University of Groningen

Prevention of bacterial endocarditis in children with congenital heart disease : A study onbacteremia after dental extraction, penicillin tolerance in viridans streptococci andexperimental endocarditis in rabbits under penicillin prophylaxis.Hess, Jakob

IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.

Document VersionPublisher's PDF, also known as Version of record

Publication date:1982

Link to publication in University of Groningen/UMCG research database

Citation for published version (APA):Hess, J. (1982). Prevention of bacterial endocarditis in children with congenital heart disease : A study onbacteremia after dental extraction, penicillin tolerance in viridans streptococci and experimentalendocarditis in rabbits under penicillin prophylaxis. [Meppel]: [S.n.].

CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).

Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.

Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.

Download date: 31-07-2020

RIJKSUNIVERSITEIT GRONINGEN

prevention of bacterial endocarditis in children with congenital heart disease

a study on bacteremia after dental extraction,

penicillin tolerance in viridans streptococci and

experimental endocarditis in rabbits

under penicillin prophylaxis

PROEFSCHRIFT

ter verkrijging van het doctoraat in de geneeskunde

aan de rijksuniversiteit te groningen

op gezag van de rector magnificus dr. l.j. engels

in het openbaar te verdedigen op

woensdag 16 juni 1982

des namiddags te 2.45 uur precies

door

jakob hess

geboren te groningen

krips repro meppel

Promotores : Prof. Dr. K.K. Bossina.

D.T. Du rack, M.D., ·D. Phil.

Referent : Ors. J. Dankert.

STELLINGEN

I.

De gevoeligheid van Streptococcus viridans voor penicilline moet war­den uitgedrukt in de Minimale Bactericide Concentratie, die bepaald is

op voedingsbodems waaraan penicillinase is toegevoegd om het zogenaamde

carry-over effect te voorkomen.

I I.

Penicilline in subminimale concentraties remt weliswaar niet de groei

van Streptococcus viridans maar speelt waarschijnlijk toch een belang­

rijke rol bij de preventie van endocarditis, omdat de adherentie.van de

bacterien aan endocard wordt verminderd.

I I I.

Het synergistische bactericide effect van penicilline in combinatie

met streptomycine treedt niet uitsluitend op bij penicilline gevoelige

Streptococcus viridans doch ook bij de penicilline resistente en de

penicilline tolerante Streptococcus viridans.

IV.

Tolerantie is bij bacterien vaker te onderkennen dan bij mensen.

V.

Kinderen, bij wie een Holter drainage is aangebracht, hebben een ver­

hoogd risico op het ontstaan van bacteriele endocarditis.

VI.

Bij de behandeling van pseudoaneurysmata van de aorta tengevolge van

aortitis is direct chirurgisch ingrijpen geindiceerd.

VII.

Endocarditis prophylaxe begint bij tandplak preventie.

VIII.

De betekenis van echocardiografie voor de diagnostiek van bacteriele endocarditis bij kinderen is gering.

IX.

Indien bij scintigrafie van de longen blijkt dat een tak van de arteria pulmonalis niet geperfundeerd wordt, dient bij kinderen naast pulmonalisangiografie ook aortografie te worden verricht.

X.

Door de toepassing van laag osmolaire rontgencontraststoffen en sterk verbeterde rontgenapparatuur bij angiografie bij zuigelingen dreigt de rontgenstralenbelasting toe te nemen.

XI.

Retrograde catheterisatie van het linker atrium is, mits lege artis uitgevoerd, een veilige procedure die te weinig bekendheid geniet.

XII.

Het lichamelijk en psychisch welzijn van het kind met een aangeboren hartafwijking is in de meeste gevallen meer gebaat bij het aanraden van sportbeoefening dan bij het ontraden ervan.

XIII.

Het enorme regeneratievermogen op de kinderleeftijd komt onder meer tot uiting in het feit dat veel kinderen ondanks hun opvoeding tot rede­lijk aangepaste volwassenen uitgroeien.

XIV.

Wetenschappelijk onderwijs zonder research is als wijn zonder alcohol.

Stellingen behorende bij het proefschrift van

J. Hess

PREVENTION OF BACTERIAL ENDOCARDITIS IN CHILDREN WITH CONGENITAL HEART DISEASE a study on bacteremia after dental extraction, penicillin tolerance in viridans streptococci and experimental endocarditis in rabbits under penicillin prophylaxis

Groningen 1982

Stoutmoediger dan het doorvorsen van het onbekende is soms het betwijfeZen van het bekende

Kaspar

Aan Inge

The research reported in this thesis was performed in the Division of Pedi­atric Cardiology (Prof. Dr. K.K. Bossina) of the Department of Pediatrics (Prof. Dr. J. Fernandes), Division of Hospital Epidemiology (Drs. J. Dankert) and the Department of Medical Microbiology (Prof. Dr. J.B. Wilterdink), the Department of Oral Surgery (Prof. Dr. G. Boering), University of Groningen, and the Division of Infectious Diseases (D.T. Durack, M.D., D.Phil.), Duke University Medical Center, Durham, U.S.A.

I wish to thank the following persons for their essential cooperation:

Prof. Dr. K.K. Bossina and D.T. Durack, M.D., D.Phil. (organization and supervision)

Drs. J. Dankert (organization, supervision and stimulating research con­ception)

Mrs. Y. Holloway (participation in weekly research discussion and english reviewal)

J.G. Oostendorp (determination of antibiotic concentrations) W. Joldersma and Mrs. D. Edwards (technical assistance) J. R. Perfect, M. D. (planning of rabbit experiments and research discussions) B.J. Dokter, D.M.D. and J.L.N. Roodenburg, D.M. D. (dental extractions) A. Bergstra (statistical advise) G.P. Messchendorp (figures) R. M. v.d. Meer (photography) Mrs. G. Bathoorn, Mrs. M. ter Brugge, Mrs. E.M. Eimers and Mrs. 0. Sherman

(typing and lay-out assistance)

This work was supported in part by grants of Beecham Research Laboratories, the Jan Kornelis de Cock Stichting, Gist-Brocades, the Nederlandse Hart­stichting and the Nederlandse organisatie voor zuiver-wetenschappelijk onderzoek (Z.W.O.).

CHAPTER I

CHAPTER I I

CHAPTER I I I

CHAPTER IV

CHAPTER V

CHAPTER VI

CONTENTS

General introduction

The incidence of post-extraction bacteremia under penicillin cover in children with cardiac disease

Penicillin prophylaxis in children with cardiac disease: post-extraction bacteremia and penicillin resistant viridans streptococci

Detection of penicillin tolerance in viridans streptococci isolated from the oral flora of children with cardiac disease and post-extraction bacteremia under penicillin prophylaxis

Significance of penicillin tolerance in vivo: prevention of Streptococcus sanguis experimental endocarditis

General discussion

Surmiary and conclusions

Samenvatting en conclusies

5

6

12

27

42

63

80

87

90

CHAPTER I

GENERAL INTRODUCTION

INTRODUCTION

The onset of bacterial endocarditis is clearly related to preexisting cardiac disease and the occurrence of bacteremia. This causual relationship was recognized in 1923 by Lewis and Grant 1

• Since 1935 it has been known that dental extraction can give rise to transient bacteremia, which is often due to viridans streptococci 2

• The evidence for a relationship between bac­teremia occurring after dental extraction and bacterial endocarditis in sus­ceptible patients was found by Taran 3 in 1944, who demonstrated that viri­dans streptococcal bacteremia after dental extraction resulted in streptococ­cal endocarditis in 4 children with rheumatic heart disease. These observa­tions are the basis for the widely recommended use of antibiotics to prevent the onset of bacterial endocarditis in susceptible patients undergoing dental treatment or other procedures that can give rise to bacteremia. However, since antibiotic prophylaxis has been employed, the incidence of bacterial endocar­ditis has not decreased significantly 4

• This apparent discrepancy might be due to several reasons. Bacteremias not only occur after major procedures, such as dental extraction, tonsillectomy, bronchoscopy, but can also occur af­ter more common events, such as tooth brushing and chewing gum 5

• In addition common bacterial infections, especially upper respiratory tract infections, may result in short-lasting significant bacteremia 6

• Therefore, it is impos­sible to prevent the occurrence of all possible bacteremias. Another reason for the constant incidence of bacterial endocarditis may be that antibiotic prophylaxis is not effective in preventing bacterial endocarditis. This could be due to the fact that the bacteremia is caused by microorganisms not sensi­tive to the antibiotic used, too low serum concentrations of the antibiotic or low concentrations of the antibiotic at the site of contamination. Direct evidence that attempted prophylaxis with antibiotics prevents bacterial endo­carditis in man, has not been reported up to this time. Such evidence can only be obtained by means of a controlled clinical trial. Such a trial has not been performed and probably never will be, as pointed out by Durack 7

, for the fol­lowing reasons: assuming a risk of developing bacterial endocarditis after dental extraction of 1 in 500 and a 90% effectiveness of the antibiotic pro­phylaxis to be tested in preventing endocarditis, one would need to include 2 groups of 5000 susceptible patients (one group treated with the antibiotic, the other with a placebo) in a double blind survey in order to obtain statis­tical significant results (X2 = 5. 8; p < 0. 03). Even if such large numbers

7

could be studied, such a trial would be rejected for obvious ethical reasons. However, direct assessment on the efficacy of antibiotics in preventing bacte­rial endocarditis has been provided by the experimental endocarditis rabbit model, originally created by Garrison and Freedman 8

, later modified by Durack, Beeson and Petersdorf 9• Various antibiotic regimens have been compared on their ability to prevent experimental endocarditis 10• 11 • The results of these studies have been critisized on the basis that man can not be compared to the rabbit. However, they can be used to compare the efficacy of various antibio­tics and in fact these studies form important grounds on which the Committee on Prevention of Bacterial Endocarditis of the American Heart Association ba­sed its recommendations 12• An indirect assessment of the efficacy of antibi­otic prophylaxis in preventing bacterial endocarditis in man may be, in case of dental extraction, the incidence of post-extraction bacteremia under anti­biotic prophylaxis. Results of earlier studies have been inconsistent, but se­veral investigators demonstrated the occurrence of early post-extraction bac­teremia under antibiotic prophylaxis 13• 16 • However, these bacteremias do not prove prophylactic failure, because killing of the microorganisms by an anti­biotic depends on several conditions. At least two conditions need to prevail: namely a bactericidal concentration of the antibiotic in the blood, and micro­organisms which are sensitive to this antibiotic. Unfortunately these two con­ditions have not been studied in conjunction with post-extraction bacteremia and the prevention of bacterial endocarditis. This is probably due to the fact that viridans streptococci, the microorganisms most frequently isolated in post-extraction bacteremia, are generally believed to be always sensitive to penicillin, the antibiotic most often used as attempted prophylaxis for pa­tients undergoing dental extraction. But penicillin resistant viridans strep­tococci are present in the oral flora r7,ia which is the source of post-ex­traction bacteremia and therefore might invade the blood stream.

A new type of penicillin resistance earlier described in Staphylococcus aureus 1 9

, has recently been reported to occur also in viridans streptococci20 , 21•

Some strains proved to be resistant to the lethal action of high concentrations of penicillin although their multiplication could be inhibited by very low concentrations. This phenomenon has been termed tolerance. Its clinical sig­nificance has been demonstrated in the case of Staphylococcus aureus endocar­ditis 22

, but is still under discussion for viridans streptococcal strains. The significance of the occurrence of penicillin resistant and penicillin

8

tolerant viridans streptococci for the efficacy of antibiotic prophylaxis of streptococcal endocarditis has so far not been studied. In this thesis studies on the efficacy of routine penicillin prophylaxis in children with cardiac dis­ease assumed to be at risk for developing bacterial endocarditis, undergoing dental extraction are described. Because certain species within the streptococ­cus viridans group are most frequently related to post-extraction bacteremia and non-acute bacterial endocarditis, there is special emphasis on this group. In chapter II the incidence of post-extraction bacteremia under penicillin pro­phylaxis and the microorganisms involved are presented and related to the se­rum penicillin concentration at the time of collecting blood for culturing. Chapter III deals with the bacterial flora of the gingival sulcus related to that of the post-extraction bacteremia. The penicillin susceptibility of the isolated microorganisms from both gingival sulcus and blood after extraction are presented and compared in order to detect a possible predictive value for performance of preextraction susceptibility testing. In chapter IV studies on the phenomenon of tolerance are presented and discussed. The presence of peni­cillin tolerant viridans streptococci in the gingival sulcus and in the blood of children after dental extraction is described and compared. In chapter V the ability of certain penicillin tolerant viridans streptococcal strains to induce bacterial endocarditis is tested in the rabbit model. The efficacy of parenteral penicillin administration in prevention of experimental endocarditis due to these penicillin tolerant strains is studied and compared to the effi­cacy of administration of penicillin plus streptomycin in the same model.

REFERENCES

1. Lewis T, Grant R T: Observations relating to subacute infective endocarditis. Heart, 10: 21, 1923.

2. Okell C C, Elliott S D: Bacteraemia and oral sepsis. With special referen­ce to the aetiology of subacute endocarditis. Lancet 2: 869, 1935.

3. Taran L M: Rheumatic fever in its relation to dental disease. N.Y. J. Dent. 14: 107, 1944.

9

4. Hayward G W: Infective endocarditis - a changing disease. Br. Med. J. 2: 706, 1973.

5. Everett E D, Hirschmann J V: Transient bacteremia and endocarditis prophylaxis. A review. Medicine 56: 61, 1977.

6. McGowan J E, Bratton L, Klein J 0, Finland M: Bacteremia in febrile children seen in a ''walk-in" pediatric clinic. N. Eng. J. Med. 288: 1309, 1973.

7. Durack D T: Experience with prevention of experimental endocarditis. In: Infective Endocarditis; edited by E L Kaplan and A V Taranta , p. 28, Dallas. Am Heart Association , 1977.

8. Garrison PK, Freedman L R: 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: 394, 1970.

9. Durack D T, Beeson P B , Petersdorf R G: Experimental bacterial endocar­ditis. I I I: Production and progress of the disease in rabbits. Br. J. Exp. Pathol. 54: 142, 1973.

10. Durack D T, Petersdorf R G: Chemotherapy of experimental streptococcal endocarditis. I: Comparison of commonly recommended prophylactic regimens. J. Clin. Invest. 52: 592, 1973.

11. Durack D T, Starkebaum M S, Petersdorf R G: Chemotherapy of experimental streptococcal endocarditis. V I. Prevention of enterococcal endocarditis. J. Lab. Chir. Med. 90: 171, 1977.

12. Kaplan E L et al: Prevention of bacterial endocarditis. A. H.A. Committee report. Circulation 56: 139A, 1977.

13. Glaser R J, Dankner A, Mathes S B, Harford C G: Effect of penicillin on the bacteremia following dental extraction. Amer. J. Med. 4: 55, 1948.

10

14. Bender I B, Pressman R S, Tashman S G: Comparative effects of local and systemic antibiotic therapy in the prevention of post-extraction bacteremia. J. Amer. Dent. Ass. 57: 54, 1958.

15. Elliott R H, Dunbar J M: Streptococcal bacteremia in children following dental extractions. Arch. Dis. Childh. 43: 451, 1968.

16. Shirger A, Martin W J, Royer R Q, Needham GM: Bacterial invasion of blood after oral surgical procedures. J. Lab. Clin. Med. 55: 376, 1960.

17. Sprunt K, Redman W, Leidy G: Penicillin resistant alpha streptococci in pharynx of patients given oral penicillin. Pediatrics 42: 957, 1968.

18. Drucker D B, Jolly M: Sensitivity of oral microorganisms to antibiotics. Brit. Dent. J. 131: 442, 1971.

19. Sabath L D, Wheeler N, Laverdiere M, Blazevic D, Wilkinson B J: A new type of penicillin resistance of Staphylococcus aureus, Lancet i, 443, 1977.

20. Horne D, Tomasz A: Tolerant response of Streptococcus sanguis to beta­lactams and other cell wall inhibitors. Antimicrob. Agents Chemother. 11: 888, 1977.

21. Holloway Y, Dankert J, Hess J: Penicillin tolerance and bacterial endo­carditis. Lancet, i: 589, 1980.

22. Rajashekarajah MD, Rice T, Rao V S, Marsh D, Ramakrishna A, Kallick C A: Clinical significance of tolerant strains of Staphylococcus aureus in patients with endocarditis, Ann. Int. Med. 93: 796, 1980.

11

CHAPTER II

THE INCIDENCE OF POST-EXTRACTION BACTEREMIA UNDER

PENICILLIN COVER IN CHILDREN WITH CARDIAC DISEASE

J. Hess, Y. Holloway and J. Dankert

Accepted for publication in Pediatrics

ABSTRACT

In order to assess the efficacy of penicillin prophylaxis as recommended by the American Heart Association to prevent the onset of bacterial endocar­ditis, the incidence of post-extraction bacteremia was determined in 82 chil­dren with cardiac disease under penicillin prophylaxis. Aerobic and anaerobic bloodcultures were taken five minutes after dental extraction, as was a blood sample to assay the serum penicillin concentration. The incidence of post­extraction bacteremia was 21%. Streptococcal species accounted for half of the number of aerobes isolated. Sixteen percent of the isolated microorganisms were strict anaerobes. Susceptibility testing of the isolates showed that 24 penicillin sensitive and 8 penicillin resistant microorganisms caused the bac­teremia. There was neither a significant difference between the serum peni­cillin concentrations of children with and without bacteremia, nor between the serum penicillin concentrations of children with bacteremia due to penicillin sensitive and children with bacteremia due to penicillin resistant microorga­nisms. It is concluded that the occurrence of post-extraction bacteremia is not prevented by penicillin prophylaxis and that the serum penicillin concen­tration at extraction is not the discriminating factor in preventing this bac­teremia. None of the children developed bacterial endocarditis. We speculate that mechanisms not thoroughly studied are involved in the prevention of bac­terial endocarditis after dental extraction.

INTRODUCTION

In order to reduce the risk of developing bacterial endocarditis it is recorrmended that susceptible patients undergoing dental extraction should receive prophylactic antibiotics 1

•

The incidence of post-extraction bacteremia occurring under such prophy­lactic regimens is an indirect assessment of the efficacy of prophylaxis. So far, the results of completed studies have been inconsistent. This is possi­bly due to the use of different antibiotics, the use of different methods both in the clinical and in the laboratory setting, and the presence or ab­sence of microorganisms not sensitive to the antibiotic employed.

Viridans streptococci, which are the microorganisms still most frequent­ly isolated in bacterial endocarditis 2

, are generally believed to be always sensitive to penicillin, and therefore this antibiotic is used most often as

13

attempted prophylaxis for patients undergoing dental extraction 1 •

The efficacy of penicillin prophylaxis as recommended by the American Heart Association 1 has not been tested in children undergoing dental mani­pulation. In the present study the incidence of post-extraction bacteremia in children at risk of developing infective endocarditis was assessed. In ad­dition serum penicillin concentration was measured in post-extraction blood­samples, and the penicillin susceptibility of microorganisms isolated from post-extraction blood cultures was determined.

MATERIALS AND METHODS

Patient population Eighty two children aged between 5 and 14 years (mean: 9.7 years) with

cardiac disease, and assumed to be at risk for bacterial endocarditis 1 were included in the study. All required extraction of primary or permanent teeth as a result of dental disease, diagnosed clinically and/or radiologically. Extraction was performed by forceps technique under local anaesthesia. None of the children had received any antibiotic in the two months preceeding the extraction. All children were out-patients who had not been admitted in the preceeding twelve months. Antibiotic prophylaxis

Antibiotic prophylaxis was based on the recommendations of the American Heart Association Committee 1 • Children younger than six years received 450. 000 Units of procaine penicillin G and 150. 000 Units of aqueous penicil­lin G, intramus�larly. Older children received 900. 000 Units of procaine penicillin G and 300. 000 Units of aqueous penicillin G, intramuscularly. The antibiotic was administered 45 minutes prior to dental extraction. Culture techniques and susceptibility testing

Five ml of blood was collected for culture 5 minutes after dental ex­traction. Blood was drawn from an antecubital vein after cleansing of the skin with chlorhexidin-gluconas (0. 5% w/v) in ethanol (75% v/v). The blood was immediately injected into glucose (1% w/v) broth {Oxoid Ltd. , U. K. ) for aerobic culture, and into pre-reduced Diagnostic Anaerobic Growth (D.A.G. ) medium for anaerobic culture 3

• Both media contained sufficient penicillinase {Mycofarm, Delft, The Netherlands) to counteract 1000 Units of penicillin. The blood cultures were incubated at 37°C for ten days, and subcultured on

14

days 3, 7 and 10. Aerobic subculture was performed onto sheep blood (5% v/v) agar (0xoid Ltd. , U. K. ), chocolate agar (BBL, Cockeysville, Maryland, U.S.A. ), mitis salivarius agar (Difeo, Detroit, U.S.A. ), M.S.F.A. agar 12

, MacConkey agar (Merck, Darmstadt, F. R.G.) and Sabouraud 's agar (Merck).

Anaerobic subculture was performed onto supplemented Brain Heart Infu­sion (BHI) agar, prepared according to Holdeman et al. 4 and a sheep blood (5% v/v) agar supplemented with haemin and menadione. The plates were placed in GasPak jars (BBL) inside a glove box, where all subcultures took place, and incubated at 37°c for 48 hours.

Aerobic microorganisms, except viridans streptococci, were identified according to Cowan 5 and anaerobic microorganisms according to Holdeman et al. 4

• Viridans streptococci were classified as previously reported 6• The

penicillin susceptibility of all microorganisms was determined by a conventi­onal disc diffusion method, using 0xoid discs containing 1. 5 Units penicillin on sheep blood agar plates and BHI plates 7

'8

• Strains were considered to be penicillin resistant if zones of inhibition smaller than 22 mm were measured in the case of Staphylococcus species, and smaller than 29 mm in the case of all other microorganisms 9

•

Determination of penicillin serum concentration Three ml of blood was drawn five minutes after extraction for determina­

tion of the serum penicillin concentration. This blood was centrifuged and serum was preserved at -7o0c. Assays were performed using an agar diffusion method. Bacillus subtilis was used as test organism. Serum (undiluted or diluted with normal human pooled serum) was pipetted into wells cut in glu­cose (0. 1%) nutrient agar base (Difeo) containing 3 to 5 x 106 B. subtilis spores per ml of medium . Penicillin was dissolved in 0. 05 M phosphate buffer (pH 6.8). Penicillin standards ranging from 0. 5 U per ml to 20 U per ml were prepared in pooled human serum. Plates were incubated at 30°C for 18 hours. Zones of inhibition were measured electronically.

Follow-up of patients The children were examined ten days after dental extraction by a pedia­

tric cardiologist and those who had had a post-extraction bacteremia had blood cultures repeated.

15

RESULTS

Post-extraction bacteremia was present in 17 (21%) of the 82 children (Table 1). Viridans streptococci were cultured from 10 children, other aero­bes from 8 and anaerobes from five. In 6 children a polymicrobial bacteremia was present. The mean serum penicillin concentration of the children with positive blood cultures did not differ from that of the children with nega­tive blood cultures.

In total 32 microorganisms were isolated from the 17 positive post­extraction blood cultures. Six of the cultures yielded two or more microor­ganisms (Table 2). Twenty seven aerobic microorganisms were isolated from 13 cultures, and 5 strictly anaerobic microorganisms were isolated from 5 cul­tures. One culture yielded both an aerobic and an anaerobic microorganism.

Susceptibility tests showed that 24 microorganisms (21 aerobes and 3 anaero­bes) were penicillin sensitive, and 8 (6 aerobes and 2 anaerobes) were peni­cillin resistant. Penicillin sensitive microorganisms were isolated from 12 children and penicillin resistant from 7. Cultures obtained from two children contained both penicillin sensitive and penicillin resistant bacteria. The mean serum penicillin concentration in blood samples taken five minutes af­ter dental extraction from children with bacteremia due to penicillin sensi­tive microorganisms did not differ from that in children with bacteremia due to penicillin resistant microorganisms (Table 3).

DISCUSSION

Microorganisms present in the gingival sulcus represent the most signi­ficant source of post-extraction bacteremia 1 0• 11• 1 2• Anaerobes account for half of the total number of microorganisms present in this area 13• Therefore the use of both aerobic and anaerobic culture techniques and the use of pre­reduced culture media has been advocated for studies assessing the incidence of post-extraction bacteremia 1 4• 15 • Blood samples in our study were injected directly and immediately into a medium suitable for aerobic microorganisms and into a recently developed pre-reduced anaerobic blood culture medium 3•

Both media contained sufficient penicillinase to counteract a high concentra­tion of penicillin. All subcultures were performed in a glove box. Using the­se techniques, we found an incidence of post-extraction bacteremia of 21% in children with cardiac disease, at risk of developing bacterial endocarditis,

16

..... -...J

Table 1. The incidence of post-extracticn bacteremia and mean serum penicillin concentration in children with cardiac disease under penicillin prophylaxis. Samples for culture and penicillin assay were collected five minutes after extraction.

Number of children Serum penicillin concentration (U per ml) mean ranqe

with negative bloodcultures 65 8.9 1.0 - 15.0

with a positive bloodculture 171 8.1 2.4 - 13.5

- due to viridans streptococci 10 7.2 2.4 - 13.5

- due to other aerobes 8 7.5 4.5 - 13.5

- due to anaerobes 5 7.3 4.4 - 10.0

total 82 8.9 1.0 - 15.0

1 Six children had polymicrobial bacteremia.

Table 2. Microorganisms isolated from post-extraction bloodcultures in relation to their susceptibility to penicillin

Microorganisms isolated Number of penicillin Number of penicillin Total number of sensitive strains resistant strains isolates

Aerobes: 21 ( 9) 1 6 ( 5) 1 27 ( 13) I - Vi ri dans streptococci 2 15 (7) 4 (3) 19 (10) - Streptococcus faecalis 1 ( 1) 1 - Micrococcus sp. 2 (2) 2 ( 2) - Staph. epidermidis 1 1 - Corynebacterium sp. 2 {2}

..... - Bacillus sp. 1 1 (X)

- Haemophilus parainfluenzae 1 (1) 1 Anaerobes: 3 (3} 2 (2) 5 ( 5)

- Bacteriodes capillosus 1 (1) 1 - Veillonella parvula 1 ( 1) 1 - Clostridium sp. 1 1 - Eunacterium ventriosum 1 1 - Propionibacterium jensenii 1 1

24 ( 12) 8 (7) 32 (17)

1 Numbers in parenthesis denote the number of patients 2 The viridans streptococci were subdivided into species:

6 bloodcultures contained more than one strain.

.... '°

Table 3. Mean serum penicillin concentration in 17 children with bacteremia in relation to the penicillin susceptibility of the microorganisms isolated from blood

penicillin-sensitive microorganisms penicillin-resistant microorganisms

Number of children

121

71

Serum penicillin concentration (U per ml) mean range

8.0 7.9

2.4 - 13.5 4.4 - 12.0

1 Cultures from 2 children contained both penicillin sensitive and penicillin resistant bacteria.

under penicillin prophylaxis according to the recommendations of the American Heart Association 1• In 13 (16%) blood cultures aerobic microorganisms and in 5 (6%) anaerobic microorganisms were detected. The incidence of post-extrac­tion bacteremia in children under penicillin prophylaxis has previously been reported and ranged from O to 53% 10'16'17'18• Several explanations may be put forward for these inconsistencies in incidence found, such as the use of different forms of penicillin, differences in the route of administration of the antibiotic, differences in blood culture technique, the use of different media with and without penicillinase, the time interval which expires between administration of penicillin and the taking of the specimens, and the presen­ce of penicillin resistant microorganisms in the gingival sulcus. Variation in the frequency and severity of periodontal disease in the children investi­gated has also been suggested to influence the incidence of post-extraction bacteremia 19 • However, Peterson and Peacock 20 demonstrated that the inci­dence of post-extraction bacteremia in children with and without periodontal disease did not differ significantly.

The microorganisms isolated from blood cultures in our study represented 12 different genera or groups, with streptococcal species accounting for half of the total. Micrococcus species, Staphylococcus species and Corynebacterium species were not considered as contaminants since these organisms are normal­ly found in the oral cavity and may enter the circulation in the same way as viridans streptococci do 14• Bacillus species and Haemophilus para influenzae have also been included since both have been cultured prior to dental extrac­tion from the gingival sulcus of children with cardiac disease (unpublished findings). Propionibacterium jensenii was included since this species is now considered to be a normal resident of the gingival sulcus 2 1•

The presence of anaerobes in post-extraction blood cultures from chil­dren has been reported in only one study so far 20• The percentage of bacte­remia in this study without antibiotic prophylaxis was 48.8%, and of the iso­lates 46% were anaerobes. It may be that penicillin prophylaxis reduces the occurrence of strict anaerobes in post-extraction bacteremia, since most oral anaerobes are generally penicillin sensitive 22 and since, in our study, only 16% of the isolates were anaerobes. However, bacteremia was due to both peni­cillin resistant and penicillin sensitive microorganisms. From 7 (42%) of the 17 positive blood cultures 8 penicillin resistant microorganisms were isola­ted and from 12 cultures 24 penicillin sensitive microorganisms even though

20

serum penicillin concentrations were sufficient to inhibit the growth of the sensitive organisms. In addition, there was no difference in the serum peni­cillin concentration of children with bacteremia due to resistant or due to sensitive microorganisms, nor between the children with bacteremia and the children without bacteremia. Thus, these findings indicate that the serum pe­nicillin concentration at dental extraction is not the discriminating factor in preventing post-extraction bacteremia.

The bacteremia due to penicillin sensitive organisms in patients under penicillin cover might be due to a too short contact time between the antibi­otic and the organisms, or to a too low level of antibiotic in the gingival sulcus. It has been reported that the penicillin concentration at this site is about 10% of that of the serum penicillin concentration 8

• In the children of our study this would have resulted in concentrations ranging from 0. 1 to 1. 5 Units per ml of gingival fluid. Such concentrations indeed do not guaran­tee that even penicillin sensitive microorganisms will be prevented from en­tering the blood stream. Furthermore, the actual level of penicillin in the gingival sulcus might be even lower due to its degradation by B-lactamase­producing organisms present in the gingival sulcus 23• In contrast, clinda­mycin has been found to give the same peak levels in the gingival crevicular fluids and in blood 8

'24 and has been suggested as an alternative for peni­

cillin in the prevention of bacterial endocarditis after dental extraction 8>

25 •

However, Durack and Petersdorf 26 demonstrated that clindamycin and other bacteriostatic agents were ineffective in preventing experimental bacterial endocarditis due to viridans streptococci in rabbits. From their data it can be concluded that an effective prophylaxis depends on achieving high blood levels with a bactericidal agent for at least six hours after the episode of bacteremia, because the antibactericidal action in blood and on the endocar­dium to eridicate microorganisms already lodged on the endocardium, is far more important than preventing the development of transient bacteremia. The­refore the finding that post extraction bacteremia due to penicillin resistant microorganisms developed in 7 (8.5%) of the 82 children investigated is alar­ming, although none of the children in our study developed bacterial endocar­ditis. So, elimination of such organisms must have been achieved by factors other than penicillin in these patients since endocarditis due to penicillin resistant viridans streptococci, Streptococcus faecalis and Haemophilus parainfluenzae has been reported 27'28•29•30

• The question remains which

2 1

factors are involved. An attractive hypothesis is that penicillin whilst not killing the microorganism affects adherence on endocardial tissue as re­cently reported for a penicillin sensitive Streptococcus sanguis 31 •

Although bacterial endocarditis is usually caused by aerobes, anaerobes originating from the oral region have also been isolated from patients with endocarditis 32• In the present study two penicillin resistant anaerobes were isolated from post-extraction blood cultures : Bacteroides capillosus and Veillonella parvula. B. capillosus is known to be resistant to penicillin 33

whereas V. parvula is normally sensitive, but penicillin resistant strains have been reported 34 •

The results of our study indicate that transient post-extraction bacte­remia can occur under parenteral penicillin prophylaxis (regimen A) recommen­ded by the American Heart Association 1

• Bacterial endocarditis did not de­velop in our group of 82 children but apparent endocarditis due to failure of penicillin prophylaxis has been reported 35

• Because the microorganisms present in post-extraction bacteremia can cause endocarditis and a high per­centage is penicillin resistant, further studies are needed to determine if penicillin plus streptomycin (regimen B) 1 or administration of a single dose of oral amoxycillin 36 would be more effective in preventing post­extraction bacteremia in practice.

REFERENCES

1. Committee on Prevention of Rheumatic fever and Bacterial Endocarditis of the American Heart Association: Prevention of Bacterial Endocarditis, Cir­culation, 56: 139A, 1977.

2. Hoeprich PD: Infective endocarditis, in Hoeprich PD (ed): Infectious Diseases, ed. 2, Hagerstown, Harper and Row, 1977, pp. 999-1011.

3. Dankert J, Holloway Y, Schaareman M: A new anaerobic blood culture medium: Laboratory evaluation. Zbl Bakt Hyg Abt Orig A 248: 392, 1980.

4. Holdeman L V, Cato E P, Moore W E C: Anaerobe Laboratory Manual, ed 4. Virginia Polytechnic Inst, State Univ. , Blacksburg (Va) U. S.A. , 1977.

22

5. Cowan ST: Cowan and Steel's manual for the identification of medical bac­teria, ed 2. Cambridge University Press, London, Cambridge, 1975.

6. Holloway Y, Schaareman M, Dankert J: Identification of viridans streptococ­ci on the Minitek Miniaturised Differentiation System. J Clin Pathol 32: 1168, 1979.

7. Drucker D B, Jolly M: Sensitivity of oral microorganisms to antibiotics. Brit Dent J 131, 442, 1971.

8. Sukchotiratana M, Linton A H, Fletcher J P: Antibiotics and the oral strep­tococci of man. J Appl Bact 38: 227, 1975.

9. Barry A L : Diffusion test procedures, in Barry A L {ed): The antimicrobial susceptibility test: principles and practices. Philadelphia, Lea and Fe­biger, 1976, pp. 180-195.

10. Bender I B, Pressman R S, Tashman S G : Comparative effects of local and systemic antibiotic therapy in the prevention of postextraction bactere­mia. J Amer Dent Ass 57: 54, 1958.

11. Burket L W, Burn C G: Bacteremias following dental extraction. Demonstra­tion of source of bacteria by means of a non-pathogen. J Dent Res 16: 521, 1937.

12. Jones J C, Cuther J L: Control of bacteremia associated with extraction of teeth. J Oral Surg Oral Med, Oral Pathol 30: 454, 1970

13. Nolte W A: The oral microflora, in Nolte WA (ed): Oral microbiology with basic microbiology and immunology, ed 3. St. Louis, The CV Mosby Co, 1977, pp 211-214.

14. Khairat 0: The non-aerobes of post-extraction bacteremia. J Dent Res 45: 1191, 1966.

15. Crawford J J, Sconyers J R, Moriarty JD et al: Bacteremia after tooth extractions studied with the aid of prereduced anaerobically sterilized culture media. Appl Microbial 27: 927, 1974.

23

16. Glaser R J, Dankner A, Mathes S B, Harford C G: Effect of penicillin on the bacteremia following dental extraction. Amer J Med 4: 55, 1948.

17. Elliott R H, Dunbar J M: Streptococcal bacteremia in children following dental extractions. Arch Dis Childh 43: 451, 1968.

18. Schirger A, Martin W J, Royer R Q, Needham GM: Bacterial invasion of blood after oral surgical procedures. J Lab Clin Med 55: 376, 1960.

19. Speck W T, Spear S S, Kongrad E, Mandel L, Gersony WM: Transient bacte­remia in pediatric patients after dental extraction. Am J Dis Childh 130: 406, 1976.

20. Peterson L J, Peacoc R: The incidence of bacteremia in pediatric patients following tooth extraction. Circulation 53: 676, 1976.

2 1. Sanyal B, Russell C: Nonsporing, anaerobic, Gram-positive rods in saliva and the gingival crevice of humans. Appl environment Microbial 35: 676, 1976.

22. Finegold SM: Necrotizing pneumonias and lung abscess , in Hoeprich P D (ed): Infectious diseases, ed 2. Hagerstown, Harper and Row, 1977, pp. 309-317.

23. Hoffman H: Chemotherapy, in Nolte W A (ed): Oral Microbiology with basic microbiology and immunology, ed. 3: St. Louis. The C V Mosby, Co, 1977, p. 101.

24. Walker C B, Gordon J M, Cornwall H A, Murphy J C, Socransky S S: Gingival crevicular fluid levels of clindamycin compared with its minimal inhibi­tory concentrations for periodontal bacteria. Antimicrob Agents Chemother 19: 867, 1981.

25. Philips I: Clinical uses and control of rifampicin and clindamycin. J Clin Pathol 24: 410, 1971.

24

26. Pelletier L L, Durack D T, Petersdorf R C: Chemotherapy of experimental streptococcal endocarditis. IV. Further observations on prophylaxis. J Clin Invest 56: 319, 1975.

27. McCarthy V P, Cho C T, Diehl A M, Ramset B W: Bacterial endocarditis due to penicillin-resistant Streptococcus viridans. Clin Ped 18: 263, 1979.

28. Parrillo J E, Borst G C, Mazur M H, Iannini P, Klempner M S, Moellering R C, Anderson M C: Endocarditis due to resistant viridans streptococci during oral penicillin prophylaxis. New Eng J Med 300: 296, 1979.

29. Weinstein L, Rubin R H: Infective endocarditis, 1973. Progr Cardiovasc Dis 16: 239, 1973.

30. Jemsek J G, Greenberg S B, Gentry L O et al: Haemophilus parainfluenzae endocarditis. Two cases and review of the literature in the past decade. Am J Med 66: 51, 1979.

31. Scheld W M, Zak 0, Vosbeck K, Kloetzlen L, Sande M A: Effect of subinhi­bitory antibiotic concentrations (sub-MIC 's) on bacterial adhesion (BA) to fibrin platelet matrices in vitro, in 12th International Congress of Chemotherapy, Florence, 1981, abstract 643.

32. Felner J M, Dowell Jr V R: Anaerobic bacterial endocarditis. N Engl J Med 238 : 1188. 1970.

33. Willis A T: Anaerobic bacteriology: clinical and laboratory practice. ed 3. London, Butterworths, 1977, p. 183.

34. Brown W J, Waatti P E: Susceptibility testing of clinically isolated anaerobic bacteria by an agar dilution technique. Antimicrob Agents Chemother 17: 629, 1980.

35. Durack D T, Kaplan E L, Bisno A L: Twenty-five cases of apparent endocar­ditis prophylaxis failure: results of a national survey. Clin Res 29: 384A, 1981.

25

36. Oakl ey C, Somerville W : Prevention of infective endocarditis. Br Heart J 45: 233 , 1981.

ACKNOWLEDGEMENTS

The authors wish to thank Mr. J.G. Oostendorp of Gist-Brocades, Delft , the Netherl ands, for the determination of serum penicill in concentrations and Mrs . M. ter Brugge for typing the manuscript.

26

CHAPTER III

PENICILLIN PROPHYLAXIS IN CHILDREN WITH CARDIAC

DISEASE : POST-EXTRACTION BACTEREMIA AND PENICILLIN

RESISTANT VIRIDANS STREPTOCOCCI

J. Hess, Y. Holloway and J. Dankert

Accepted for publication in J. Inf . Dis .

ABSTRACT

Antibiotic prophylaxis is recoITVTiended to reduce the risk of developing bacterial endocarditis in patients with cardiac disease undergoing dental ma­nipulation. The incidence of post-extraction bacteremia in 82 children with dental foci and cardiac disease given parenteral penicillin prophylaxis was assessed by collecting blood five minutes after extraction. Penicillin concen­tration was determined in blood samples obtained at the same time. Pre-extrac­tion swabs were taken from the gingival sulcus. Penicillin susceptibility of microorganisms isolated from blood and the gingival sulcus was determined by both disc and dilution method. Post-extraction bacteremia, occurring in 21% of the patients was due to penicillin sensitive microorganisms only in 10, and to both penicillin sensitive and penicillin resistant microorganisms in 7 chil­dren. In four cases these penicillin resistant strains were viridans strepto­cocci. Penicillin resistant viridans streptococci were present in the gingival sulcus flora of 39% of the children, but a relationship between the presence of such strains in the gingival sulcus and the occurrence of post-extraction bacteremia could not be demonstrated. There was no difference in the serum pe­nicillin concentrations found in children with bacteremia due to penicillin sensitive or penicillin resistant microorganisms, nor between the concentra­tions in children with and without bacteremia. Therefore, pre-extraction sen­sitivity testing of the gingival sulcus flora does not seem to have predictive value in detecting patients at risk of developing a post-extraction bacteremia. We conclude that bacteremia will occur in some patients despite administration of parenteral penicillin regimens.

INTRODUCT ION

Viridans streptococci, which are the microorganisms still most frequently involved in the occurrence of bacterial endocarditis 1

, are widely believed to be always sensitive to penicillin 2

• Therefore this antibiotic is used most often as attempted prophylaxis for susceptible patients undergoing dental pro­cedures. Nevertheless penicillin resistant viridans streptococci can be isola­ted from the oral flora of patients, whether or not they have recently recei­ved penicillin 3

•4

• Because the oral flora is the source of post-extraction bacteremias 5

, this study was initiated in order to assess a possible rela­tionship between the presence of penicillin resistant organisms in the oral

28

flora and the incidence of post-extraction bacteremia in children with cardiac disease under parenteral penicillin prophylaxis pri or to dental extraction. Furthermore, we studied the relationship between post-extraction serum peni­cillin concentration and the occurrence of post-extraction bacteremia due to penicillin sensitive or resistant microorganisms.

MATERIALS AND METHODS

Patient population Eighty two children between the ages of 5 and 14 years (mean 9. 7 years)

who were assumed to be at risk for bacterial endocarditis, because they had cardiac disease, were included in the study. All required extraction of pri­mary or permanent teeth because of dental disease, which diagnosis was based on clinical and/or radiological findings. The extraction was performed by for­ceps technique under local anesthesia. None of the children had received anti­biotics in the preceeding two months and all were out-patients who had not been admitted to hospital for any reason in the preceeding twelve months. Antibiotic prophylaxis

Children younger than 6 years received 450,000 U procaine penicillin G and 150,000 U aqueous penicillin G intramuscularly ; children of 6 years and older received 900,000 U procaine penicillin G and 300,000 U aqueous penicil­lin G intramuscularly. The antibiotics were administered 45 minutes prior to dental extraction. Culture technique and susceptibility testing

To assess the predominant aerobic flora on the gingival sulcus a swab was taken before administration of penicillin and immediately taken to the labora­tory. Direct plating was done onto sheep blood (5% v/v) agar (Oxoid Ltd. , U. K. ), chocol ated agar (BBL, Cockeysville, Maryland, U.S.A. ), mitis sal ivarius agar (Difeo, Detroit, U.S.A. ), MSFA agar 6 , MacConkey agar (Merck, Darmstadt, F. R.G. ) and two Sabouraud 's agar plates (Merck, Darmstadt, F. R.G. ). The swab was then dipped into 100 ml of Todd Hewith broth (Oxoid Ltd. , U. K. ) and squeezed out against the side of the bottle. This broth was incubated at 37°c for 24 hours and then plated out as described above. All plates were incubated under 10% co2 at 37°c for 24 to 48 hours, except for one Sabouraud 's agar plate which was incubated at 2s0c . Blood was collected for culture 5 minutes after dental extraction . One sample of approximately 5 ml was drawn from a vein in the

29

antecubital fossa of each child after cleansing the skin with chlorhexidin­digluconas (0. 5% w/v) in ethanol 75% (v/v). The blood was immediately injec­ted into glucose ( 1% w/v) broth for aerobic culture . Sufficient penicillina­se (Mycofarm, Delft, The Netherlands) was added to this medium to counteract 1000 U of penicillin. These cultures were incubated at 37°c for 10 days and subcultured on days 3, 7 and 10.

Microorganisms were identified according to the criteria described by Cowan and Steel 7

• The penicillin susceptibility of the aerobic microorganisms was determined by a conventional disc method, using Oxoid discs containing 1. 5 U penicillin on sheep blood agar plates 4

'8

• Strains were considered to be penicillin resistant if zones of inhibition were smaller than 29 lllTI for staphylococci , and smaller than 22 mm for other microorganisms 9

• All mor­phologically distinct colonies of microorganisms isolated were tested. A to­tal of 294 strains of viridans streptococci from the gingival sulcus were tested. In the last 35 children studied, the microbiological investigation was extended. The 136 strains of viridans streptococci isolated from these children were identified using the Minitek Miniaturised Differentiation Sy­stem (BBL) as previously described 1 0 • In addition, penicillin susceptibility of these strains was also tested by a dilution method. The Minimum Inhibito­ry Concentration (M IC) was determined by the method of Schauf et al . 1 1 using serum (1% v/v) broth prepared according to Cowan and Steel 7

• Standard two­fold dilutions of penicillin were made in 1 ml amounts of serum broth ranging from 0. 4 to 800 U/ml. M IC was determined after 18 hours of incubation at 37°C. Strains were considered to be sensitive if no growth occurred in the presence of 0. 8 U/ml penicillin 1 2 •

Determination of penicillin concentration in serum Five minutes after extraction another 3 ml blood sample was drawn for

determination of the serum penicillin concentration . Serum was separated and preserved at -7o0c. As says were performed by an agar diffusion method using Bacillus s ubtilis as the test organism . Serum (undiluted or diluted with normal pooled human serum) was pipetted into wells cut in glucose (0. 1%) nu­trient agar base (Difeo) containing 3 to 5 x 1D6 Bacillus subtilis spores per ml of medium . Penicillin was dissolved in 0. 05 M phosphate buffer (pH 6. 8). Penicillin standards ranging from 2. 5 U per ml to 20 U per ml were prepared in pooled human serum. Plates were incubated for 18 hours at 30°C and the zones of inhibition were measured electronically.

30

Fol l ow up of pati ents

The chi l dren were exami ned ten days after extracti on by a ped i atri c car­

di ol ogi st and those who had had post-extracti on bacteremi a had bl ood cul tures

repeated .

RESULTS

Post-extracti on bacteremi a was present i n 17 ( 21%) chi l dren ( tabl e 1 ) .

V i ri dans streptococci were cul tured from the bl ood of 10 chi l dren . Other mi ­

croorgan i sms caused bacteremia i n 7 chi l dren . The mean serum peni ci l l i n con­

centrati on of ch i l dren wi th posi ti ve bl ood cul tures di d not di ffer s i gn i fi ­

cantly from that of chi l dren with negati ve bl ood cul tures , nor d id thi s con­

centration di ffer i n chi l dren wi th bacteremi a due to peni ci l l i n sens i ti ve or

those wi th peni ci l l i n res i stant mi croorgani sms . The predominant organi sms cul ­

tured from both gi ngi val su l cus ar,d bl ood were v ir idans streptococci ( tabl e 2 ) .

Peni ci l l i n res i stance was demonstrated i n 46 ( 16%) out of 294 strai ns of v i r i ­

dans streptococci . Pen i ci l l i n res i stant strai ns were present i n the gi ngi val

s ul cus fl ora of 32 ( 39% ) of the chi l dren . Peni ci l l i n res i stance was al so pre­

sent i n 48 ( 36% ) of 132 strains of other mi croorgan i sms i sol ated from the g i n­

g i va l su l cus . Of the 32 strains i sol ated from bl ood , 19 were v i ri dans strep­

tococci . Four ( 2 1%) of these from three ( 9%) chi l dren were peni ci l l i n resi stant.

We found no rel ati ons h i p between the presence of peni ci l l i n res i stant v i r i -

dans streptococci i n the su l ci and the occurrence o f post-extraction bactere­

mi a . Three ( 9%) of the 32 ch i l dren wi th pen ic i l l i n res i stant vi ri dans s trep­

tococci i n thei r gi ngi val s u l cus fl ora had a post-extracti on bacteremi a , one

caused by a res i stant stra i n (Fi gure 1 ) . Seven ( 14% ) of the 50 chi l dren from

whom only peni ci l l i n sens i ti ve vi ri dans streptococci were cul tured from the

g i ngi val su l cus fl ora , had posi ti ve bl ood cul tures after extracti on , two cau­

s ed by res i s tant s tra i n s . The i ncidence of vi r idans streptococcal bacteremi a

i n these two groups di d not di ffer s i gn i fi cantly. I n the case of other mi cro­

organi sms no rel ationsh i p between the presence of pen i ci l l i n res i stant mi cro­

organi sms i n the gi ngi val su l cus fl ora and the occurrence of bacteremi a cou l d

be demonstrated ei ther. Identi fi cati on of the 136 vi ri dans streptococci i so l a­

ted from the l ast 35 chi l dren s howed that S .mi ti or and uncl assi fi abl e vi r i -

dans streptococci were most frequently cul tured from the gi ngi val sul cus fl o-

ra ( tabl e 3 ) . These strai ns al so had the h i ghest percentage of pen i ci l l i n

31

w N

Table 1. Incidence of post-extraction bacteremia due to penicillin sensitive and penicillin resistant microorganisms and mean serum penicillin concentration in 82 children with cardiac disease.

Blood culture Number of Serum peni cillin concentration (U per ml) children (mean) (range)

Negative 65 8.9 (1.0 - 15.0)

Positive 17 1 8. 1 (2. 4 - 13.5)

[

peoicilli o seosi ti ,e 7 9. 2 (2. 4 - 13.5) - due to viridans streptococci

penicillin resistant 3

I 9.1 (6.0 - 12.0)

[

peoicilli o seosi ti ,e 9 8.0 (4.5 - 13.5) - due to other microorganisms

penicillin resistant 4 I 7.2 (4.4 - 12.0)

1 Six children had polymicrobial bacteria.

Table 2. Penici l l i n susceptibi l i ty (di sc method ) of m icroorgani sms i sol ated from b lood and gingival sul cus i n 82 chi l dren.

number of strai ns i sol ated from

Microorganisms gingival su l cus (n = 82) bl ood (n = 17 )

penici l l i n penici l l i n total penici l l i n penici l l i n total sensit ive resi stant sensit ive resi stant

- Vi ri dans streptococci 248 46 294 15 4 19 - Other streptococci 8 4 12 0 1 1 - Staphyl ococcus speci es 21 1 8 39 3 0 3

w - Other gram positive aerobes 25 4 29 3 0 3 w - Haemophi l us species 4 13 17 0 1 1 - Enterobacteri aceae speci es 0 2 2 0 0 0 - Pseudomonas aerugi nosa 0 1 1 0 0 0 - Other gram negative aerobes 24 6 30 0 0 0 - Gram positive anaerobes 0 0 0 3 0 3 - Gram negative anaerobes 2 0 2 0 2 2

Total 332(78% } 94(22%} 426 24(75%) 8 (25% ) 32

SULCUS GING/VALIS

Number of children (N) with penicillin l]- sensitive

�- resistant strains

@-- . �- -�N.S.�\.@ 2

BLOOD

,ff N.S. =not significant

Figure 1. The incidence of post-extraction bacteremia due to penicillin sensi tive and penicillin resistant viridans streptococci i n relation to the presence of penicillin sensitive and resistant viridans streptococci in the gingi val sulcus flora.

34

Table 3. Penicillin resistance of viridans streptococci isolated from the gingival sulcus of 35 chil dren.

Species Total number of Number (%) of resistant strains found by strains Disc method 1 MIC 2

S. mitior 66 10 (15. 1%) 23 (34. 8% ) S. sanguis 13 1 ( 7. 6%) 2 (15.3%) S. salivarius 11 0 0 S. mutans 6 0 0 S. milleri 2 0 0 S. viridans 38 4 (10. 5%) 10 (26. 3%)

Total 136 15 (11. 6%) 35 (26.2%)

1 Zone smaller than 22 mm diameter of growth inhibition round a disc containing 1. 5 U penicillin 2 Visible growth in a tube containing 0. 8 U penicillin per ml broth

w a,

Table 4. Serum penicillin concentration and MIC values of penicillin for viridans streptococci isolated from 5 children with post-extraction bacteremia.

Patient

1 2 3

4 5

1 N.D. = not done 2 unclassified

Serum peni ci 1 1 in concentration (U per ml)

6 . 6

6 . 0

9 .4

N . D . 1

2 . 4

Organisms MIC (U per ml)

S. mitior 0 . 4

S. sanguis 400 .0

S. viridans2 0 . 4

S. viridans 2 0 . 4

S. viridans 2 0 . 8

S. vi ridans 2 0 . 4

S. viridans2 0 . 4

resistance as demonstrated by both disc and dilution method. Penicillin resis­tance was also seen in the small number of S.sanguis isolated. Five (14%) of the last 35 children had viridans streptococcal bacteremia and seven viridans streptococcal strains were isolated from their blood (one child with 3 strains isolated). These strains were all penicillin sensitive by disc method, but one strain proved to be resistant by MIC (table 4).

D ISCUSSION

The incidence of post-extraction bacteremia in children without antibi­otic prophylaxis has been variously reported to range from O to 65% 1 3 ' 1� ' 1 8

,

and with penicillin prophylaxis from O to 53% 13 • 19 • 2 1 • We found an incidence of 21%. Several explanations may be put forward for these wide variations: use of different forms of penicillin, differences in the route of administra­tion, difference in blood culture techniques, use of different media with and without penicillinase, and the time interval between administration of peni­cillin and the collection of the specimens. Another possible reason is vari­ation in the frequency and severity of periodontal disease in the children investigated 1 9 • However, Peterson and Peacock 1 7 demonstrated that the inci­dence of postextraction bacteremia in children with and without periodontal disease did not differ significantly. The finding of penicillin resistant mi­croorganisms isolated from gingival sulcus and blood does not explain the oc­currence of post-extraction bacteremia , since occurrence of bacteremia was not related to the occurrence of penicillin resistant microorganisms on the gingival sulcus, and sometimes bacteremia was also due to penicillin sensiti­ve microorganisms. Furthermore , post-extraction bacteremia also occurred in children from whom no penicillin resistant microorganisms could be isolated from the gingival sulcus. Admittedly such strains may have been overlooked in those patients in whom only penicillin sensitive strains were recovered, as they might have been present in very low numbers.

The high percentage of penicillin resistant viridans streptococci isola­ted from the children in this study was not due to the previous use of peni­cillin since none of the children had received any antibiotics in the two months preceeding the extraction. The fact that the serum penicillin concen­tration of children with and without bacteremia did not differ significantly and that penicillin sensitive microoganisms were also recovered from blood

37

indicate that the serum penicil l in concentration is not the discriminating fac­tor in preventing post-extraction bacteremia. Although we did not determine pe­nicil l in concentration at the gingival sulcus, it has been reported that peni­cil l in concentration at this site is about 10% of that of the serum concentra­tion 8

• In the children of our study this would have resul ted in concentrations ranging from 0.1 to 1. 5 U. per ml gingival fl uid. Such concentrations indeed do not guarantee that even penicil l in sensitive microorganisms wil l not enter the bl oodstream. Furthermore, penicil lin concentrations might stil l be even l ower than expected due to inactivation of penicil lin by e-l actamase producing microorganisms present on the gingival sulcus 22 • It could al so be that the contact time between penicil lin and the penicil l in sensitive microorganisms was too short to be effective.

The cl inical significance of bacteremia due to penicil l in resistant vi­ridans streptococci is not known. However, the occurrence of endocarditis due to such strains has been demonstrated 2 3

• None of the chil dren with post­extraction bacteremia in our study developed bacterial endocarditis. Mul tipli­cation of penicil l in sensitive strains is likely to have been prevented by penicil l in, but penicil lin resistant strains must have been el iminated from the bl ood by other factors. Cl earance might have been enhanced by a synergis­tic antibacterial effect of serum and penicillin 2 4

•2 5

• Another mechanism pos­sibl y preventing the onset of endocarditis in chil dren with bacteremia due to penicil l in resistant strains might have been that penicil l in, whilst not kil ­ling the microorganisms, affected their virul ence, perhaps by interfering with factors invol ved in adherence 2 6

•

In conclusion, we found penicil l in resistant microorganisms in a high percentage of chil dren . However, presence of penicil l in resistant microorga­nisms in the gingival sul cus did not predispose to the occurrence of post­extraction bacteremia . Therefore pre-extraction sensitivity testing of the gingival sul cus flora, as previousl y recommended by others 2 7

, does not seem to offer a worthwhile prediction for patients at risk of devel oping post­extraction bacteremia despite penicil l in prophyl axis. Parenteral penicil l in prophyl actic regimens do not prevent post-extraction bacteremia in children at risk. Further studies are needed to determine if penicil l in plus strepto­mycin 2 8 would be more effective in preventing bacteremia in practice.

38

REFERENCES

1. Weinstein L, Rubin R H: Infective endocarditis - 1973. Progr. Cardiovasc . Dis. 16: 239, 1973.

2 . Garrod L P, Lambert H P, 0 'Grady F: In: Antibiotic and chemotherapy 4th edition. Churchill Livingstone, Edinburgh, 1973.

3. Sprunt K, Redman W, Leidy G . Penicillin resistant alpha streptococci in pharynx of patients given oral penicillin. Pediatrics 42: 957, 1968.

4. Drucker D B, Jolly M: Sensitivity of oral microorganisms to antibiotics. Brit. Dent. J. 131: 442, 1971.

5. Crowley M C, Beppler W A, Ramfjord S P: The bacterial flora of the normal gingival sulcus. J. Periodont. 35: 502, 1964 .

6. Linke H A B: New medium for the isolation of Streptococcus mutans and its differentiation from other oral streptococci. J. Clin. Microbiol. 5: 604, 1977.

7. Cowan S T: In: Cowan and Steel's Manual for the identification of medical bacteria, 2nd edition. Cambridge University Press, London, Cambridge, 1975.

8. Sukchotiratana M, Linton A H, Fletcher J P: Antibiotics and the oral strep­tococci of man. J. Appl. Bact . 38: 277, 1975.

9. Barry A L. Diffusion test procedures. In: The antimicrobial susceptibili­ty test: principles and practices, edited by A. L . Barry. Lea and Febiger, Philadelphia, 1976.

10. Holloway Y, Schaareman M, Dankert J: Identification of viridans strepto­cocci on the Minitek Miniaturised Differentiation System. J. Clin. Pathol. 32: 1168, 1979.

11. Schauf V, Deveikis A, Riff L, Serota A: Antibiotic killing kinetics of Group B Streptococci . J. Pediat. 89: 194, 1976.

39

1 2 . Phi l l i ps I , Warren C , Harri son J M , Sharpl es P , Bal l L C , Parker M T

Ant ib ioti c suscepti bi l i ti es of streptococci from the mouth and bl ood

of pati ents treated wi th peni ci l l i n or l i ncomyci n and cl i ndamyci n .

J . Med . Mi crobiol . 9 : 393 , 1976 .

13 . E l l i ott R H , Dunbar J M : Streptococcal bacteremi a i n chi l dren fol l owi ng

dental extractions . Arch . Di s . Chi l dh . 43 : 451 , 1968.

14 . Okel l C C , El l i ott S D : Bacteremia and oral seps i s . Lancet i i : 869 , 1935 .

1 5 . Berry F A , Yarbrough S , Yarbrough N , Rus sel l C M , Carpenter M A , Hendl ey

J O D : Transi ent bacteremi a duri ng dental mani pul ation i n chi l dren .

Ped i atri cs 51 : 476 , 1973 .

16 . Fai gel H C , Gaski l l W F : Bacteremi a i n pedi atri c pati ents fol l owi ng

dental mani pul ati ons . C l i n . Ped . 14: 562 , 1975 .

17 . Peterson L J , Peacock R : The i nci dence of bacteremi a i n pedi atri c

pati ents fol l owi ng tooth extraction . Ci rcu l ation 53 : 676 , 1976 .

18 . Speck W T , Spear S S , Kongrad E , Mandel L , Gersony W M : Trans i ent

bacteremi a in pedi atri c pati ents after dental extracti on . Am. J. Di s .

Ch i l d . 130 : 406 , 1976 .

19 . Gl aser R J , Dankner A , Mathes S B , Harford C G : Effect of peni ci l l i n

on the bacteremi a fol l owi ng dental extraction . Amer . J . Med . 4 : 55 , 1948.

20 . Bender I B, Pressman R S , Tashman S G: Comparative effects of l ocal and

systemi c anti bi oti c therapy in the preventi on of post-extraction bacte­

remi a . J . Amer. Dent. Ass . 57 : 54 , 1958.

21 . Sh i rger A, Marti n W J , Royer R Q, Needham G M : Bacteri al i nvasi on

of bl ood after oral surgi cal procedures . J . Lab . C l i n . Med . 5 5 : 376 , 1960 .

22 . Hoffman H : Chemotherapy . I n : Oral Mi crobi ol ogy wi th bas i s mi crobio l ogy

and immunol ogy , edi ted by W . A . Nol te , 3rd edi ti on . C . V . Mosby Company ,

St . Loui s , 1977 .

40

23 . McCarthy V P , Cho C T , Di ehl A M , Ramsey B W : Bacteri a l endocard i t i s

due to pen ic i l l i n-resi stant Streptococcus vi ri dans . Cl i n . Ped . 18 : 263 ,

1979 .

24 . Dutcher B S , Reynard A M , Beck M E , Cunni ngham R K : Potenti ati on of

anti bi oti c bacteri c idal ac i t i v i ty by normal human serum . Antimi crob .

Agents Chemother. 13 : 820 , 1978 .

25 . Lori an V , Atki nson B : Effect on serum on grampos it ive cocci grown i n

the presence of pen ic i l l i n . J . I nf . Di s . 138 : 865 , 1978 .

26 . Al kan M L , Beachey E H : Excretion of l i potei choi c acid by group A

streptococci : i nfl uence of peni c i l l i n on excretion and l oss of abi l i ty

to adhere to human oral mucosal cel l s . J . Cl i n . I nvest . 61 : 671 , 1978 .

27 . Sti rl and R M , Shotts N : Anti b i oti c-res i stant streptococci i n the

mouths of ch i l dren treated wi th pen ic i l l i n . Lancet i : 405 , 1967 .

28 . Commi ttee on Preventi on of Rheumati c Fever and Bacteri al Endocardi t i s

o f the Ameri can Heart Associ ation . Preventi on o f Bacterial Endocard i ti s .

C i rcul ation , 56 : 139A , 1977 .

ACKNOWLEDGEMENTS

The authors wi sh to thank Dav id T. Durack , M . D . , P h . D . for rev i ewi ng the

manuscri pt , Mr . J . G . Oostendorp of Gi s� Brocades Del ft for determi nati on

of the serum pen ic i l l i n concentrati ons , Mrs . M . Schaareman and Mr. W . Jol ­

dersma for the ir techni cal ass i stance and Mrs . M . ter Brugge for typi ng the

manuscri pt .

41

CHAPTER IV

DETECTION OF PENICILLIN TOLERANCE IN VIRIDANS STREPTO­

COCCI ISOLATED FROM THE ORAL FLORA OF CHILDREN WITH

CARDIAC DISEASE AND POST-EXTRACTION BACTEREMIA UNDER

PENICILLIN PROPHYLAXIS

J. Hess, Y. Holloway and J. Dankert

Submitted for publication

ABSTRACT

The presence of viridans streptococci, tolerant to the lethal action of penicillin, in the oral fl ora and in blood after dental extraction under pe­nicillin prophylaxis in 35 children with cardiac disease and dental foci was investigated. Of the 134 strains isolated from the gingival sulcus before pe­n icillin administration, 21 (16%) were penicillin tolerant. These strains we­re isolated from 14 (40%) children, of whom 4 developed post-extraction bac­teremia due to such strains. Of the remaining 21 children, only 1 had post­extraction bacteremia, due to a penicillin resistant strain. The incidence of post-extraction bacteremia due to penicillin tolerant strains was signi­ficantly related to the presence of such strains on the gingival sulcus (p < 0.05), while the mean penicillin concentrations in blood 5 min after ex­traction between the two groups did not differ significantly.

Some variables in methodology for detection of penicillin tolerance in viridans streptococci were also investigated. The use of different media did not influence M ICs and MBCs of penicillin for non-tolerant and tolerant strains, but growth phase did: mean MBCs were higher for bacteria in the logarithmic than for those in the stationary growth phase. Addition of penicillinase to the subculture medium for MBC determination resulted in an increase of the number of penicillin tolerant strains, detected. Timed killing kinetics sho-wed that the reduction of CFUs in tolerant strains was far slower than in non-tolerant strains. The benefits of the use of penicillin as endocarditis prophylaxis for children at risk undergoing dental extraction should be care­fully weighed, taking into consideration its bacteriostatic action on tolerant viridans streptococci.

INTRODUCTION

Most of the viridans streptococci that frequently cause bacterial endo­carditis 1 are thought to originate from the oral cavity 2 • It is widely be­lieved that these microorganisms are nearly always sensitive to penicillin, and therefore this antibiotic is used for attempted prophylaxis of endocar­ditis for susceptible patients undergoing dental procedures 3

• In a previous study we demonstrated the presence of penicillin resistant viridans strepto­cocci in the gingival sulcus flora of children with cardiac disease given pa­renteral penici llin prophylaxis and the occurrence of post-extraction

43

bacteremia due to such organisms 4 • Post-extraction bacteremia due to viridans streptococci occurred in 12% of these children 5 and twenty one percent of the strains isolated from the blood were penicillin resistant. A relationship be­tween the presence of penicillin resistant strains in the gingival sulcus and the occurrence of post-extraction bacteremia could not be demonstrated although 39% of the children had penicillin resistant viridans streptococci in the gin­gival sulcus flora 4

•

Recently species of the viridans streptococci group have been shown to be tolerant to the lethal action of penicillin 6 • Tolerance is characterized by a wide discrepancy between minimal bactericidal and minimal inhibitory con­centration. Since tolerant viridans streptococci have been demonstrated to be present in the gingival sulcus 7

, we studied the occurrence of post-extraction bacteremia due to such organisms and a possible relationship with the presence of such strains in the gingival sulcus flora of children with cardiac disease given parenteral penicillin prop�ylaxis. We also investigated variables in methodology for the detection of penicillin tolerance, because the tolerant response of group B streptococci to penicillin has been reported to be influ­enced by the growth medium used 8

, the bacterial growth phase 9 and the pH of the medium 10

PATIENTS AND METHODS

Thirty-five children between the ages of 5 and 14 years (mean 8.7 years ) with cardiac disease, who were assumed to be at risk for infective endocardi­tis were included in the study. All patients required extraction of primary or permanent teeth because of dental disease, which diagnosis was based on clinical and radiological findings. The extraction was performed by forceps technique under local anesthesia. None of the children had received antibio­tics in the preceeding two months.

Antibiotic prophylaxis. Children younger than 6 years received 450. 000 U procaine penicillin G and 150. 000 U aqueous penicillin G intramuscularly ; chil­dren of 6 years and older received 900. 000 U procaine penicillin G and 300. 000 U aqueous penicillin G intramuscularly. Antibiotic prophylaxis was administered 45 minutes prior to dental extraction.

Follow-up of patients. The children were examined ten days after extraction by a pediatric cardiologist and those with post-extraction bacteremia had blood

44

cultures repeated. Determination of penicillin serum concentration in post-extraction blood

samples. Five minutes after extraction a blood sample (3 ml) was drawn for de­termination of the serum penicillin concentration. Blood was centrifuged and serum was preserved at -7o0

c . Assays were performed using an agar diffusion method with Bacillus subtilis as test organism. Serum (diluted or undiluted with nonnal pooled human serum) was pipetted into wells cut in glucose (1%) nutrient agar base (Difeo, Detroit, Mich.) containing 3 to 5 x 106 Bacillus subtilis spores per ml of medium. Penicillin was dissolved in 0. 05 M phospha­te buffer (pH 6. 8), and standards ranging from 2. 5 U to 20 U per ml were pre­pared in pooled human serum. Plates were incubated for 18 hr at 30°C and the zones of inhibition were measured electronically.

Culture techniques. The gingival sulcus of the children was swabbed imme­diately before administration of penicillin . Swabs were plated directly onto sheep blood (5% v/v ) agar (Oxoid Ltd. , U. K . ) , mitis salivarius agar (Difeo), and MSFA agar 1 1 plates, all of which were incubated under 10% CO2 at 37°C for 24 to 48 hr.

Blood (5 ml) was collected for culture five min after dental extraction. Blood samples were drawn from a vein in the antecubital fossa after cleansing of the skin with chlorhexidine digluconate (0. 5% w/v) in ethanol 75% (v/v) and immediately injected into glucose ( 1% w/v) broth and diagnostic anaerobic growth (DAG) medium 1 2

, both containing sufficient penicillinase (Mycofarm, Delft, the Netherlands) to counteract 200 U penicillin. After incubation at 37°C subcultures were performed on sheep blood agar plates on days 3, 7 and 10. All morphologically distinct colonies of viridans streptococci .isolated were identified using the Minitek Miniaturised Differentiation System (BBL, Cockeysville, Mas s.) as previously described 1 3 and tested for penicillin susceptibility.

Susceptibility testing. The Minimum Inhibitory Concentration (MIC) and the Minimum Bactericidal Concentration (MBC) were determined according to Schauf et al. 1 4 using Mueller-Hinton (MH) broth (Oxoid Ltd). Inocula were prepared by making a hundred-fold dilution in fresh MH broth from an 18 h culture in the same medium. One drop (0. 05 ml) of this suspension was added to glass tubes containing standard two-fold dilutions of penicillin in 1 ml amounts of MH broth. Penicillin concentrations ranged from 0. 01 to 800 U per ml. The final concentration of microorganisms in each tube was approximately

45

105 to 5 x 105 colony fanning units (CFU) per ml. Tubes without penic illin were included i n each series of dilutions to serve as controls. The MIC was read after 18 hr of i ncubation at 37°C and was taken to be the lowest concen­tration of penicillin showi ng no visible turbidity with the unaided eye. The MBC was determined by spreading 0. 1 ml from all tubes (taki ng care not to touch the side of the tubes with the pipette) in duplicate onto sheep blood agar plates each containing sufficient penicillinase to counteract 500 U of penicillin . After incubation under 10% co2 at 37°C for 48 hr , the colonies were counted and the MBC was taken to be the least concentration of penicil­l in which provided 99.9% killing of the initial inoculum (10 CFU per plate or less). A strai n was defined as sensitive to penicillin if the MIC was lower than 0. 8 U per ml and tolerant if the MBC/MIC ratio was equal to or greater than 32.

Effect of bacterial growth phase on MIC and MBC. To examine the effect of the bacterial growth phase .on the MIC and MBC of penicillin for v iridans streptococci , the suscepti bility of 82 non-tolerant and 13 tolerant strai ns was tested on 4 hr (logarithmi c growth phase) and 18 hr (stationary growth phase) old cultures. The i nocula for testing 4 hr old bacteria were prepared from colonies of overni ght cultures on sheep blood agar. Approximately five colonies were suspended in 10 ml of MH broth. After i ncubation for 4 hr at 37°c in 10% CO2, one drop (0. 05 ml) of this suspension was added to glass tubes with 1 ml amounts of MH broth containing standard two-fold di lut ions of penicill in . Tubes without penicillin served as controls. Colony counts

5 5 performed with each experiment showed that inocula ranged from 10 to 5 x 10 CFU per ml. MIGs were read and MBCs were determined as described for the stan­dard susceptibi lity test. From the same strains MIC and MBC were also deter­mined using bacteria from 18 hr old cultures in MH broth. Inocula were prepa­red and incubation and subculturing were performed according to the standard susceptibi lity testing method.

Effect of adding penicillinase to subculture medium on MBC/MIC ratio. To determi ne the i nfluence of penicillin carry-over on the MBC/MIC rat io of penicillin for viridans streptococci in 15 strains (7 tolerant and 8 non-to­lerant) subcultures from all tubes after MIC reading were made on both plain blood agar medium and on blood agar medium containing sufficient pen ic illinase to counteract 500 U penicillin.

Effect of medi a on MIC and MSC. To study the effect of different medi a

46

on M IC and MBC of penicillin , the susceptibility of 10 viridans streptococcal strains was tested using MH broth, serum broth (SB), prepared according to Cowan and Steel 1 5 , Trypticase soy broth (TSB , BBL), Tryptose phosphate broth (TPB,Difco) and TH broth. Susceptibility testing was performed according to the standard method. Subcultures to assess MBC were done on blood agar contai­ning sufficient penicillinase to counteract 500 U penicillin.

Timed killing kinetics. Killing kinetics of two penicillin sensitive strains (strain 1: M IC 0 . 01 U and MBC 0. 01 U; strain 2: M IC 0. 02 U and MBC 0. 03 U) and three penicillin tolerant strains (all three strains had M ICs of 0. 01 U and MBCs of 400 U) were performed on logarithmic (4 hr) and stationary (18 hr) phase cultures in MH broth containing 1 U penicillin per ml. Inocula were prepared by suspending five colonies from an overnight blood agar plate in 10 ml of MH broth. Logarithmic phase cells were obtained after incubation of this MH broth for 4 hr at 37°c in 10% co2. One ml of the suspension was transferred into each of the following 100 ml of fresh MH broth (growth con­trol), 100 ml of fresh MH broth containing penicillin ( 1 U per ml) and 100 ml of fresh MH broth to which penicillin ( 1 U per ml) was added 4 hr later. Co­lony counts with each test showed that the inocula were 106 to 5 x 106 CFU per ml. Aliquots taken at 48 hr were serially ten-fold diluted in MH broth. Each dilution was subcultured by spreading 0. 1 ml on sheep blood agar plates containing sufficient penicillinase to counteract 500 U of penicillin and in­cubated for 48 hr under 10% co2 at 37°c to determine surviving organisms (CFUs). Stationary phase cells were obtained after 18 hr incubation of MH broth into which five colonies from an overnight blood agar plate had been inoculated. One ml of a 1: 100 dilution of this suspension was transferred into each of the following 100 ml of fresh MH broth (growth control) , 100 ml of fresh MH broth containing penicillin (1 U per ml) and 100 ml of fresh MH broth to which peni­cillin (1 U per ml) was added 4 hr later. Colony counts of each experiment showed that inocula were 5 x 105 to 106 CFU per ml. Killing kinetics were per­formed as described for the logarithmic phase cells.

Statistics. Calculations for determination of significant differences we­re done by the chi square test and the Student ' s t test.

RESULTS

Post-extraction bacteremia. Viridans streptococcal bacteremia under

47

-I=> CX)

Table 1 . M I C and MBC of penici l l i n for vi ridans streptococci i sol ated from 5 chi ldren wi th post-extracti on bacterem ia

Patient Microorganisms M I C (U/m l ) MBC { U/ml ) MBC/M IC ratio

1 S . m it ior 0 . 1 6 . 3 63 2 S . sanguis 400 400 1 3 S . vi ri dans X 0 .25 6 .3 126

S . vi ridans X

0 .2 25 125 S. viridans

X 0 . 8 400 500

4 S . viri dans X

0 .4 200 500 5 S. vi ri dans

X 0 .4 25 62.5

X unclassi fied

Table 2. MBC/MIC ratio of penicil lin for 134 viridans streptococci isolated from the gingival sulcus fl ora of 35 chil dren with cardiac disease at risk for bacterial endocarditis

Microorganisms Number of strains MBC/MIC ratio 1- 15 16-31 �2

s . mitior 67 54 4 9

s . sangui s 12 10 0 2 s . sal ivarius 8 8 0 0

s . mutans 7 7 0 0

s . mill eri 2 2 0 0

s . vi ri dans X 38 25 3 10

Total 134 106 7 21

X uncl assified

49

SULCUS GING/VALIS

- - - - - - - - - - - - - - - - - -

e � p�0.05 ----"7. X = 3. 9

N=

BLOOD

Number of children (NJ

m with penicillin tolerant � strains

/1 without penicillin LJ tolerant strains

Figure 1. The incidence of pos�-extraction bacteremia due to penicillin tolerant and non-tolerant viridans strep­tococci in relation to the presence of penicillin tolerant and non-tolerant viridans streptococci in the gingival sulcus flora.

50

(J') ......

Table 3 . Effect of bacterial growth phase on M ICs and MBCs of penicil lin for nontolerant and tol erant viridans streptococci strains isolated from the sul cus gingivalis fl ora of chi l dren with cardiac disease .

Vi ri dans Streptococci

non-tolerant (n=82)

tol erant (n= l 3)

1 geometric mean

Growth phase

l oga ri thmi c

stationary

logarithmic

stationary

MIC (U/m l) mean1 range

0 .07 0 .01-0 .5

0 . 08 0 .01-0 .5

0 .04 0 . 01-0 . 2

0 . 04 0 .01-0 . 1

MBC ( U/ml mean1 range

0 . 32 1 0 . 01- 1 .0

0 . 142 0 .01- 1 .0

216 1 .0 -400

96 0 .01-400

2 P <0 . 05 when the MBCs of l ogarithmic phase bacteria were compared with those of stati onary phase bacteria (Student ' s t test)

Table 4. Effect of adding penicillinase to subculture medium on MBC/MIC ratio for 15 viridans streptococci strains

MBC/M IC 1 ratio

1-31 32-500

>500

without

Number of strains subcultured for MBC on blood agar medium

penicillinase with penicillinase

13 8 ( N. S.) 2 1 (N. S .) 0 6 {p<0.02) 2

1 M IC and MBC determinations were performed using bacteria in the stationary growth phase.

2 p valve by chi-square test.

52

parenteral penicillin prophylaxis occurred in five (14%) of the 35 children. All but one of the seven different streptococcus viridans strains isolated from these five children were sensitive when tested on MIC, but all were re­sistant when tested on MBC (table 1). The MBC/MIC ratio ranged from 1 to 500. A ll but one strain were penicillin tolerant (MBC/MIC ratio equal to or greater than 32). The mean serum penicillin concentration of children with bacteremia was 6. 1 U per ml (range 2. 4 to 9. 4 U per ml) and did not differ from that of children without bacteremia (6. 0 U per ml ; range 1. 0 to 10. 0 U per ml).

In total, 134 different viridans streptococcal strains were isolated from the sulcus gingivalis flora. Susceptibility testing showed that 110 (82%) had MIGs lower than 0.8 U per ml and that 24 (18%) had MIGs equal to or grea­ter than 0. 8 U per ml (penicillin resistant). A discrepancy of the MBC/MIC ratio equal to or greater than 32 was found in 21 (16%) of the strains (table 2). These tolerant strains were isolated from 14 children of whom four deve­loped post-extraction bacteremia due to penicillin tolerant strains. Children in whom no penicillin tolerant strains were isolated from the sulcus gingivalis did not develop bacteremia due to penicillin tolerant strains after dental extraction, but in this group there was one child with post-extraction bacte­remia due to a penicillin resistant, non-tolerant strain. The occurrence of tolerant viridans streptococci in the sulcus gingivalis flora was significant­ly related to the incidence of post-extraction bacteremia due to tolerant strains (p < 0. 05) (figure 1). Mean serum penicillin concentration of children with tolerant strains in their sulcus gingivalis flora was not significantly different from that in children without tolerant strains (5. 1 U per ml and 6. 8 U per ml, respectively).

Effect of bacterial growth phase on MIC and MBC. The growth phase of the inoculum used from 82 penicillin sensitive and 12 penicillin tolerant viridans streptococci had no effect on the MIGs of penicillin (table 3). Mean MBCs of non-tolerant strains were significantly higher for logarithmic than for stationary phase cultures. The mean MBC of tolerant strains in their logarith­mic phase was higher than the mean MBC of stationary phase cells, but this difference was not statistically significant.

Effect of adding penicillinase to subculture medium on MBC/MIC ratio. MBC/MIC ratio ' s for the 15 test organisms when MBCs were determined on plain blood agar revealed that two strains were penicillin tolerant (table 4). When MBCs were determined on blood agar plates containing penicillinase to

53

(JI �

Table 5. Mean MICs and MBCs of penicillin for 10 (5 non-tolerant and 5 tolerant viridans streptococci viridans grown in five defferent media. MBCs were determined by subculturinq onto bl ood agar plates containing penicillinase.

Non-tolerant Strains Tolerant Strains

Medium MIC (U/ml) MBC (U/ml) MIC (U/ml) MBC (U/ml)

mean 1 ( range ) mean 1 (range) mean 1 (ranqe) mean 1 (range)

MH2 0. 34 (0. 01-1.6) 0. 36 (0. 02-1. 6 ) 0. 01 (0. 0 1-0. 02) 400 -SB 0. 32 (0. 01-1. 6 ) 0. 62 (0. 1 -1.6) 0. 07 (0 . 01-0. 1 ) 321.2 (6.4-400) TSB 0. 33 (0. 01-1.6) 0.51 (0. 02-1.6) 0. 02 (0. 01-0. 05) 400 -TPB 0. 33 (0. 01-1.6) 0.51 (0. 02-1.6) 0. 01 (0. 01-0. 02) 400 -TH 0. 34 (0. 01-1. 6) 0.50 (0. 02-1 .6) 0. 03 (0. 01-0.05) 400 -

1 geometric mean 2 MH : Mueller Hinton broth ; SB : serum broth ; TSB: Trypticase soy broth ; TPB : Tryptose phosphate broth ;

TH: Todd-Hewitt broth.

8

� 5 LL. u 4 �

- - -1; . __ -o -- -----; 'o J. '

' 'O

- - - if ---o..,_

--, '/.,,, '

,o

2 ��- ..-------.-----h 0 4 24 48 0 4 24 48

HOURS HOURS

Figure 2. Timed killing curves of 2 penicillin sensitive (o--o) and 3 penicillin tolerant (o----o) strains of viridans streptococci. The data presented are mean values. (Left) curves obtained from logarithmic phase bacteria (4 hr). (Right) curves obtained from statio­nary phase bacteria (18 hr). t, moment of addition of 1 U penicillin per ml MH broth. C, control.

55

counteract the carry-over of penicillin, seven strains were penicillin tole­rant of which six had a MBC/MIC ratio greater than 500 in contrast with nought on plain blood agar (p < 0. 02). Addition of penicillinase did not increase the MBCs of the 8 non-tolerant strains.

Effect of media on MIC and MBC. Comparison of the mean values and ranges of the MICs of the five non-tolerant viridans streptococcal strains did not show significant differences when determined in five different media (table 5), nor did the mean values and ranges of the MBCs of the five strains differ. Mean MICs of the five tolerant strains grown in the different media showed small differences, but these were not significant. MBCs of the tolerant strains were high in all media.

Timed killing kinetics. Logarithmic phase and stationary phase cells of two penicillin sensitive and three penicillin tolerant viridans streptococcal strains were exposed to penicillin in concentrations 65 and 100 times higher than their respective MICs. Complete killing of the two sensitive strains was not achieved earlier in the logarithmic phase than in the stationary phase cultures (figure 2). In the tolerant strains there was a 1000-fold reduction in viable counts after 48 hr in the case of stationary phase bacteria and on­ly a 200-fold reduction in the case of logarithmic phase bacteria. Timed kil­ling assays performed on the sensitive and tolerant strains exposed to peni­cillin after having grown for 4 hr, did not show substantial differences for logarithmic or stationary phase inocula. The reduction in viable counts in tolerant strains after 48 hr was only 100 to 200-fold.

DISCUSSION

Viridans streptococcal strains isolated from the gingival sulcus flora of children at risk for bacterial endocarditis showed three patterns of res­ponse to penicillin. Most strains were penicillin sensitive (MIC and MBC < 0. 8U), a surprisingly high percentage (18%) was penicillin resistant (MIC and MBC � 0.8U) and a substantial percentage (16%) was penicillin tolerant (discrepancy of MBC/MIC ratio � 32). Tolerance to penicillin has been recog­nized for several bacterial species during the last decade 1 6 • The true fre­quency of tolerance is uncertain because the methodology differed in the va­rious reports . The importance of the medium for the detection of penicillin tolerance in group B streptococci has been shown 8

; several strains were

56

penicillin tolerant when tested in TPB , but non-tolerant when tested in TH or MH broth. A similar effect of media on tolerance to other beta-lactam antibi­otics has also been demonstrated for Staphylococcus aureus 1 7 , where tolerance was found frequently in strains grown in MH broth. A possible explanation for these inconsistent findings could be a selective suppression of the lytic ef­fects of beta-lactam antibiotics by a pH decline resulting in an apperent to­lerant response to such antibiotics 1 0 • 1 8 • The variation in occurrence of to­lerance in various media might therefore be due to differences in pH decline during bacterial growth although the pH-related tolerant response to penicil­lin was not detectable after 24 hr of incubation of group B streptococci 1 0 •

In our study MIGs and MB Gs of penicillin determined after 18 hr in a small number of tolerant and non-tolerant viridans streptococcal strains grown in five different media did not differ significantly. Measurement of the pH of inoculated media not containing penicillin showed a strong pH decline from 6. 9-7.3 to 5 . 1-5.7 after 18 hr of incubation in SB , TSB and TPB , while the pH of MH and TH did not show such a decline. In addition, no strains were tolerant when tested in one medium and non-tolerant when tested in another me­dium. This suggests, that the protection of the streptococci against penicillin by low pH is not absolute. The MB Gs of the same strains were less consistent if penicillinase was not added to the subculture medium for MB G determination (results not shown). This may be due to the carry-over of penicillin with sur­viving bacteria from clear tubes to the subculture medium, resulting in growth inhibition 19 • The addition of penicillinase to the subculture medium to coun­teract the carried-over penicillin resulted in an increase of the number of penicillin tolerant strains, when compared to the number of strains tested on medium without penicillinase. This finding is supported by the results obtai­ned in the timed killing experiments. Strains not tolerant when MB Gs were determined on subculture medium without penicillinase, but tolerant when tes­ted with penicillinase present showed only a slight reduction of viable GFU ' s after 48 hr of exposure to penicillin at a concentration one hundred times higher than their MIGs. We therefore conclude that MB G testing of viridans streptococci should be performed on subculture media to which penicillinase is added 2 0 •

Penicillin-induced lysis of bacteria is thought to be greatest in growing multiplying cultures 9 ' 2 1 and to require active participation of the autolysin enzyme system 22• Therefore, our finding that MB Gs of viridans streptococcal

57

strains were higher in logarithmic than in stationary phase bacteria, was un­expected. However, a plausible hypothesis for this finding might be that in rapidly dividing bacteria during exposure to penicillin differences in the activity of the autolytic enzym system are greater. In normally growing bacteria autolysins exhibit balanced activity during cell division and cell wall turn over 23 • In bacteria exposed to penicillin, the autolytic activity is increased 2 2

and lipoteichoic acid and other cell wall constituents are released into the growth medium, resulting in a suppression of the autolysins 2 1 ' 22 probably in­ducing uncoupling of their balanced activity. The utmost expression of the unbalanced activity of autolysins in bacteria when exposed to penicillin may result in a tolerant response to penicillin, although tolerance to penicillin is not an all-or-none phenomenon 2 1• This implies that uncoupling of the ba­lanced autolysin activity may occur gradually in various strains, especially in rapidly dividing bacteria. Thus, MBC/MIC discrepancies are more prominent in the logarithmic growth phase than in the stationary growth phase of bac-teria. In addition, tolerance to penicillin has been reported most often in logarithmic growing bacteria 10 • 2� - 26 • The finding that timed killing kine-tics of the five strains tested in different growth phases did not differ is probably due to the fact that CFUs were determined only after 4, 24 and 48 hr. More experiments with a greater number of strains and more frequent CFU de­terminations should be performed to detect the influence of growth phase on killing rate and extent.

We found penicillin tolerant viridans streptococci on the gingival sul­cus in nearly half of the children studied. The clinical significance of pe­nicillin tolerance has been discussed 16 but bacterial endocarditis due to tolerant viridans streptococci has been reported 27 • In this study the rela­tionship between the presence of penicillin tolerant viridans streptococci in the gingival sulcus flora of children with cardiac disease at risk for bacte­rial endocarditis and the occurrence of such strains in post-extraction bac­teremia under penicillin prophylaxis was assessed. This means that children with penicillin tolerant viridans streptococcal strains on the gingival sul­cus are more at risk of developing post-extraction bacteremia than children without such strains, despite attempted penicillin prophylaxis, since peni­cillin serum concentrations did not differ. Cases of apparent penicillin pro­phylactic failures resulting in viridans streptococcal endocarditis after dental extraction have been reported 28 • The presence of penicillin tolerant

58

strains in the oral flora and in post-extraction bacteremias may be one cau­se for failure of attempted penicil lin prophyl axis, because penicill in acts as a bacteriostatic agent on such strains. We suggest that other antibiotics should be evaluated for ability to kill penicillin tolerant viridans strep­tococci.

REFERENCES

1. Kaplan E L, Rich H, Gersony W, Manning J A, 1979. A collaborative study of infective endocarditis in the 1970 s. Circulation 59: 327-335.

2. Kaplan E L, 1977. Infective endocarditis in the pediatric age group. An overview. In E.L. Kaplan and A.V. Taranta (ed). Infective endocarditis. American Heart Association, Dallas, Texas. p. 51-54.

3. Kaplan EL, Anthony B F, B isno A, Durack D, Houser H, Millard D, San­ford J, Shulman S T, Stillerman M, Taranta A V, Wenger N, 1977. Preven­tion of bacterial endocarditis. Circulation 56: 139A-143A.

4. Hess J, Holloway Y, Dankert J. Penicillin prophylaxis in children with cardiac disease: post-extraction bacteremia and penicillin resistant vi­ridans streptococci. J. Inf. Dis. accepted for publication.

5. Hess J, Holloway Y, Dankert J. The incidence of post-extraction bacteremia under penicillin cover in children with cardiac disease. Pediatrics, accep­ted for publication.

6. Horne D, Tomasz A, 1977. Tolerant response of Streptococcus sanguis to beta­lactams and other cell wall inhibitors. Antimicrob. Agents Chemother. 11: 888-896.

7. Holloway Y, Dankert J, Hess J, 1980. Penicillin tolerance and bacterial en­docarditis. Lancet i: 589.

8. Kim K S, Yoshimori R N, Imagawa D T, Anthony B F, 1979. Importance of medium in demonstrating penicil l in tol erance by group B streptococci. Antimicrob.

59

Agents Chemother. 16: 214-216.

9. Kim K S, Anthony B F, 1981. Importance of bacterial growth phase i n deter­mining minimal bactericidal concentrations of penicillin and methic illin. Antimicrob. Agents Chemother. 19: 1075-1077.

10. Horne D, Tomasz A, 1981. pH-dependant penicillin tolerance of group B streptococci . Antimicrob. Agents Chemother. 20: 128-135.

11. Linke H A B, 1977. New medium for the i solation of Streptococcus mutans and i ts di fferentiati on from other oral streptococci. J. Cli n. Microbial. 5: 604-609.

12. Dankert J, Holloway J, Schaareman M A, 1980. A new anaerobi c blood culture medi um: laboratory evaluation. Zbl. Bakt. Hyg. I. Abt. Orig. A 248 : 392-401.

13. Holloway Y, Schaareman M, Dankert J, 1979. Identification of viridans strep­tococci on the Minitek Miniaturised Di fferentiati on System . J. Cli n. Path . 32: 1168-1173.

14. Schauf V, Deveikis A, R i ff L, Serota A, 1976. Antibiotic-k illing ki netics of group B streptococci . J. Pediatr. 89: 194-198.

15. Cowan S T, 1975. Cowan and Steel 's Manual for the identification of medical bacteri a. Cambridge Universi ty Press, London and Cambridge, 2nd edit ion.

16. Editorial, Penicillin-tolerant bacteria, 1980. Lancet i : 856-857.

17. Norden C W, Keleti E, 1981. Antibiotic tolerance in strains of Staphylococcus aureus. J. Anti microb. Chemother. 7: 599-605.

18. Lopez R, Ronda-Lain C, Tapia A, Waks S B, Tomasz A, 1976. Suppressi on of the lytic and bactericidal effects of cell wall-inhibitory antibiotics. Anti ­microb. Agents Chemother. 10: 697-706.

19. Russell A D, Ahonkhai Y, Rogers D T, 1979. A review. Microbiologi cal

60

applications of the inactivation of antibiotics and other microbial agents. J. Appl. Bact. 46: 207-245.

20. Dankert J, Holloway Y, Joldersma W, Hess J. Importance of penicillin in subculture medium for detection of penicillin tolerant viridans strepto­cocci. Submitted for publication.

21. Shockman G D, Daneo-Moore L, Cornett J B, Mychajlonka M, 1979. Does pe­nicillin kill bacteria? Rev. Inf. Dis. 1: 787-796.

22. Tomasz A, 1979. From penicillin-binding proteins to the lysis and death of bacteria: A 1979 view. Rev. Inf. Dis. 1: 343-467.

23. Rogers H J, Autolysins. In R.C. W. Berkeley, G. W. Gooday and D.C. Ellwood (ed. }. Microbial polysaccharides and polysaccharases. Academic Press, Inc., London, 1979, p. 237-268.

24. Best G K, Best N H, Koval A V, 1974. Evidence for participation of auto­lysins in bactericidal action of oxacillin on Staphylococcus aureus Antimicrob. Agents Chemother. 6: 825-830.

25. Sabath L D, Wheeler N, Laverdiere M, Blazevic D, Wilkinson B, 1977. A new type of penicillin resistance of Staphylococcus aureus. Lancet i: 443-447.

26. Kim A S, Anthony B F, 1981. Penicillin tolerance in group B st�eptococci isolated from infected neonates. J. Inf. Dis. 144: 411-419.

27. Pulliam L, Inocuchi S, Hadley W K, Mills J, 1979. Penicillin tolerance in experimental streptococcal endocarditis. Lancet ii: 957.

28. Durack D T, Kaplan E L, Bisno A L, 1981. Twenty-five cases of apparent endo­carditis prophylaxis failure: results of a national survey. Clin. Res. 29 : 384A .

61

ACKNOWLEDGEMENTS

We thank Willem Joldersma for excellent technical assistance and Mr. J. G . Oostendorp of Gist Brocades, Delft, for determination of the serum peni­cillin concentrations. We also thank David T. Durack, M.D. , D. Phil. , for re­viewing the manuscript. This work was supported in part by a research grant from the Dutch Heart Foundation.

62

CHAPTER V

SIGNIFICANCE OF PENICILLIN TOLERANCE IN VIVO : PRE­

VENTION OF STREPTOCOCCUS SANGUIS EXPERIMENTAL ENDO­

CARDITIS

J. Hess, J. Dankert and D . Durack

Submitted for publication

ABSTRACT

To determine whether in vitro tolerance among viridans streptococci in­fluences in vivo therapy, we studied four strains of dextran-producing Strep­tococcus sanguis serotype II. All four strains were inhibited in vitro by 0. 1 ug penicillin or l ess; one was non-tolerant, one intermediate, and two were tolerant to the lethal action of penicillin. The combination of penicillin and streptomycin killed all strains completely within 24 hours. Sera from rab­bits injected with penicillin were inhibitory for all strains, but were bac­tericidal only for the non-tolerant strain. The incidence of endocarditis in untreated rabbits with left heart catheters was 100 percent after inoculation of each of the four strains. Despite attempted prophylaxis with procaine peni­cillin G 250 mg/kg, endocarditis developed in 44 of 70 rabbits (63%) injected with the tolerant strains, contrasting with 2 of 22 (9%) for the non-tolerant strain (p = 0. 0001). Combined penicillin plus streptomycin prevented endocar� ditis completely. We conclude that penicillin tolerance can be a crucial de­terminant of the response of viridans streptococci to penicillin in vivo.

INTRODUCTION

Tolerance to the lethal action of beta-lactam antibiotics, which has been much studied in staphylococci 1 , also occurs among the viridans streptococci 2

• 3 •

We have recently reported that strains of viridans streptococci isolated from the oral flora of children, and from their blood shortly after dental extrac­tions, are often tolerant to penicillin 3

• To assess whether tolerance in vitro has any significance for chemotherapy in vivo, we studied the ability of peni­cillin to prevent experimental endocarditis caused by tolerant or non-tolerant strains of Streptococcus sanguis serotype I I. We also studied the effect of combined penicillin and streptomycin in vitro and in vivo.

METHODS

Test organisms. Four strains of S.sanguis serotype II were used in these experiments. Strains 1, 2 and 4 were isolated from the sulcus gingivalis of three children. Strain 3 was cultured from the blood of a patient with strep­tococcal endocarditis. All four strains produced dextran when grown on sucrose­containing media according to the method of Holloway et al � .

Susceptibility testing. The minimal inhibitory concentrations (MIC) and

64

minimal bactericidal concentrations {MBC) of penicillin and streptomycin for the four strains were determined by a standard two-fold tube dilution method. Inocula were prepared by making a four hundred-fold dilution in fresh Todd­Hewitt broth {Oxoid Ltd, England) from an 18 h culture in the same medium. Of this suspension 0. 05 ml was added to tubes containing serial two-fold dilu­tions of penicillin G {Gist B rocades, Delft, the Netherlands) or streptomycin {Specia, Paris, France) standards in Mueller-Hinton broth (Difeo Laboratories, Detroit, Mich.), providing a final inoculum of 1-5 x 105 colony-forming units (CFU) per ml. Control tubes without antibiotics were included in each series of dilutions. After 18 hrs incubation at 37°c, the M IC was read as the lowest concentration of antibiotic that showed no visible turbidity. The MBC was de­termined by spreading 0. 1 ml from each clear tube in duplicate onto the surfa­ce of sheep blood (5% v/v) agar plates {Oxoid Ltd. , England) which contained 50 Units per ml penicillinase (Mycofarm, Delft, the Netherlands). After incu­bation in 10% co2 at 37°c for �8 h the colonies were counted; the MBC was read as the lowest concentration of antibiotic that killed 99.9% of the ini­tial inoculum . This corresponded to 10 surviving colonies or less per plate.

Antibiotic kill kinetics and bacteridical synergy. The effects of peni­cillin and streptomycin on the four strains were measured by means of time­kill curves performed in Mueller-Hinton broth using a modification of the method of Wolfe and Johnson 5

• Penicillin was tested at a concentration of 1. 0 ug per ml, streptomycin at 10 ug per ml and the combination of penicil­lin and streptomycin at concentrations of 0. 5 and 5 ug per ml, respectively. Inocula were prepared from overnight cultures in Todd-Hewitt broth to provi­de a final suspension of approximately 5 x 105 CFU per ml. All tubes, inclu­ding tubes without antibiotic as growth controls, were incubated in 10% CO2 at 37°C. 0. 1 ml portions from serial hundred-fold dilutions taken before incubation and 4, 24 and 48 hours later were plated onto sheep blood agar containing 50 Units per ml penicillinase, and the number of colonies counted after incubation in 10% CO2 for 48 hours. The combination of penicillin and streptomycin was considered to be synergistic if either of these criteria were fulfilled: 1) increased rate of killing, that is 99% or greater diffe­rence in the number of organisms killed by penicillin plus streptomycin than by penicillin or streptomycin alone at 4, 24 and 48 h; or 2) sterilization of the inoculum by penicillin plus streptomycin, but not by either drug alo­ne, within 48 h.

65

Assay for serum penicillin concentration. The modified agar well diffu­sion method of Bennett et al. 6 was used to measure penicillin concentrations in serum from blood samples drawn from one of the rabbit ' s ear veins 0. 5, 1, 3 and 6 h after intramuscular injection of procaine penicillin G (PPG } 250 mg/kg.

Serum inhibitory (SIT} and bactericidal titers (SBT). Serial two-fold di­lutions of serum separated from blood drawn 30 minutes after injection of PPG alone or PPG plus streptomycin (SM) were made in Microtiter wells (Flow Labo­ratories, McLean, Va. , ) using Mueller-Hinton broth. Approximately 5 x 105 CFU of streptococci were added to each well, followed by incubation for 24 hours. After the S IT was read, 20 ul from each well was spread onto sheep blood agar plates containing 50 Units per ml penicillinase and incubated to determine the

SBT, which was taken as the highest dilution that killed 99.9% or more of the initial inoculum.

Production of bacterial endocarditis . Male new Zealand White rabbits weighing 1. 5 - 2. 5 kg were anesthetized with 1 mg/kg ketamine plus 1 mg/kg xylazine. Through an incision in the neck a polyethylene catheter of external diameter 0. 8 mm and internal diameter 0. 4 mm was passed down the right carotid artery toward the aortic valve. It was fixed in place with the distal end of the catheter located at the aortic valve or in the left ventricle by means of two ligatures around the carotid artery. The other end of the catheter was sea­led with a heated spatula and the skin was closed with silk sutures. After 24 hours antibiotics were injected intramuscularly ; 0. 5 h later rabbits were in­oculated with approximately 107 CFU of the test organisms via an ear vein. These inocula were prepared from a 18 h culture in tryptic soy broth (Difeo ) as fol­lows: after centrifugation, the streptococci were washed three times with 0. 015

M phosphate buffer pH 7. 40 in isotonic saline (PBS} , resuspended at an optical density of 1.0 at 540 nm, and diluted in PBS for intravenous injection. The ac­tual inoculum size was measured by spreading 0. 1 ml portions from serial hun­dred-fold dilutions in PBS onto blood agar plates for incubation and counting.

Administration of Antibiotics. The antibiotic regimen used in rabbit expe­riments was a single dose of PPG 250 mg/kg (Wyeth Laboratories, Inc. , Philadel­phia, PA) injected into a thigh muscle 30 minutes before inoculation of orga­nisms. In one group of animals, streptomycin 15 mg/kg (Eli Lilly, Indianapolis, IN ) was given by injection into the other thigh immediately before injection of PPG.

66

Evaluation of infection. Forty-eight hours after injection of streptococci the rabbits were sacrificed by intravenous injection of pentobarbitone. The hearts were removed w ith antisepti c precautions and the vegetations were exci ­sed, homogenized in glass tissue grinders and suspended in 1. 0 ml of trypti c soy broth (Di feo). 0. 1 ml of this homogenate was removed to make serial ten­fold dilutions ; the remainder and 0. 1 ml portions of the dilutions were i ncor­porated i nto blood agar plates that contai ned 50 Uni ts per ml of penicillinase (Whatman Bi ochemi cals Ltd. , Maidstone, Kent) and incubated for 48 h at 37°c. Then the number of colonies was counted . If no growth occurred in the plate containing the undiluted homogenate (which represented approxi mately 90% of the origi nal vegetation) , the animal was counted as steri le. Fisher 's exact test was used to calculate the significance of di fferences between groups.

RESULTS

Antibiotic susceptibilities. The MICs and MBCs of penici llin and strepto­mycin for the four strains are shown in Table 1. Strains were defi ned as sen­sitive to penicillin i f the MIC was lower than 0.2 ug/ml and tolerant to peni­c illin i f the MBC/MIC ratio was 32 or greater. By these criteria all four strains were sensitive to penicillin ; strain 1 was non-tolerant, strains 3 and 4 were tolerant, and strain 2 was intermedi ate in respect of tolerance. The MIC of streptomycin for the four strains ranged from 2 to 16 ug/ml.

Time-ki ll curves and bacterici dal synergy. In penicillin, the count of strain I fell from 106 to 101 (greater than 99. 99%) CFU per ml over 48 hours (Fig. la). The ti me-kill curve for strai � 2 in 1. 0 ug per ml peni cillin (10 t imes hi gher than its MIC) showed a reduction in CFU 's at 24 h which was just greater than 99. 9%. In the presence of 0. 5 ug per ml peni cillin the observed reduction in CFU was less than 99. 9% over 24 h. For strains 3 and 4, the num­ber of organisms fell by less than 99. 9% over 48 hours in 1. 0 ug per ml of pe­nicillin (Fi g. lb).

Streptomycin caused an initial reduction in count, but the organi sms began to multiply agai n after 4 hours. For the tolerant strain 4, 5 x 103 CFU remai­ned ali ve after 48 h i n penicillin, but penicillin plus streptomycin sterilized the culture withi n 24 hours (Fi g. lb). Similar results were obtained with strains 2 and 3.

Serum penicillin concentrations. The concentration of peni cillin in rabbit

67

CX)

Table 1. MIC and MBC of penicilli n and streptomycin for the four strains of Streptococcus sanguis I I used in these experiments.

MIC µg/ml MBC µg/ml MBC/MIC M IC MBC mean (range) mean (range) ratio µg/ml µg/ml

Strain 1 0.01 (0. 006-0. 03) 0. 02 (0. 006-0. 3) 1: 2 8 8 2 0. 01 (0. 06 -0. 15) 2 (1. 2-2. 4) X 1:20 16 16 3 0. 006 (0. 006) 800 (500-2000)0 1 : 105 2 8 4 0.006 (0. 006) 32,000 ( 10 ,000-40 , 000) 8 1: 107 8 8

· 7 determinations ; x 8 determinations ; 0 6 determinations ; 0 13 determinations

8

�1---o 7

6 \

_ 5

u. 4 u

3

2 1S:

:: � 0 4 24 48 0 4 24 48

HOURS HOURS

la lb

Figure 1. Time-kill curves for penicillin and streptomycin , alone and in combination for Streptococcus sanguis. la: curves for non-tolerant strain 1. lb : curves for tqlerant strain 4. o---o control ........... penicillin 1.0 µg/ml A------£ streptomycin 10 µg/ml ---- penicillin 0.5 µg/ml and streptomycin 5 µg/ml

69

Tab l e 2 . Penicillin concentrations at various i nterva ls after intramuscular administration of procaine penicil lin G 250 mg/kg to rabbits .

Hours after Number of Serum penicil lin inj ection sampl es concentration µg/m l

mean + SD

0 . 5 5 9 .3 + 3. 2 1 5 13. 2 + 4. 8 3 4 5 . 9 + 2. 8 6 3 3.4 + 0 .5

70

Table 3 . Reciprocals of geometric mean inhibitory (SIT) and bactericidal titers (SBT) in sera sampl ed from 3 rabbits 0.5 h after injection of procaine penicillin (PPG) or PPG plus streptomycin (SM) for strains 1-4 of Streptococcus sanguis .!..!_.

SIT SBT Strain PPG PPG + SM PPG PPG + SM

1 32 128 21 53 2 53 213 2 85 3 213 192 2 149 4 107 96 8 96

1 reciprocal of geometric mean titer

71

Table 4. I ncidence of infection in rabbits after attempted prophylaxis with procaine penicillin G (PPG) or PPG plus streptomycin (SM).

Strain No Treatment PPG PPG + SM

1 8/8 ( 100%) 2/22 ( 9%) o not done 2 4/4 ( 100%) 5/18 (27%) 0/7 (0%) 3 8/8 (100%) 12/30 (40%) 0 0/7 (0%) 4 10/10 ( 100% ) 32/40 (80% ) 0 0/20 (0%)

Total 30/30 (100%) 51/110 (47%)

0 The difference between strain 1 and 3 p = 0. 02

0/34

The difference between strain 1 and 4 p = 5 x 10-8

The difference between strain 3 and 4 p = 0.001

(0%)

The difference between strain 1 and 2 N.S. (Fisher ' s exact test)

72

5 //r:0,94

/ e 4 /

/

3 /

/ - / u 2 CJ) /

� 1 / -

/

0 ,... -1 //20

0 40 60 80 100 /.

-2 / 0 0 0

-3 I NFECTION RATE (°lo)

Figure 2. Relation between percentage of rabbits with infected vegetations despite attempted pro­phyl axis and the MBCs (•) for penicil lin of the strains of Streptococcus sanguis II ino­culated. There was no rel ation to MICs (o) .

73

sera peaked at 13. 2 ± 4 . 8 ug/ml one hour after i ntramuscular i njection of 250 mg/ml PPG and decreased to 3. 4 ± 0. 5 ug/ml six hours after i njection (Table 2).

Serum inhibitory and bacterici dal ti ters. The geometri c mean SIT of sera from three rabbits 30 minutes after injecti on of PPG for strain 1 was 1:32, and the geometri c mean bacteri cidal titer was 1: 21 (Table 3) . These sera were actu­all y more inhibitory for the two fully-tolerant strai ns than for the non-tol e­rant strain, by two- or three-fold. The inhibi tory activity of sera sampled 30 minutes after administrati on of PPG plus SM was simi lar to or higher than that after PPG alone against all strains. Serum bactericidal activity was l ow or ab­sent against the tolerant strai ns after treatment with PPG alone, but after treatment with PPG and SM sera showed good bactericidal activity against these strains (Table 3).

Prophylaxis of experi mental endocardit is. The infection rate in 30 untrea­ted control rabbits was 100% (Tabl e 4). The success of a si ngle dose of PPG in preventi on of endocarditis varied stri k ingly between the four strains. Strepto­coccal endocardi tis developed despite attempted prophylaxis i n 9, 27, 40 and 80% of rabbits i njected with strains 1,2,3 and 4 respectively. The differences between strains 1 and 3, strai ns 1 and 4 and strains 3 and 4 were signif icant at the 0. 05 level or less by Fi sher 's exact test. There was a close relation­ship between the rate of infection and MBCs (r = 0,94), but no relationship with MIGs (Fi gure 2).

None of 34 animals i njected wi th strai ns 2, 3 or 4 developed streptococcal endocarditis after prophyl axis with combi ned PPG plus streptomycin (Table 4).

DISCUSSION

The phenomenon of bacteri al tolerance to antibiotic action has been widely discussed during the last five years. Sabath and colleagues 1 introduced the term "tol erant" to characterize strai ns of Staphyl ococcus aureus that were i n­hibi ted by low concentrati ons of penicillin, but were resistant to its lethal action. Rajashekarajah and colleagues 7 reported that some pati ents with endo­carditis and bacteremi a due to tolerant strains of Staphyl ococcus aureus see­med to respond less favorably to therapy than patients infected with non-tol e­rant strains. Nevertheless , the clinical s ignificance of tolerance remai ns controversial 8

• Goldman and Petersdorf 9 found no difference between the res­ponse of tolerant and non-tolerant strains of Staphylococcus aureus to

74

methicillin in prevention and treatment of experimental endocarditis. The phe­nomenon of in vitro tolerance has also been reported among viridans streptococ­ci 2 , 3

, but its significance for in vivo therapy, if any, has not been esta­blished.

Demonstration of tolerance in vitro is highly independent upon the condi­tions used in the test. Thus, changes in media or inoculum size can determine whether a strain appears to be tolerant or non-tolerant 1 0 • For the streptococ­ci used in this study, consistent values for MBCs could only be obtained by including beta-lactamase in the medium used for subculture. Even the low con­centration of peAicillin due to carry-over in the volume used for subculture can result in inhibition of tolerant strains 1 1 •

Dextran produced by some viridans streptococci may promote adherence to endocardium 1 2 • To control for this factor as far as possible, we used only dextran-positive strains in this study. The incidence of endocarditis in non­treated rabbits was 100 percent for all four strains, indicating that they all were fully capable of adhering to endocardium.

Attempted penicillin prophylaxis for experimental S. sanguis II endocar­ditis was only partially effective for tolerant strains despite the presence of high serum inhibitory activity at the time when the test organisms were in­troduced. Although the mean serum penicillin concentrations after one and six hours were 13. 2 ug/ml and 3. 4 ug/ml, respectively, substantial serum bacteri­cidal activity was present only against the penicillin-sensitive strain 1. At­tempted prophylaxis was much more effective for this strain . Although serum bactericidal activity against the tolerant strains was low or non-existent, the incidence of endocarditis despite attempted prophylaxis for these strains va­ried. The frequency of prevention failure was directly related to the MBCs, but not to the M ICs of these strains.

One half hour after administration of PPG serum penicillin concentrations were much lower than the MBCs, and there was a corresponding lack of serum bac­tericidal activity for strains 2 and 3. Nevertheless, endocarditis due to these strains occurred significantly less frequently in rabbits treated with PPG than in the control groups. Recently, Bernard and colleagues 13 have argued on in­direct evidence that vancomycin prophylaxis of experimental endocarditis due to a vancomycin-tol erant strain of S. sanguis may succeed because of inhibition of adherence rather than bacterial killing. Such a mechanism could be involved in the case of penicillin prophylaxis of penicillin-tolerant S. sanguis I I

75

strains: although attempted penicillin prophylaxis often failed in rabbits chal­lenged with tolerant strains 3 and 4, it is important to note that the frequen­cy of infection was not significantly different from untreated controls. There­fore, mechanisms other than complete bactericidal action may be operative in prevention of endocarditis by penicillin in this system.

Three decades ago Hunter 1 4 and later others 5 ' 1 5 - 1 8 showed that the com­bination of penicillin plus streptomycin acted synergistically against penicil­lin-sensitive viridans streptococci. More recently Parillo et al. 1 9 showed that the same was true for two penicillin-resistant strains. Our time-kill curves for the tolerant strains grown in the presence of either penicillin plus streptomy­cin showed a much greater reduction in CFU ' s after 24 h than found in the pre­sence of penicillin or streptomycin alone. Thus, the combination of penicillin plus streptomycin acts synergistically against tolerant as well as non-tolerant strains of viridans streptococci, whether or not they are inhibited by low con­centrations of penicillin.

In previous in vivo studies penicillin combined with streptomycin was very effective in prevention of both right and left-sided experimental endocarditis due to a penicillin-sensitive strain of S. sanguis II. In this study the combi­nation of penicillin plus streptomycin provided better bactericidal activity than penicillin alone against the tolerant strains and succesfully prevented en­docarditis due to these strains.

We conclude that penicillin prophylaxis is less effective against tolerant than non-tolerant strains of viridans streptococci, even when they are fully sen­sitive to penicillin by MIC testing. For these organisms, penicillin ' s lack of in v i vo efficacy resembles that of bacteriostatic antibiotics 1 5

•1 7

, Furthermore, in vitro tolerance as determined by MICs, MBCs and killing kinetics, is reflec­ted by the results of studies on serum inhibitory and bactericidal activity af­ter antibiotic administration. Since tolerant S. viridans strains are frequent­ly present in the sulcus gingivalis of children 3

, which is the source of bac­teremia after dental extraction, attempted prophylaxis with penicillin G may carry some risk of failure. In a recent survey, Durack et al found 38 examples of apparent failure of penicillin prophylaxis for streptococcal endocarditis 2 0 •

This indicates that failure of penicillin alone is not rare in clinical practi­ce. Because the combination of procaine penicillin G plus streptomycin is high­ly effective in vitro and in vivo against all strains, this combination should provide a wider margin of safety for prophylaxis of streptococcal endocarditis.

76

REFERENCES

1. Sabath L D, Wheeler N, Laverdiere M, Bl azevic D, Wil kinson B J, 1977. A new type of penicil l in resistance of Staphyl ococcus aureus. Lancet i: 443-447.

2. Horne D, and Tomasz A, 1977. Tol erant response of Streptococcus sanguis to beta-l actams and other cel l wal l inhibitors. Antimicrob. Agents Chemo­ther. 11: 888-896.

3. Hol l oway Y, Dankert J, Hess J, 1980. Penicil lin tol erance and bacterial endocarditis. Lancet i: 589.

4. Hol l oway Y, Schaareman M, Dankert J, 1979. Identification of viridans strep­tococci on the minitek miniaturized differentiation system. J. Cl in. Patho l . 32: 1168-1173.

5. Wolfe J C, Johnson W D, Jr, 1974. Penicil l in-sensitive streptococcal endo­carditis. In vitro and cl inical observations on penicil l in-streptomycin therapy. Ann. Intern. Med. 81: 178-181.

6. Bennett J V, Brodie J L, Benner E J, Kirby W M M, 1966. Simplified accurate method for antibiotic assay of cl inical specimens. Appl . Microbial . 14: 170-177.

7. Rajashekarajah M D, Rice T, Rao V S, March D, Ramakrishna B, Kal l ick C A, 1980. Cl inical significance of tol erant strains of Staphyl ococcus aureus in patients with endocarditis. Ann. Int. Med. 93: 796-801.

8. Kaye D, 1980. The cl inical significance of tol erance of Staphylococcus aureus. Ann. Int. Med. 93: 924-926.

9. Goldman P L and Petersdorf R G, 1979. Significance of methicil lin tolerance in experimental staphylococcal endocarditis. J. Antimicrob. Agents and Chemother. 15: 802-806.

10. Norden C W, Keleti E, 1981. Antibiotic tol erance in strains of Staphyl ococcus aureus. J. Antimicrobial . Chemother. 7: 599-605.

77

11. Hess J, Holloway Y, Dankert J. Detection of penicillin tolerance in viri­dans streptococci isolated from the oral flora of children with cardiac disease and post-extraction bacteremia under penicillin prophylaxis. Submitted for publication.

12. Ramirez-Ronda C H, 1978. Adherence of glucan-positive and glucan-negative streptococcal strains to normal and damaged heart valves. J. Clin. Invest. 62: 805-814.

13. Bernard J P , Francioli P, Glauser M P, 1981. Vancomycin prophylaxis of ex­perimental Streptococcus sanguis endocarditis. Inhibition of bacterial adhe­rence rather than bacterial killing. J. Clin. Invest. 68: 1113-1116.

14 . Hunter T H, 1952. The treatment of some bacterial infections of the heart and pericardium. Bull N.Y. Acad. Med. 28: 2 13-228.

15. Durack D T, Petersdorf R G, 1973. Chemotherapy of experimental streptococcal endocarditis. I. Comparison of commonly recommended prophylactic regimens. J. Clin. Invest. 52: 592-598.

16. Durack D T, Pelletier L L, Petersdorf R G, 1974. Chemotherapy of experimen­tal streptococcal endocarditis. I I. Synergism between penicillin and strep­tomycin against penicillin-sensitive streptococci. J. Clin. Invest. 53: 829-833.

17. Pelletier L L, Durack D T, Petersdorf R G, 1975. Chemotherapy · of experimen­tal Streptococcus viridans endocarditis. 129: 572-576.

18. Sande M A, Irvin R G, 1974. Penicillin- aminoglycoside synergy in experi­mental Streptococcus viridans endocarditis. 129 : 572-576.

19. Parillo J E, Borst G C, Manux M N, Jannini P, Klepmner M S, Moellering R C, Anderson S E, 1979. Endocarditis due to resistant viridans streptococci du­ring an oral penicillin chemo-prophylaxis. N. Engl. J. Med. 300: 296-300.

20. Durack D T, Kaplan E L, Bisno A L, 1982. Apparent failures of endocarditis prophylaxis: Analysis of 51 cased submitted to a national registry. Sub­mitted for publication.

78

ACKNOWLEDGEMENTS

We thank the Netherlands Organization for the Advancement of Pure Research (Z.W.O. ), the Dutch Heart Foundation and the Jan Kornelis de Cock Stichting for financial support. We also thank Willem Joldersma and Deborah Edwards for tech­nical assistance, and Yvette Holloway and John Perfect for stimulating contri­butions to these experiments.

79

CHAPTER VI

GENERAL DISCUSSION

GENERAL DISCUSSION

Viridans streptococci are recovered from blood cultures of approximately 50% of children with bacterial endocarditis 1

• This percentage has not changed significantly during the past four decades. In about one third of these cases the origin appears to have been the upper respiratory tract and oral cavity 2

•

Although a diversity of microbial species colonize the oral cavity, and occur in the blood after dental extraction in patients without prophylaxis 3

, and under prophylaxis 4 , it is striking that viridans streptococci seem to be more specifically virulent for the endocardium than other species. Studies on bacterial adherence to endocardial tissue showed that viridans streptococ­cal strains are much more adherent than for example Escherichia coli and

Klebsiella pneumoniae strains 5• In addition, there are also differences in

the ability to adhere within the viridans streptococcus group. The ability of Streptococcus mutans to adhere is far greater than that of Streptococcus milleri 6 • This difference appears to be related to the production of extra­cellular dextran, a polysaccharide produced by some bacterial strains. There­fore the occurrence of such strains in blood after dental manipulation pro­bably poses a greater risk of inducing endocarditis in patients with cardiac disease, than strains that do not produce dextran. This riks will further in­crease if dextran producing strains also appear to be resistant to the lethal action of the antibiotic given as endocarditis prophylaxis. We demonstrated that viridans streptococcal strains which are both dextran- producers and re­sistant or tolerant to the lethal action of penicillin are frequently present on the gingival sulcus and in blood after dental extraction under penicillin cover in children at risk. As viridans streptococci are generally believed to be always sensitive to penicillin , these children had received penicil­lin prophylaxis according to the general recommendations for prevention of viridans streptococcal endocarditis 7

• In a recent study on registration of "proven bacterial endocarditis prophylactic failures", of the 25 patients who developed bacterial endocarditis despite attempted prophylaxis, the ma­jority had viridans streptococcal endocarditis after attempted penicillin prophylaxis 8

• In our study, however, none of the children developed endo­carditis. It is probable, therefore, that other mechanisms also prevent the onset of endocarditis due to bacteria, resistant to penicillin. It is known that blood itself has some bactericidal effect upon microorganisms, an effect that can be potentiated by penicilllin, notwithstanding the fact that the

81

involved bacteria themselves are not sensitive to penicillin 9• Furthermore,

it has rece11tly been reported that prevention by vancomycin of experimental endocarditis due to Streptococcus sanguis appeared to be the result of impair­ment of adherence rather than bacterial killing 9

• Thirdly, some bacteria, ex­posed to antibiotic concentrations far below their MICs may be less adherent to endocardial tissue 1 1 • These last two mechanisms may be based upon the same feature. The real significance of these mechanisms is not fully under­stood and further research is indicated.

Conclusions on the efficacy of antibiotic prophylaxis for bacterial endo­carditis in patients undergoing dental extractions based upon the incidence of post-extraction-bacteremia can be argued. In case of attempted penicillin prophylaxis in dental extraction, bacteria can enter the bloodstream and can be still alive shortly after extraction, but will probably be killed a little later. However, if penicillin has no bactericidal action against these bacte­ria, they may persist and may induce endocarditis. In a study on 32 children who received penicillin pTus streptomycin as attempted prophylaxis, 5 (16%) had post-extraction bacteremia due to viridans streptococci despite the fact that in timed killing experiments these microorganisms were rapidly killed by the synergistic action of penicillin and streptomycin. This indicates that conclusions on the efficacy of antibiotic prophylaxis are only justified if the incidence of post-extraction bacteremia is related to lethal activity of the antibiotics used as attempted prophylaxis against the bacteria isola­ted. In view of this, the presence of penicillin resistant viridans strepto­coccal strains on the sulcus gingivalis before dental extraction and the oc­currence in blood after dental extraction under penicillin prophylaxis in children at risk for bacterial endocarditis is alarming 1 2 • A finding that needs perhaps even more consideration when evaluating the efficacy of penicil­lin prophylaxis is that of the presence of penicillin tolerant strains of vi­ridans streptococci in the oral cavity 1 3 • 1 4 •

The clinical importance of penicillin tolerant viridans streptococci in the onset of endocarditis is not clear. In the rabbit study we demonstrated that endocarditis was evoked by penicillin tolerant strains in the same per­centage as by penicillin sensitive strains 1 5• The reason why penicillin to­lerant strains are only seldom detected in patients with endocarditis may be that susceptibility testing in routine diagnostic microbiological laboratories is performed with disc diffusion tests or MIC determination. On disc diffusion

82

tests penicillin tolerant strains show zones of growth inhibition comparable to penicillin sensitive strains 1 6

• In the broth dilution test only MIGs in combination with MBCs will reveal the presence of penicillin tolerant strains, although addition of penicillinase to prevent carry-over of penicillin to the subculture medium for MBC determination is necessary to give consistent results 1 7

Therefore strains tolerant to penicillin will be frequently classified as sen­sitive. The reason that patients with endocarditis due to tolerant strains do not frequently relapse, may be that antibiotic therapy of Streptococcus viri­dans endocarditis generally includes both penicillin and streptomycin 1 8 , a combination demonstrated to have a rapid killing effect because of their sy­nergistic action upon penicillin resistant and penicillin tolerant strains 1 4 •

Therefore therapy will be effective in case of tolerant strains classified as penicillin sensitive strains. However, this discourages recommendations of single penicillin treatment for penicillin-sensitive Streptococcus viridans endocarditis 1 9

, when penicillin susceptibility is based upon routine disk diffusion test and/or on MIC only.

Because penicillin is still recommended as the drug of choice in antibi­otic prophylaxis for Streptococcus viridans endocarditis 7 in this study we investigated the bactericidal effect of penicillin against viridans strepto­cocci. In timed killing experiments penicillin tolerant viridans streptococcal strains exposed to penicillin in concentrations far above their MIGs were not killed after 48 hours at all, in contrast with penicillin sensitive strains 1 4 •

The onset of experimental endocarditis due to penicillin strains was frequent­ly not prevented in rabbits under penicillin prophylaxis 1 5 • Therefore peni­cillin prophylaxis may carry a substantial failure risk. The most recent re­commendations of the American Heart Association supply another prophylactic regimen, namely the combination of penicillin plus streptomycin, especially for patients "at high risk" 7

• Although 5 of the 32 children given the com­bination of penicillin plus streptomycin as attempted prophylaxis had post­extraction bacteremia, the in vitro findings showed that penicillin resistant and penicillin tolerant viridans streptococci were killed rapidly after expo­sure to both antibiotics. In addition, the onset of endocarditis due to peni­cillin tolerant viridans streptococcal strains in the experimental rabbit stu­dy was prevented in all animals by penicillin plus streptomycin administration.

In conclusion, post-dental extraction bacteremia due to viridans strepto­cocci in children under penicillin prophylaxis appears to be mainly caused by

83

penicillin tolerant strains and can be predicted by the presence of such strains in the oral cavity. Since penicillin acts as a bacteriostatic agent on such strains, we strongly recommend to consider children with cardiac dis­ease at risk to develop viridans streptococcal endocarditis {f. e. after den­tal extraction ) as patients "at high risk'' which means appliance of penicil­lin plus streptomycin prophylaxis. Further research will be necessary to eva­luate the efficacy of other antibiotics in the prevention of viridans strepto­coccal endocarditis. Because bacterial adherence to endocardial tissue 5 , 6

and serum bactericidal activity probably 9 also play an important role in the prevention of endocarditis the influence of other antibiotics upon these me­chanisms need to be studied as well.

REFERENCES

1 . Johnson D H, Rosenthal A, Nadas A S: A forty year review of bacterial endocarditis in infancy and childhood. Circulation 51 : 58, 1975.

2. Kaplan E L: Infective endocarditis in the pediatric age group. An Overview. In: Infective endocarditis, edited by E. L. Kaplan and A.V. Taranta, p. 51, Dallas, Am. Heart Association, 1977.

3. Crowley M C, Beppler W A, Ramfjord S P: The bacterial flora of the normal gingival sulcus. J. Periodont. 35: 502, 1964.

4. Hess J, Holloway Y, Dankert J: The incidence of post-extraction bacteremia under penicillin cover in children -with cardiac disease. Pediatrics, accep­ted for publication.

5. Holmes K R, Ramirez-Ronda C H: Adherence of bacteria to the endothelium of heart valves. In: Infective endocarditis edited by E. L. Kaplan and A.V. Taranta, p. 12, Dallas, Am. Heart Association, 1977.

6. Ramirez-Ronda, C H: Adherence of glucan-positive and glucan-negative strep­tococcal strains to normal and damaged valves. J. Cl in. Invest. 62 : 805, 1978.

84

7. Committee on Prevention of Rheumatic Fever and Bacterial Endocarditis of the American Heart Association: Prevention of bacterial endocarditis, Cir­culation, 56: 139A, 1977.

8. Durack D T, Kaplan E L, Bisno A L: Twenty-five cases of apparent endocar­ditis prophylaxis failure: results of a nati onal survey. Clin. Res. 29: 384A, 1981.

9. Lorian V: Effects of subminimum inhibitory concentrations of antibiotics on bacteremia. In: Antibiotics Laboratory Medicine, edited by V. Lorian, p. 342, Baltimore, London, Williams and Wilkins, 1980.

10. Bernard J P, Francioli P, Glauser M P: Vancomycin prophylaxis of experi­mental Streptococcus sanguis endocarditis. J. Clin. Invest. 68: 1113, 1981.

11. Scheld W M, Zak 0, Vosbeck K, Kloetzlen L, Sande M A: Effect of subinhi­bitory antibiotic concentrations ( Sub-MICs) on bacterial adhesion ( BA) to fibrin platelet matrices in vitro. In: 12th International Congress of Chemotherapy, Florence, 1981, abstract 643.

12. Hess J, Holloway Y, Dankert J : Penicillin prophylaxis in children with

cardiac disease: post-extraction bacteremia and penicillin resistant vi­ridans streptococci. J. Inf. Dis. accepted for publication.

13. Holloway Y, Dankert J, Hess J : Penicillin tolerant and bacterial endocar­ditis. Lancet , i: 589, 1980.

14. Hess J, Holloway Y, Dankert J: Detection of penicillin tolerance in viri­dans streptococci isolated from the oral flora of children with cardiac disease and post-extraction bacteremia under penicillin prophylaxis. Sub­mitted for publication.

15. Hess J, Dankert J, Durack D: Significance of penicillin tolerance: Failu­re of penicillin in prevention of experimental Streptococcus sanguis II endocarditis. Submitted for publication.

85

16. Dankert J, Hol loway Y, Joldersma W, Hess J: Screening for penicillin tol erance in viridans streptococci by a simple disc method. Submitted for publication.

17. Dankert J, Holloway Y, Joldersma W, Hess J: Importance of penicillinase in subculture medium for detection of penicill in tolerant viridans strep­tococci . Submitted for publication.

18. Bryant R E, Kimbrough R C: Treatment of infective endocarditis. In: Infec­tive endocarditis, edited by S. H. Rahimtoola, p. 327, New York, Grune and Stratton, 1978.

19. Karchmer A W: Single antibiotic therapy for streptococcal endocarditis. JAMA, 241: 1801, 1979.

SUMMARY AND CONCLUSIONS

Viridans streptococcal bacteremia occurring after dental extraction may result in viridans streptococcal endocarditis in patients with cardiac disease. This bacteremia originates from the bacterial flora in the oral cavity. To reduce the risk of developing viridans streptococcal endocarditis it is recom­mended that susceptible patients undergoing dental extraction should receive prophylactic antibiotics. Because viridans streptococcal strains are generally believed to be always sensitive to penicillin, this antibiotic is used most often as attempted prophylaxis for patients undergoing dental extraction. How­ever, the incidence of viridans streptococcal endocarditis in children has not decreased significantly since penicillin prophylaxis has been employed. To assess the efficacy of parenteral penicillin prophylaxis, based upon the re­commendations of the American Heart Association, the incidence of bacteremia 5 minutes after dental extraction was determined in 82 children with cardiac disease under penicillin prophylaxis (Chapter II). Post-extraction bacteremia occurred in 17 (21%) children , from whom 32 different bacteria were isolated.

Susceptibility testing by disc method showed that 24 strains were penicillin sensitive and 8 penicillin resistant. Nineteen (59%) of the isolated strains belonged to the viridans streptococcus group. Of the children with post-extrac­tion bacteremia 4 had viridans streptococcal bacteremia due to penicillin re­sistant strains (Chapter III). Swabs of the sulcus gingivalis, the source of post-extraction bacteremia, taken prior to penicillin administration, showed that 39% of the children had penicillin resistant viridans streptococci at that site. There was no relationship between the presence of such strains on the gingival sulcus and the occurrence of post-extraction bacteremia due to such strains. Penicillin concentrations tietermined in blood obtained 5 min after extraction, did not differ in children with bacteremia due to penicil­lin sensitive or penicillin resistant microorganisms, nor between the concen­trations in children with and without bacteremia. Because bacterial strains tolerant to the lethal action of penicillin, characterized by a wide discre­pancy between MBC and MIC values, have been found to occur also within the

Streptococcus viridans group, we wanted to determine whether the presence of such strains on the sulcus gingivalis increased the risk of developing post­extraction bacteremia (Chapter IV). Of the 35 children included in this study 5 ( 14%) had viridans streptococcal bacteremia which was due to 7 different strains. Of these 6 were penicillin tolerant (MBC/MIC ratio � 32). Penicillin

87

tolerant viridans streptococcal strains were isolated from the gingival sul­cus of 14 (40%) children. Four of these developed post-extraction bacteremia due to tolerant strains. This incidence of bacteremia due to tolerant strains was significantly related to the presence of such strains on the gingival sulcus (p < 0. 05). The mean penicillin concentrations in blood 5 min after extraction of children with and without penicillin tolerant strains on the gingival sulcus did not differ significantly. To obtain more insight into the phenomenon of penicillin tolerance in viridans streptococci, the influence of the bacterial growth phase, different media and the addition of penicillinase to the subculture medium for MBC determination on MBC/MIC ratio was studied. Bacterial growth phase had no effect on MICs but mean MBCs were higher for or­ganisms in the logarithmic growth phase than for those in the stationary growth phase. Comparison of MICs and MBCs determined in five different media did not reveal significant differences for nontolerant or tolerant strains. Timed kil­ling kinetics showed that the reduction of CFUs in tolerant strains after ex­posure to penicillin in a concentration of 65 to 100 times the respective M ICs was far slower than in nontolerant strains. There was no difference be­tween organisms tested in the logarithmic growth phase or the stationary growth phase. Addition of penicillinase to the subculture medium for MBC determination resulted in an increase in the number of penicillin tolerant strains, as com­pared to subculture medium without penicillinase. To assess whether in vitro tolerance has any significance in vivo, we studied the ability of penicillin to prevent experimental endocarditis caused by one non-tolerant and three to­lerant strains of dextran-producing Streptococcus sanguis I I, in a rabbit mo­del {Chapter V). Timed killing kinetics showed that penicillin was not lethal for the three tolerant strains, but the combination of penicillin and strepto­mycin was synergistic, killing all three strains completely within 24 hours. Inhibitory activity against 4 strains was present in the serum of rabbits 30 minutes after intramuscular injection of procaine penicillin G 250 mg/kg, but bactericidal activity was only present against the nontolerant strain . Serum sampled from rabbits 30 minutes after injection of procaine penicillin G 250 mg/kg plus streptomycin 15 mg/kg showed a high bactericidal activity against all four strains. The incidence of endocarditis in control rabbits was 100% for each of the four strains inoculated. After prophylaxis with procaine pe­nicillin G the incidence of endocarditis was 56% for the tolerant strains ver­sus 9% for the nontolerant strain (p = 0. 0001). After injection of procaine

88

penicillin G plus streptomycin the incidence of endocarditis was 0% for all tolerant strains.

These results lead to the following conclusions: - Post-extraction bacteremia in children at risk of developing bacterial en­

docarditis is not prevented by parenteral penicillin prophylaxis. - The serum penicillin concentration at extraction is not the discriminating

factor in preventing bacteremia. - Penicillin resistant and tolerant viridans streptococcal strains are fre­

quently present in the oral cavity flora and in blood after dental extrac­tion.

- The presence of penicillin tolerant viridans streptococcal strains in the oral cavity of children undergoing dental extraction implies a significant higher risk for the occurrence of post-extraction bacteremia due to tolerant strains.

- Penicillin prophylaxis often cannot prevent experimental endocarditis caused by penicillin tolerant strains of Streptococcus viridans, whereas the com­bination of penicillin plus streptomycin is always effective.

- Children with cardiac disease at risk for viridans streptococcal endocardi­tis should be considered as patients "at high risk", which means, according to the recommendations of the American Heart Association, appliance of penicillin plus streptomycin prophylaxis.

89

SAMENVATTING EN CONCLUSIES

Streptococcus viridans bacterien, die tengevolge van tand- of kiesextrac­tie in het bloed geraken, kunnen bij patienten met een hartafwijking aanlei­ding geven tot het ontstaan van endocarditis. Deze bacterien zijn afkomstig uit de flora van de mondholte. Om het risico van endocarditis bij daarvoor vatbare patienten te verkleinen, wordt aanbevolen v66r een tand-of kiesextrac­tie een antibiotische prophylaxe toe te dienen. Hiervoor wordt meestal penicil­line gebruikt, omdat men in het algemeen aanneemt dat Streptococcus viridans altijd gevoelig is voor penicilline. Sinds echter deze penicilline prophylaxe wordt toegepast, is de frequentie van endocarditis tengevolge van Streptococcus viridans niet afgenomen. Ter evaluatie van de werkzaamheid van parenterale pe­nicilline prophylaxe, zoals aanbevolen door de American Heart Association, werd bij 82 kinderen met een hartafwijking onderzocht of er 5 minuten na een element­extractie onder penicilline prophylaxe bacterien in het bloed aanwezig waren (Hoofdstuk II). Dit bleek het geval te zijn bij 17 (21%) kinderen, bij wie in totaal 32 verschille�de bacteriestammen uit het bloed werden gekweekt. Hiervan waren 24 gevoelig voor penicilline en 8 resistent (disc methode). Negentien (59%) van de geisoleerde bacterien waren viridans streptococcen. Van de kinde­ren met positieve bloedkweken bleken in 4 gevallen penicilline resistente viri­dans streptococcen aanwezig te zijn (Hoofdstuk III). V66r de toediening van pe­nicilline werd een uitstrijkje gemaakt van de sulcus gingivalis, de bron van de post-extractie bacteriaemie. Bacteriologisch onderzoek hiervan toonde aan dat bij 39% van de kinderen penicilline resistente viridans streptococcen aanwezig waren. Er bleek overigens geen relatie te bestaan tussen de aanwezigheid van dergelijke stammen in de sulcus gingivalis en het voorkomen ervan in bloed na extractie. De penicilline concentraties die bepaald werden in bloed dat 5 minu­ten na de extractie werd afgenomen, was niet verschillend bij kinderen met pe­nicilline gevoelige- en penicilline resistente bacterien in het bloed. Er was evenmin een verschil in penicilline concentraties bij kinderen bij wie wel en bij wie geen bacterien uit het bloed geisoleerd werden. Aangezien aangetoond is dat ook binnen de Streptococcus viridans groep stammen kunnen voorkomen die to­lerant zijn voor de lethale werking van penicilline, hetgeen gekenmerkt wordt door een grote discrepantie tussen MBCs en MICs is verder nagegaan of de aanwe­zigheid van dergelijke stammen in de sulcus gingivalis het risico voor het krij­gen van een post-extractie bacteriaemie vergroot (Hoofdstuk IV). Hiervoor wer­den 35 kinderen onderzocht , van wie bij 5 (14%) in totaal 7 viridans streptoccen

90

uit het bloed na extractie geisoleerd werden. Hiervan bleken 6 penicilline to­lerant (MBG/MIG ratio � 32). Bij 14 (40%) van de kinderen werden penicilline tolerante viridans streptococcen uit de sulcus gingivalis geisoleerd, hetgeen significant correleerde met het voorkomen van tolerante stammen in het bloed na extractie (p < 0. 05). De gemiddelde penicilline concentraties in het bloed 5 minuten na extractie waren bij de kinderen met en zonder penicilline tole­rante stammen in de sulcus gingivalis niet verschillend.

Om meer inzicht te krijgen in de fenomenologie van de penicilline tole­rantie bij viridans streptococcen, werd het effect van de bacteriele groei­fase van verschillende groeimedia en van penicillinase toevoeging aan de me­dia voor de MBG bepalingen op de MBG/MIG ratio ' s onderzocht. De bacteriele groeifase bleek geen invloed te hebben op de MIGs, maar wel op de MBGs: de gemiddelde MBGs voor bacterien in hun logarithmische groeifase waren signifi­cant hoger dan voor bacterien in de stationaire groeifase. Er waren geen sig­nificante verschillen tussen de MIGs en MBGs die in 5 verschillende groeime­dia werden bepaald, voor zowel niet-tolerante als tolerante stammen. Aan de hand van afstervingscurven bleek dat na blootstelling aan penicilline in een concentratie die 65 tot 100 maal hoger was dan de MIGs de reductie van bacte­riekolonien van tolerante stammen veel geringer was dan bij niet-tolerante stammen. Er was geen verschil tussen de afstervingscurven van bacterien in hun logarithmische- en stationaire groeifase. Toevoeging van penicillinase aan het groeimedium voor MBG bepaling resulteerde vergeleken met groeimedia zonder pe­nicillinase, in een toename van het aantal penicilline tolerante stammen.

De betekenis van penicilline tolerante stammen in vivo werd onderzocht aan de hand van de doelmatigheid van penicilline toediening ter preventie van experimentele endocarditis bij konijnen veroorzaakt door een niet-tolerante en drie tolerante Streptococcus sanguis II stammen die allen dextraan vormden (Hoofdstuk V). Op grond van afstervingscurven bleek dat penicilline geen bac­tericide werking had op de 3 tolerante stammen, maar dat de combinatie van penicilline en streptomycine binnen 24 uur volledig bactericide was voor de­ze stammen. Een half uur na intramusculaire toediening van procaine penicil­line G 250 mg per kg lichaamsgewicht was er een inhibitoire activiteit tegen alle 4 stammen in het serum van konijnen aantoonbaar, maar er bleek alleen tegen de niet-tolerante stam bactericide activiteit te bestaan. Na toediening van dezelfde dosis procaine penicilline G gecombineerd met streptomycine, 15 mg per kg lichaamsgewicht, was er na 30 min. duidelijke bactericide

91

activiteit in het serum aanwezig tegen al l e 4 stammen. Bij de control edieren, die geen antibiotische prophyl axe kregen, trad in 100% van de geval l en na in­ocul atie met elk van de stammen endocarditis op. Na prophyl axe met procaine penicil l ine G was de frequentie van endocarditis tengevol ge van tol erante stammen 56% versus 9% tengevolge van de niet-tol erante stam (p = 0. 0001). Na prophyl axe met de combinatie procaine penicilline G en streptomycine was de endocarditis frequentie tengevol ge van de tol erante stammen 0%.

Uit de resul taten van dit onderzoek komen de vol gende conclusies naar voren: * Het optreden van post-extractie bacteriaemien bij kinderen die een verhoogd

risico hebben om bacteriel e endocarditis te krijgen, wordt door parenteral e penicil line prophyl axe niet voorkomen.

* De penicil l ine concentratie in het serum tijdens de extractie speel t geen bepal ende rol bij de preventie van de bacteriaemie.

* Bij kinderen zijn vaak penicil l ine resistente- en tolerante viridans strep­tococcen aanwezig in de mondfl ora en in bl oed na tand- of kiesextracties.

* Indien er bij kinderen penicil l ine tol erante viridans streptococcen in de mondfl ora aanwezig zijn, dan bestaat er bij tand- of kiesextracties een significant grater risico dat dergel ijke tol erante stammen in de bl oedbaan geraken.

* Penicil l ine prophyl axe kan het ontstaan van experimentel e endocarditis ten­gevolge van penicil l ine tol erante viridans streptococcen dikwijl s niet voor­komen; de combinatie penicil l ine en streptomycine is al tijd effectief.

* Kinderen die tengevol ge van een hartafwijking een verhoogde kans hebben op endocarditis tengevol ge van viridans streptococcen, dienen beschouwd te worden als patienten ' 'at high risk", hetgeen, volgens de aanbevel ingen van de American Heart Association, neerkomt op toediening van gecombineerde pe­nicil l ine en streptomycine prophylaxe.

92