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Journal of Dental Research An in Vitro Study of Antiseptics and Antibiotics Used in Endodontics

Olivier Pita Fajarda, Louis I. Grossman and Joan McShane J DENT RES 1956; 35; 656 DOI: 10.1177/00220345560350042401 The online version of this article can be found at:

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International and American Associations for Dental Research

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OLIVIER PITA FAJARDA, LOUIS I. GROSSMAN, AND JOAN McSHANE From the Faculty of Dentistry, University of Uruguay and the School of Dentistry, University of Pennsylvania, Philadelphia, Pa.

THE object of this study is to ascertain the action, in vitro, of some antiseptics and antibiotics which are used for the destruction of microorganisms in infected root canals. The laboratory procedures are divided into three parts: (1) preliminary tests to determine the efficacy of the agents in general and to establish whether they are adapted to clinical practice; (2) a qualitative study by the disc method; and (3) a quantitative study by the serial dilution method. In our laboratory, the flora of several infected root canals were found to be of a mixed type when several teeth were compared, but in most cases the root canals contained only one type of organism. The alpha streptococcus was found most frequently. In some cases 2 types of organisms were found, but 1 type exceeded the other in numbers. Mixed flora were nearly always observed in exposed root canals, or in the case of contamination during the course of endodontic treatment. A number of pulpless teeth with periapical areas were found to be sterile. With the advent of antibiotics, it has become necessary to study the bacterial flora, as most antibiotics are selective in their action. In our experiments, 4 types of microorganisms, Str. fecalis, Staph. albus, C. albicons, and E. coli, were used because they are most commonly found in infected root canals. After some preliminary tests, Antistine, Asterol, sodium caprylate, decyl ammonium sulfate, fradicin, neomycin, Perazil, rimocidin, Terramycin, and thiolutin were selected for the tests. Of these, Antistine, Asterol, sodium caprylate, decyl ammonium sulfate, and Perazil are not antibiotics. Jawetz, Gunnison, and Speck,' who studied the antagonism caused by Aureomycin, Terramycin, or chloramphenicol on the one hand, with penicillin on the other, showed that it occurs only if any of the former is given either with. or before the latter, but not if penicillin is administered first. This seems to show that it is not a mutual antagonism or incompatibility of the drugs, but rather an interference by chloramphenicol, Terramycin, or Aureomycin with the activity of penicillin. Interference of effect is observed not only among antibiotics but also when an antibiotic is used with some other agent, such as penicillin with boric acid, or with sulfadiazine under certain conditions.
Received for publication April 28, 1954. Revised Feb. 10, 1956. 656

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On the other hand, several studies have demonstrated a synergistic effect among certain antibiotics such as between penicillin and streptomycin, and penicillin and bacitracin, and also between penicillin and some sulfonamides.

Agents.-Antistine is a synthetic antihistaminic (2 phenyl-benzylaminomethyl-imidazoline hydrochloride) with relatively low toxicity. Asterol is the dihydrochloride of 2 methylamine-6-benzothiazole considered to have a high degree of activity against fungi, including C. albicans. Sodium caprylate, the sodium salt of caprylic acid, is relatively nontoxic. Fradicin is an antibiotic derived from Streptomyces fradiae. Neonmycin is an antibiotic having a cycloethanol type structure. Perazil is an antihistaminic which has a piperazine nucleus as a base instead of the ethylene group. Rimocidin is an antibiotic derived from Streptomyces rimosus. Terramycin is an antibiotic derived from Streptomyces rimosus. Thiolutin is an antibiotic derived from Streptomyces albus.

Method A.-Cultures were prepared from a 24-hour growth of organisms grown in brain heart infusion broth for Str. fecalis, S. albus, and E. coli, and in Sabouraud's medium for C. albicans. The agents to be tested for their antibacterial effect were prepared in dilutions of 5 and 50 mg. per cubic centimeter of solvent. In addition, dilutions of 100 mg. per cubic centimeter of solvent were made in those cases where the chemical agents were to be used against Str. fecalis or C. albicans because of the greater resistance of these microorganisms. In those cases where the antibiotic or chemical agent was not soluble in water, it was first dissolved in a little propylene glycol and then in water. The amount of propylene glycol used was negligible from an antibacterial standpoint as determined by test. Graduated pipettes were used for transferring solutions of the chemicals or dilutions of the microorganisms. To each test tube was transferred 0.1 c.c. of the chemical solution and 0.1 c.c. of the suspension of microorganisms. Fresh pipettes were used for each transfer. As controls, 40 tubes of culture medium were used containing the same amount of culture medium and the same amount of bacterial suspension, but without the addition of chemicals. The tubes were incubated at 370 C. except the Sabouraud 's medium, which was incubated at room temperature. In those cases where. there was no evidence of growth, subcultures were made. Results: All cultures with Str. fecalis were positive when the chemicals were used in a concentration of 5 mg. per cubic centimeter. In a 50 mg. per cubic centimeter concentration, Antistine, decyl ammonium sulfate, and Terramycin cultures were negative while all others were positive. All cultures were positive against C. albicans except thiolutin in a concentration of 100 mg. per cubic centimeter. Neomycin, Perazil, Terramycin, and thiolutin in both 5 mg. and 50 mg. concentrations were effective against Staph. atbus. All the rest showed growth. Against E. coli, Antistine, decyl ammonium sulfate, neomycin, Perazil,

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Terramycin, and thiolutin showed no growth in both 5 mg. and 50 mg. per cubic centimeter concentration. Cultures with Asterol, sodium caprylate, fradicin, and rimocidin were positive on examination. Method B. In order to study the action of the chemical objectively, agar pour-plates were made containing 10 per cent of sterile horse blood, and the agar was then inoculated with a suspension of organisms against which the chemical solution was to be tested. Discs of sterile filter paper which had been submerged in a solution of the chemical for 24 hours previously and then dried thoroughly were placed on the surface of the agar immediately after its inoculation with the microorganisms. The plates were then incubated for 48 hours at 370 C., except Sabouraud's medium, which was kept at room temperature. Zones of inhibition were apparent in most cases after 24 hours and observations were made both after 24 and 48 hours. In addition to the chemicals previously tested, aerosporin, endomycin, humulon, lupulon, hexahydrolupulon, aspergillic acid, and gliotoxin were added to the tests. Since one cannot determine microscopically whether a zone of inhibition is total or only partial, the zones were divided into small areas, working from the border of the disc to the periphery of the zone of inhibition. Samples were taken from each of these areas, transferred to culture medium, and incubated. Smears were then made for microscopic examination. Results: It was found that in addition to partial or total inhibition, some chemicals produced two well-defined zones of inhibition, e.g., neomycin against Str. fecaldis, gliotoxin against E. coli, aerosporin against E. coli, Perazil against staphylococcus, etc. In some cases a third zone appeared which may be considered a zone of stimulation. Sometimes this zone was situated between the zones of inhibition, at other times it was outside the zones of inhibition. Method C.-After determining the qualitative effect of the chemicals, an effort was made to determine their quantitative effect. Only those chemical agents which appeared to be relatively effective were included in these tests. The tests were carried out by the serial dilution method in the following manner: Seven tubes were each filled with 4.5 c.c. of brain heart infusion broth; 0.5 c.c. of a 24-hour culture of the selected organisms was added to the first tube, which was shaken to distribute the organisms evenly; 0.5 c.c. was then removed from the first tube and transferred to the second tube, and this procedure was continued until each of the tubes was inoculated. Then 0.5 of this inoculated medium was transferred to a tube containing molten agar, and a Petri plate was poured. This was repeated in triplicate for each tube for a total of 21 plates. Incubation was carried out at 20 C., 37 C., and 450 C. for 48 hours. It was found that 370 C. was the optimum temperature for growth, followed by 200 C. and 450 C., respectively. Little difference was found between brain heart infusion broth and Sabouraud's medium for growing C. albicais. The number of colonies which developed upon these plates was counted over a Frosh counter. Where the colonies were numerous, only onefifth of each dish was counted and multiplied by 5. An average of the 3 plates was taken in each case. In some cases, counting was impossible owing to the large number of colonies.

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1. A number of antibiotics and other chemical agents were tested against 4 types of microorganisms commonly found in infected root canals, Str. fecalis, Staph. atbus, E. coli, and C. atbicans. 2. Fradicin, thiolutin, sodium caprylate, and Asterol were effective against C. atbicans, the most effective being fradicin. 3. Of those agents tested, decyl ammonium sulfate and neomycin were most effective against E. coli. 4. Terramycin and Perazil were effective against both Staph. albus and Str. fecalis. 5. The possibility of combining 2 or more agents in order to produce a wider antibacterial spectrum than each has commends itself. Such factors as compatibility, stability, possible irritant effect, etc., would need to be taken into consideration. 6. The more commonly used antibiotics, such as penicillin, streptomycin, or bacitracin, were not included in these tests as their antimicrobial effect has been studied by others.
1. Jawetz, E., Gunnison, J. B., and Speck, R. S.: New England J. Med. 245: 966, 1951.

Antibiotic Synergism and Antagonism,

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