8.Glauser, M. P. & Francioli, P (1982). Successful prophylaxis against experimental streptococcalendocarditis with bactenostatic antibiotics. Journal of Infectious Diseases 146, 806-10.Glauser, M. P., Bernard, J. P., Moreillon, P & Francioli P. (1983a). Successful single-doseamoxicillin prophylaxis against experimental streptococcal endocarditis: Evidence for twomechanisms of protection. Journal of Infectious Disease 147, 568-75.Glauser, M. P , Francioli, P., Meylan, P., Moreillon, Ph. & Heralef E. (19836). Antibioticprophylaxis for patients with prosthetic valves. Lancet i, 237-8.Hess, J., Dankert, J. & Durack D. (1983). Significance of penicillin tolerance in vivo, preventionof experimental Streptococcus sanguis endocarditis. Journal of Antimicrobial Chemotherapy11, 555-64.Holloway, Y., Dankert, J. & Hess, J. (1980). Penicillin tolerance and bacterial endocarditis(letter). Lancet i, 589.Lowy, F. D., Chang, D. S., Neuhaus, E. G, Home, D S., Tomasz, A. & Steigbigel, N. H.(1983). Effect of penicillin on the adherence of streptococcus sanguis in vitro and in therabbit model of endocarditis. Journal of Clinical Investigation 71, 668-75.Pelletier, L. L., Jr., Durack, D. T. & Petersdorf, R. G. (1975). Chemotherapy of experimentalstreptococcal endocarditis. IV. Further observations on prophylaxis. Journal of ClinicalInvestigation 56, 319-30Petersdorf, R G. (1978). Antimicrobial prophylaxis of bacterial endocarditis: prudent caution orbacterial overkill? American Journal of Medicine 65, 220-3Sabath, L. D. & Anhalt, J. P. (1980). Assay of antimicrobics In Manual of ClinicalMicrobiology, 3rd edn. (Lennette, E. H., Blows, A., Hausler, W. J. Jr. & Truant, J P , Eds),pp. 485-90. American Society for Microbiology, Washington, D.C.Scheld, W. M., Zack, O., Vosbeck, K. & Sande, M. A. (1981). Bacterial adhesion in thepathogenesis of infective endocarditis: effect of subinhibitory antibiotic concentrations onstreptococcal adhesion in vitro and the development of endocarditis in rabbits. Journal ofClinical Investigation 68, 1381-4Working Party of the British Society for Antimicrobial Chemotherapy. (1982). The antibioticprophylaxis of infective endocarditis. Lancet ii, 1323-6.

Successful prophylaxis of experimental streptococcal endocarditis with single doses of sublethal concentrations of penicillin

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