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Acquired Resistance of Bacteria to Antibiotic Compounds
ACQUIRED RESISTANCE OF BACTERIA TO ANTIBIOTIC COMPOUNDS John A . Kolmer, M.D., Philadelphia
resistance of bacteria to anti biotic compounds is of interest not only from the standpoint of its mech anism but also because it is of great prac tical importance in treatment. This is especially true of penicillin and strepto mycin, about which considerable infor mation has been gained. Whether or not bacteria may acquire resistance to tyrothricin, bacitracin, subtilin, polymyxin (aerosporin) and other antibiotics re mains to be more fully determined by experimental and clinical investigations but, theoretically at least, this appears to be possible. Acquired resistance of bacteria to anti biotic compounds may be fundamentally related to natural resistance in the sense that in any culture of a highly susceptible species there may occur mutants of in creased resistance.1 These not only may be responsible for failures in treatment but apparently are capable of acquiring additional resistance through exposure to a compound in vitro or in vivo. Suscep tibility and resistance, however, are usually only relative terms; resistance means that a particular strain of micro organism requires more antibiotic for its crippling or complete destruction than most other strains of the same species. Tests of the susceptibility of micro-
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T h is article has been authorized for publication by the Council on D ental Therapeutics. T h e opinions expressed are those of the author and _d o not neces sarily reflect the opinions of the Council. Professor of medicine, , School of ^ M edicine and School of Dentistry, Tem ple U niversity; director of the Research Institute of Cutaneous Medicine, Phila delphia. i. T h e term “ mutants” is employed throughout this article as a synonym for naturally resistant cells occurring in a bacterial population although the trans missible change is not always permanent.
organisms to penicillin or streptomycin, or both, frequently are required when treatment with antibiotics is considered. Unfortunately, as ordinarily conducted, they may be of but limited value because of technical conditions. Tests conducted in different laboratories with the same culture may give widely varying and con fusing results, for many factors influence tests of the susceptibility of bacteria to penicillin and especially to streptomy cin .2'6 It is particularly important that the tests be capable of detecting the presence of naturally resistant mutants. This re quires that susceptibility tests be con ducted with relatively large numbers of micro-organisms since there are usually but few naturally resistant mutants among millions of highly susceptible bac terial cells. Thus when tests are con ducted with single colonies of any micro organism, resistant mutants may readily escape detection. A mixed culture of nu merous colonies (the more the better), along with a large test dose or inoculum, is required to reduce this possible source of error to a minimum. Furthermore, as ordinarily conducted, susceptibility 2. Kolm er, J. A ^ Penicillin Therapy Including Strep tom ycin, Tyrothricin and O ther Antibiotic Therapy, Second edition. N ew Y o rk : D . Appleton-Century C om pany, Inc., 1947» P* 52 3. Lenert, Tulita, and Hobby, Gladys, Observations on the Action of Streptom ycin in V itro ( I) . Proc. Soc. Exper. Biol. & M ed. 65:235 (June) 1947. 4. Berkman, S.; H enry, R . J., and Housewright, R . D ., Studies on Streptomycin. I. Factors Influencing the Activity of Streptomycin. J. Bact. 53:567 (M a y )
1947*
5. Rhym er, I., and Wallace, G. I., T h e N ature of a Streptomycin In h ib ito r O ccurring in B rain Tissue and Plant Extracts. /. Bact. 54:521 (October) 1947. 6. Green, S. R .; Iverson, ^ W . P., and Waksman, S. A., Effect of O rga n ic Acids on Streptomycin A c tivity. Proc . Soc. E xper. Biol. & M ed. 6 7 :2 8 s (M a rc h ) 1948.
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tests show only the bacteriostatic activi ties of penicillin or streptomycin, whereas it is advisable to determine susceptibility to the bactericidal effects of these agents as a more reliable guide to dosage and administration. It is apparent that stand ardized tests for determining the sus ceptibility of micro-organisms to both the bacteriostatic and the bactericidal effects of penicillin and streptomycin are urgently required to facilitate the pro phylaxis and treatment of acute and chronic infections. M echanism of Antibacterial A ctivity
Knowledge of the way in which peni cillin and streptomycin destroy bacteria in vitro and in vivo is fundamental to an understanding of the mechanism of ac quired resistance. Although much infor mation and numerous theories have ac cumulated bearing on the antibacterial activity of penicillin, the precise mode of action is still unknown and even less is known about the mechanism by which streptomycin acts. Penicillin is believed to be more active against young cells and those about to divide than against resting cells. The same may be true of strepto mycin, with the suggestion that the anti bacterial activity of both depends to a large extent upon genesistasis. Further more, penicillin, at least, apparently in jures respiratory enzymes or other enzy matic systems, especially the hydrogenase system, causing inhibition of respiration and of endogenous oxygen uptake, and resulting in regressive biochemical changes.7’ 8 Gale and his colleagues,9’ 10 working with staphylococci, have also ob 7. Chain, E .j Duthie, E . S., and Callow, D ., Bac* tericidal and Bacteriolytic Action of Penicillin on the Staphylococcus. Lancet 1:652 (M a y 26) 1945* 8. Kram pitz, L . O ., and W erkm an, C. H ., O n the M o d e of Action of Penicillin. A rch. Biochem . 12:57 (January) 19479. Gale, E . F., and Taylor^ E ; S., Action o i Peni cillin in Preventing the Assim ilation o f G lutam ic A cid by Staphylococcus Aureus. N ature 158:676 (N ovem ber 9) 1946. 10. Gale, E . F., and Rodwell, A . W ., A m in o A cid Metabolism of Penicillin-resistant Staphylococci. /. Bact. 5 5 :161 (February) 1948.
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served that penicillin prevents the assim ilation of free glutamic acid, which pre cedes the loss of respiration and lysis of cells coincident with the loss of viability. Under the circumstances it appears that the antibacterial activity of peni cillin, and doubtless that of streptomycin, is due to interference with some essential metabolite or metabolic process of sus ceptible cells. This may result only in bacteriostasis, with the assumption that in vivo the crippled micro-organisms are finally disposed of by immunologic proc esses, particularly phagocytosis by the cells of the lymphoid-reticulo-endothelial system. On the other hand higher con centrations of these antibiotics are bac tericidal, causing complete destruction of the cells through lysis or disintegration. Curiously enough, Paine and Finland11 have recently reported that mutants have been found in cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus morganii which depended upon the presence of strepto mycin in culture media as an essential metabolite or growth factor; conse quently, they designated these mutants as “dependent” strains. Similar results have been reported by Miller 12 with two resistant mutants of meningococcus which required the presence of strepto mycin for growth not only in, vitro but likewise in vivo, since they did not pro duce infections in mice until the animals were treated with streptomycin. So far there have been no reports on the occurrence in vitro or in vivo of micro-organisms dependent on penicillin as an essential metabolite or growth fac tor. Nor have there been any reports of infections due to streptomycin-dependent mutants of micro-organisms, probably because media containing streptomycin have not been used for bacteriologic ex11. Paine, T . F., and Finland, Maxw ell, Strepto mycin-sensitive, -dependent, and -resistant Bacteria. Science 1 0 7 :143 (February 6) 1948. 12. M iller, C . P., Developm ent of Bacterial Resist ance to Antibiotics. J .A .M .A . 135:749 (Novem ber 22) 1947*
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The Journal of the American Denial Association
aminations of patients. Hall and Spink,13 however, have described a strain of Brucella abortus that acquired resistance to streptomycin during treatment and grew better in the presence of the com pound. It is likely that if and when de pendent mutants produce infection, the administration of streptomycin may not only fail therapeutically but actually pro duce an enhancement of the infection. Mechanism of A cq uired Resistance
Mutants naturally resistant to strepto mycin occur much more frequently than mutants naturally resistant to penicillin. This probably accounts for the fact that bacteria acquire resistance to streptomy cin much more frequently and more rap idly than to penicillin. Furthermore, nat urally resistant mutants occur more fre quently in some bacterial species than in others. For example, staphylococci as a group show more mutants naturally re sistant to penicillin and acquire resist ance to the compound both in vitro and in vivo much more readily and frequently than most other bacterial species. Al though Treponema pallidum is highly susceptible to penicillin, Tung and Fra zier14 were unable to increase the re sistance of the Reiter strain in vitro. Kolmer and Rule 15 observed no detect able increase of resistance of the virulent Nichols-Hough strain after three trans fers through rabbits treated with sub curative doses of penicillin. Gezon16 has observed that beta hemolytic strep tococci of group B acquire resistance to 13. H all. W . H., and Spink, W . W., In V itro Sen sitivity of Brucella to Streptomycin: Development of Resistance D u rin g Streptomycin Treatment. Proc. Soc. Exper. Biol. & M ed . 64:403 (A p ril) 1947. 14. Tung, T., and Frazier, C. N., Penicillin Sensi tivity an d M o rp h olo gy of the Reiter Strain of Trepo nema Pallidum After Cultivation in M e d ia Containing Penicillin. A m . J . S y p k .t Gonor. & Ven. Dis. 30:5205 (M a y ) 1946. 15. Kolm er, J. A., and Rule, A . M ., Acquired Resistance of Treponem a Pallidum to Penicillin. Proc. Soc. Exper. Biol. & M ed. 63:240 (Novem ber) 1946. 16. Gezon, H . M ., Antibiotic Studies on Beta H em o lytic Streptococci. I. Penicillin Resistance Acquired by G rou p A Organism s. Proc. Soc. Exper. Biol & M ed. 67 :2 08 (February) 1948.
penicillin in vitro much more frequently than streptococci of groups A and C. The exact mechanism of acquired re sistance of bacteria to antibiotics is still unknown. According to Demerec17’ 18 the mechanism consists in the elimination of susceptible micro-organisms, leaving nat urally resistant mutants which undergo further enhancement of resistance upon repeated exposures to the compound. Demerec has interpreted this as being due to the summation of the effects of several independent genetic factors for resistance which undergo consecutive mutation. In other words, according to this hypothesis, acquired resistance consists of the selec tion of naturally resistant mutants which undergo a further increase of resistance through “training” in vitro or in vivo. From this point of view the mechanism is to be interpreted as essentially one of bacterial genetics. Certainly considerable evidence has accumulated in support of the theory,19’ 20 although it must be ad mitted that it is difficult to determine whether or not only mutants are in volved. Some investigators maintain that this selective process is not as important as the physiologic response of bacteria to antibiotic compounds.21 A m in o -A cid s
As previously stated, Gale and his col leagues9’ 10 concluded that penicillin im pairs the ability of staphylococci and other gram-positive micro-organisms to assimilate certain free amino acids. In 17. Demerec, M ., Production of Staphylococcus Strains Resistant to Va rious Concentrations of Peni cillin. Proc. N at. Acad. Sc. 3 1 :16 (January) 1945. 18. Demerec, M ., Genetic Aspects of Changes in Staphylococcus Aureus Producing Strains Resistant to Various Concentrations of Penicillin. A n n . Missouri Botan. Garden 3 2 :131 (M a rc h ) 1945. 19. Kle in , M . , and Kiram elm an, L. J., T h e Role of Spontaneous Variants in the Acquisition of Strepto m ycin Resistance by the Shigellae. /. B a d . 52:471 (October) 1946. 20. Alexander, H . E . } and Leidy, G., M o d e of Action of Streptomycin on Type b H . Influenzae. I. O rigin of Resistant Organism s. J. Exper. M ed. 85:329 (A p ril) * 94721. Seligman, E., and Wassermann, M ., Induced Resistance to Streptomycin. /. Im m unol. 5 7 :351 (D e cember) 1947.
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their opinion acquired resistance is due to a reduced dependence on assimilation with an acquired ability to synthesize glutamic acid and other amino acids from ammonia and glucose in the pres ence of thiamine. In other words, accord ing to their observations, acquired resist ance consists in the selection of mutants that are less dependent on the process of assimilation of free amino acids until, eventually, all micro-organisms are sup pressed except those that grow by syn thesis instead of by assimilation of these substances. Since gram-positive bacteria usually become gram-negative upon acquiring a high degree of resistance in vitro, these investigators have logically concluded that the capacity for assimila tion of free amino acids is associated with the presence of the gram-positive com plex in the cell wall. Whether or not this mechanism is in volved in acquired resistance of meningo cocci, gonococci, and other gram-nega tive micro-organisms to penicillin or of acquired resistance of gram-positive and gram-negative bacteria to streptomycin cannot be stated. Nevertheless, the results indicate that the mechanism of acquired resistance of micro-organisms to anti biotics may be similar in principle to that involved in acquired resistance to the sulfonamide compounds. Woods22 has shown that sulfonamides compete with bacteria for para-aminobenzoic acid which they require in enzyme systems necessary for their survival and growth. Since susceptible micro-organisms are regarded as unable to synthesize paraaminobenzoic acid, this competition re sults in their crippling or complete de struction. Upon repeated exposure to sulfonamide compounds in vitro or in vivo, however, the bacteria may acquire the ability to synthesize sufficient amounts of para-aminobenzoic acid to meet their requirements, which results in acquired
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resistance. Fildes23 has suggested that a rational approach to chemotherapeutic research consists in the endeavor to alter the structure of a known essential metab olite of bacterial cells in such a way that the resulting compound may block an enzyme system of which the metabolite normally occupies an important func tional part. Penicillinase
As is now well known, many different gram-positive and gram-negative micro organisms produce penicillinase, which undoubtedly accounts, at least partly, for their natural resistance to penicillin in vitro and in vivo. But micro-organisms may be naturally resistant to penicillin without the detectable production of this inhibiting agent.24 Furthermore, it does not appear that staphylococci which acquire resistance to penicillin in vitro owe this resistance to an acquired ability to produce penicillinase, although this may be true of some strains of staphylo cocci acquiring resistance in vivo.2* At all events it is practically certain that natural and acquired resistance of bac teria to penicillin is not due solely to the production of penicillinase. Furthermore, bacteria apparently do not produce a similar inhibitor for streptomycin.21’ 26 C h an ge s A c co m p a n yin g A cquired Resistance
When bacteria acquire resistance to penicillin or streptomycin in vivo during treatment they are not likely to show de tectable changes in morphology or in tinctorial or biochemical properties. Con
23. Fildes, P., A R ational Approach to Research in Chemotherapy. Lancet 1:955 (M a y 25) 1940. 24. Bondi, Amedeo, Jr., and Dietz, Catherine, R e lationship of Penicillinase to the Action of Penicillin. Proc. Soc. E xper. Biol. & M ed. 56:135 (June) 1944. 25. Spink, W . W ., and Ferris, V ., Penicillin I n hibitor F ro m Staphylococci W h ich H a s Developed R e sistance to Penicillin in the H u m a n Body. Proc. Soc. Exper. Biol. & M ed. 5 9 :188 (June) 194526. Graessle, O . E., and Frost, B. M ., Induced in 22. Woods, D . D ., T h e Relation of A m in o Acids V itro Resistance of Staphylococci to Streptomycin and Penicillin. Proc. Soc. E xper. Biol. & M ed. 6 3 :171 to the M echanism of the Action o f Sulfonamide. Brit. (October) 1946. J . Exper. Path. 2 1 :7 4 (A p ril) 1940.
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sequently, resistance acquired in vivo is not usually detectable in the course of routine bacteriologic examinations al though it is sometimes suspected because of slower growth and the production of smaller colonies. But pronounced resist ance acquired in vitro through “training” frequently results in such marked pleo morphic and tinctorial changes that staphylococci assume coccobacillary or diphtheroid forms and gram-positive micro-organisms become gram-negative. Pronounced physiologic or biochemical changes may also occur; for instance, reduced growth and smaller colonies in culture media, reduction or loss of ability to grow anaerobically, and reduction or loss in the capacity for producing pig ments or hemolysins or for fermenting sugars, reducing nitrates or producing hydrogen sulfide. Staphylococci that be come resistant in vitro may acquire the ability to synthesize nicotinic acid .21’ 26' 31 Curiously enough, however, bacteria that have undergone these profound changes while acquiring resistance gradually re turn to their original state when culti vated in antibiotic-free broth media .28’ 32 Induced Susceptibility
Even more surprising is the recent re port of Voureka 33 that some strains of penicillin-resistant staphylococci and streptococci were readily reversed to a susceptible state by growing them in mixed culture with other penicillin-sus 27. Silver, H . K ., and Kem pe, C . H ., Resistance to Streptomycin: A Study of the M echanism of Its Development. /. Im m u n o l. 57:263 (Novem ber) 1948. 28. Klim e k, J. W .; Cavallito, G. J., and Bailey, J. H ., Induced Resistance of Staphylococcus Aureus to V a rious Antibiotics. /. Bact. 55:139 (February) 1948. 29. Bellamy, W . D ., and K lim e k, J. W ., Som e Properties of Penicillin-resistant Staphylococci. /. B act . 55:153 (February) 1948. 30. Bellamy, W . D ., and Klim ek, J. W ., T h e Rela tion Between Induced Resistance to Penicillin and O xygen Utilization. J. Bact. 5 5 :i4 7 (February) 1948.
31. Dufrenoy, J ., and Pratt, R ., Cytochemical Mech anism of Penicillin Action. III. Effect on Reaction to the Gram Stain. J . Bact. 54:283 (September) 1947« 32. Bailey, J. H ., and Cavallito, C . J., T h e R e versal of A ntibiotic Action. J. Bact. 55:175 (Febru ary) 1948. 33. Voureka, A ., Sensitisation of Penicillin-resistant Bacteria. Lancet 1:62 (January 10) 1948.
ceptible or penicillin-resistant micro-or ganisms. Indeed, it is stated that this re versal can be induced if the bacterial association occurs in the refrigerator, where growth is suspended, or by using autolysates of sensitizing micro-organ isms. Voureka offered no explanation of this phenomenon—whether, for exam ple, the sensitizing agent adsorbs the re sistant micro-organisms, or whether sen sitized staphylococci lose their capacity to produce penicillinase— but her work certainly opens up a new and interesting field in the problem of both natural and acquired resistance. This transference of susceptibility to penicillin from one micro-organism to another apparently has been confirmed by Winner,34 who has reported that the susceptibility of penicillin-resistant staphylococcus was increased about two hundred times by growing it for four hours in contact with a susceptible strep tococcus. Barber,35 however, has pointed out that since many penicillin-sensitive staphylococci kept in the laboratory do not breed true, such claims are unreliable if based on the testing of single colonies. By studying 50 colonies from each of the two cultures used by Voureka, Barber was unable to find any significant in crease in the number of colonies rendered more susceptible to penicillin through exposure to the Milne strain of strepto coccus for “sensitizing” purposes. Under the circumstances Barber has expressed the opinion that the results reported by Voureka and Winner were due merely to spontaneous mutation. O f further interest in this connection is whether or not bacteria with an ac quired resistance to either penicillin or streptomycin also display acquired re sistance to the other drug. Graessle and Frost26 have reported that one of two strains of penicillin-resistant staphylo34. W inner, H . I., Quantitative Sensitisation of a Penicillin-resistant Staphylococcus. Lancet 1:674 (M a y 1) 1948. 35. Barber, M a ry, Sensitisation of Penicillin-resistant Staphylococci. Lancet 1:730 (M a y 8) 1948.
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cocci showed increased resistance to streptomycin, but that strains of strepto mycin-resistant micro-organisms showed no cross resistance to penicillin. Klimek and his associates,28 however, have ob served this cross resistance. Sullivan and his colleagues36 found that a strain of Staphylococcus aureus with acquired resistance to streptothricin was more resistant to streptomycin than the parent culture. Meleney and John son37 have reported that aerobic and anaerobic cocci resistant to penicillin were susceptible to bacitracin and organ isms resistant to bacitracin were suscep tible to penicillin. Unfortunately, and of great impor tance in relation to treatment as well as to public health and epidemiology, patho genic micro-organisms acquiring resist ance to penicillin or streptomycin in vivo or in vitro usually retain their virulence. Thus all but one of seven strains of men ingococci with acquired resistance to penicillin were found to be virulent for mice.38 Tubercle bacilli with acquired resistance to streptomycin have also been found virulent for mice and guinea pigs.39’ 40 Salmonella, with acquired re sistance to streptomycin in vitro were stated, however, to have lost their viru lence for animals.26 Clinical Aspects
How the dangers of acquired resistance to antibiotic compounds are to be over come is still conjectural, especially in the 36. Sullivan, Marguerite» and others, Reciprocal Sen sitivities of Staphylococcus Aureus to Streptomycin, Streptothricin and Penicillin. Science 104:397 (O cto ber 25) 1946. 37. Meleney, F. L., and Johnson, Balbina, Bacitracin Therapy. J .A .M .A , 133:675 (M a rc h 8) 1947. 38. M iller, C . P., and Bohnhoff, Marjorie, Studies on Action of Penicillin. V . Virulence of Penicillin Resistant Strains of Meningococcus. Proc. Soc. E xper. Biol. & M ed. 60:356 (December) 1945. 39. Youm ans, G. P., and W illiston, E . H ., Effect of Streptomycin on Experimental Infections Produced in M ic e with Streptomycin Resistant Strains of M . Tuberculosis var. H om inis. Proc. Soc. Exper. Biol. & M ed. 6 3 :131 (October) 1946. 40. Karlson, A . G.; Feldman, W . H ., and Hinshaw, H . C., Persistence^ of Resistance of Tubercle^ Bacilli to Streptomycin D u rin g Passage T h ro u gh Guinea Pigs. Proc. Soc. Exper . Biol. & M e d . 6 4 :6 (January) 1947.
case of streptomycin. Fortunately the lit erature contains few reports presenting convincing evidence of resistance to peni cillin acquired by bacteria during treat ment. Resistance is usually acquired slowly and the dosages ordinarily pre scribed are sufficient for the destruction of naturally resistant variants. Miller,12 discussing infections that apparently be come resistant to penicillin during treat ment, said: In such instances, one must be sure that the infection is being maintained by the same strain which initiated it, for secondary infec tion with a penicillin-resistant micro-organism often accounts for failure of the drug to con trol an infection rather than the actual devel opment of penicillin resistance.
Whether or not bacteria, including oral spirochetes, may acquire resistance in the course of topical treatment with peni cillin cannot be stated, as this phase of the problem has not been sufficiently in vestigated. Available data, however, indi cate that bacteria may acquire resistance to penicillin through topical treatment with the compound. Under the circum stances I believe that topical solutions should contain at least 1,000 to 2,000 units per cubic centimeter and ointments at least 500 to 2,000 units per gram for dental use, and that applications should be frequent enough not only for prompt therapeutic results but for complete de struction of any naturally resistant mu tants that may be present. As previously stated, tests for suscep tibility of micro-organisms to penicillin or streptomycin should be capable of detecting the presence of any naturally resistant mutants in order that dosage and intervals of administration of these compounds may be so arranged as to bring about their complete destruction as promptly as possible. The danger of inadequate dosage over long periods has been demonstrated clinically and like wise by Silver and Kempe 27 who found
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that Escherichia coli and Aerobacter aerogenes acquired resistance to strepto mycin when exposed to subinhibitory concentrations of the compound in vitro. Miller and Bohnhoff,41 however, have stated that gonococci exposed to bacterio static concentrations of penicillin in vitro did not acquire resistance. Furthermore, Eagle42 has reported that certain micro organisms are killed more rapidly in vitro at low concentrations of penicillin than at high concentrations. Nevertheless there is no escape from the conclusion that in antibiotic therapy it is always advisable to secure complete destruction of the infecting micro-organisms as promptly as possible. This is particularly important in streptomycin therapy. The problem of acquired resistance is far more difficult and complex in the antibiotic treatment of chronic infections than of acute infections because of the need for prolonged therapy. Bigger43 has suggested that since bacteria are espe cially likely to be killed while they are multiplying, intermittent treatment might be more effective than continuous treatment. That is, it might be well to alternate periods of effective blood con centrations and penicillin-free intervals, since resting micro-organisms ( “persisters” ) would resume multiplication in the penicillin-free intervals and thereby be come more vulnerable to succeeding in jections of the compound. 41. M ille r, G. P., and Bohnhoff, M arjorie, Studies on the Action of Penicillin. IV . Development of Peni cillin Resistance by Gonococcus. Proc. Soc . Exper. Biol. & M ed. 60:354 (Decem ber) 1945. 42. Eagle, Harry, A Paradoxical Zone Phenomenon in the Bactericidal Action of Penicillin in Vitro. Science 107:4 4 (January 9) 1948. 43. Bigger, J. W ., Treatment of Staphylococcal I n fections W ith Penicillin. Lancet 2:497 (October 14) 1944.
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C om b ination Therapy
One promising approach to the prob lem of acquired resistance is synergistic . and additive chemotherapy, commonly designated as combination therapy. Ex tensive experimental data and some clin ical data indicate that treatment of infec tions with such combinations as penicillin and sulfonamides, streptomycin and sulfonamides, streptomycin and sulfones (for tuberculosis), penicillin and strep tomycin, penicillin and oxophenarsine hydrochloride (Mapharsen) (for syphi lis) and penicillin and bismuth (for syph ilis) may be more effective than treat ment with single compounds and may reduce the chances of acquired resistance by materially shortening the duration of treatment.44'48 Whether or not penicillinsensitive micro-organisms may render penicillin-resistant micro-organisms sensi tive in vivo, as reported by Voureka and Winner to occur sometimes in vitro, is not known at the present time, but such action appears doubtful. If the observa tions of Voureka and Winner are con firmed, however, the incidence of ac quired resistance of micro-organisms in mixed infections may be thereby reduced. 44. Eagle, H ., M agnuson. H . J., and Fleischman, R., T h e Synergistic Action of Penicillin and M apharsen (Oxophenarsine Hydrochloride) in the Treatm ent of Experimental Syphilis. J. Ven. Dis. Inform . 2 7 :3 (January) 1946. 45. Kolm er, J. A., and Rule, A . ' M ., Studies on Penicillin in the Treatm ent of Experimental Syphilis of Rabbits. I I . T h e Synergistic or Additive A ctivity of M apharsen (Oxyphenarsine H ydrochloride) and Po tassium Bism uth Tartrate. Arch. D erm at. & Syph. 5 6 : 179 (August) 1947 . 46. Kolm er, J ; A., and Rule, A . M ., T h e Synergistic or A dditive A ctivity of Chemotherapeutic Compounds. A m . J . M . Sc. 215:136 (February) 1948. 47. Kolm er, J. A., T h e Present Status of Synergistic and Additive Chemotherapy. Texas State /. M ed. 4 4 : 81 (June) 1948. 48. Kle in , M ., and Kim m elm an, L . J., T h e C orre lation Between the Inhibition of D ru g Resistance and Synergism in Streptomycin and Penicillin. /. B a d . 5 4 : 363 (September) 1947.
A — An average of one out of eight Americans received hospital care in 1947, the American Hospital Association reports. Eighteen million were admitted to the nation’s 6,173 hospitals. T en years ago the figure was 9,221,517. Average stay in hospital was eight days compared to 9.1 in 1946 .— Insurance E conom ics Surveys, Septem ber 1948. Hospitalization Averages.
resistance of bacteria to anti biotic compounds is of interest not only from the standpoint of its mech anism but also because it is of great prac tical importance in treatment. This is especially true of penicillin and strepto mycin, about which considerable infor mation has been gained. Whether or not bacteria may acquire resistance to tyrothricin, bacitracin, subtilin, polymyxin (aerosporin) and other antibiotics re mains to be more fully determined by experimental and clinical investigations but, theoretically at least, this appears to be possible. Acquired resistance of bacteria to anti biotic compounds may be fundamentally related to natural resistance in the sense that in any culture of a highly susceptible species there may occur mutants of in creased resistance.1 These not only may be responsible for failures in treatment but apparently are capable of acquiring additional resistance through exposure to a compound in vitro or in vivo. Suscep tibility and resistance, however, are usually only relative terms; resistance means that a particular strain of micro organism requires more antibiotic for its crippling or complete destruction than most other strains of the same species. Tests of the susceptibility of micro-
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T h is article has been authorized for publication by the Council on D ental Therapeutics. T h e opinions expressed are those of the author and _d o not neces sarily reflect the opinions of the Council. Professor of medicine, , School of ^ M edicine and School of Dentistry, Tem ple U niversity; director of the Research Institute of Cutaneous Medicine, Phila delphia. i. T h e term “ mutants” is employed throughout this article as a synonym for naturally resistant cells occurring in a bacterial population although the trans missible change is not always permanent.
organisms to penicillin or streptomycin, or both, frequently are required when treatment with antibiotics is considered. Unfortunately, as ordinarily conducted, they may be of but limited value because of technical conditions. Tests conducted in different laboratories with the same culture may give widely varying and con fusing results, for many factors influence tests of the susceptibility of bacteria to penicillin and especially to streptomy cin .2'6 It is particularly important that the tests be capable of detecting the presence of naturally resistant mutants. This re quires that susceptibility tests be con ducted with relatively large numbers of micro-organisms since there are usually but few naturally resistant mutants among millions of highly susceptible bac terial cells. Thus when tests are con ducted with single colonies of any micro organism, resistant mutants may readily escape detection. A mixed culture of nu merous colonies (the more the better), along with a large test dose or inoculum, is required to reduce this possible source of error to a minimum. Furthermore, as ordinarily conducted, susceptibility 2. Kolm er, J. A ^ Penicillin Therapy Including Strep tom ycin, Tyrothricin and O ther Antibiotic Therapy, Second edition. N ew Y o rk : D . Appleton-Century C om pany, Inc., 1947» P* 52 3. Lenert, Tulita, and Hobby, Gladys, Observations on the Action of Streptom ycin in V itro ( I) . Proc. Soc. Exper. Biol. & M ed. 65:235 (June) 1947. 4. Berkman, S.; H enry, R . J., and Housewright, R . D ., Studies on Streptomycin. I. Factors Influencing the Activity of Streptomycin. J. Bact. 53:567 (M a y )
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5. Rhym er, I., and Wallace, G. I., T h e N ature of a Streptomycin In h ib ito r O ccurring in B rain Tissue and Plant Extracts. /. Bact. 54:521 (October) 1947. 6. Green, S. R .; Iverson, ^ W . P., and Waksman, S. A., Effect of O rga n ic Acids on Streptomycin A c tivity. Proc . Soc. E xper. Biol. & M ed. 6 7 :2 8 s (M a rc h ) 1948.
Kolmer
tests show only the bacteriostatic activi ties of penicillin or streptomycin, whereas it is advisable to determine susceptibility to the bactericidal effects of these agents as a more reliable guide to dosage and administration. It is apparent that stand ardized tests for determining the sus ceptibility of micro-organisms to both the bacteriostatic and the bactericidal effects of penicillin and streptomycin are urgently required to facilitate the pro phylaxis and treatment of acute and chronic infections. M echanism of Antibacterial A ctivity
Knowledge of the way in which peni cillin and streptomycin destroy bacteria in vitro and in vivo is fundamental to an understanding of the mechanism of ac quired resistance. Although much infor mation and numerous theories have ac cumulated bearing on the antibacterial activity of penicillin, the precise mode of action is still unknown and even less is known about the mechanism by which streptomycin acts. Penicillin is believed to be more active against young cells and those about to divide than against resting cells. The same may be true of strepto mycin, with the suggestion that the anti bacterial activity of both depends to a large extent upon genesistasis. Further more, penicillin, at least, apparently in jures respiratory enzymes or other enzy matic systems, especially the hydrogenase system, causing inhibition of respiration and of endogenous oxygen uptake, and resulting in regressive biochemical changes.7’ 8 Gale and his colleagues,9’ 10 working with staphylococci, have also ob 7. Chain, E .j Duthie, E . S., and Callow, D ., Bac* tericidal and Bacteriolytic Action of Penicillin on the Staphylococcus. Lancet 1:652 (M a y 26) 1945* 8. Kram pitz, L . O ., and W erkm an, C. H ., O n the M o d e of Action of Penicillin. A rch. Biochem . 12:57 (January) 19479. Gale, E . F., and Taylor^ E ; S., Action o i Peni cillin in Preventing the Assim ilation o f G lutam ic A cid by Staphylococcus Aureus. N ature 158:676 (N ovem ber 9) 1946. 10. Gale, E . F., and Rodwell, A . W ., A m in o A cid Metabolism of Penicillin-resistant Staphylococci. /. Bact. 5 5 :161 (February) 1948.
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served that penicillin prevents the assim ilation of free glutamic acid, which pre cedes the loss of respiration and lysis of cells coincident with the loss of viability. Under the circumstances it appears that the antibacterial activity of peni cillin, and doubtless that of streptomycin, is due to interference with some essential metabolite or metabolic process of sus ceptible cells. This may result only in bacteriostasis, with the assumption that in vivo the crippled micro-organisms are finally disposed of by immunologic proc esses, particularly phagocytosis by the cells of the lymphoid-reticulo-endothelial system. On the other hand higher con centrations of these antibiotics are bac tericidal, causing complete destruction of the cells through lysis or disintegration. Curiously enough, Paine and Finland11 have recently reported that mutants have been found in cultures of Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa and Proteus morganii which depended upon the presence of strepto mycin in culture media as an essential metabolite or growth factor; conse quently, they designated these mutants as “dependent” strains. Similar results have been reported by Miller 12 with two resistant mutants of meningococcus which required the presence of strepto mycin for growth not only in, vitro but likewise in vivo, since they did not pro duce infections in mice until the animals were treated with streptomycin. So far there have been no reports on the occurrence in vitro or in vivo of micro-organisms dependent on penicillin as an essential metabolite or growth fac tor. Nor have there been any reports of infections due to streptomycin-dependent mutants of micro-organisms, probably because media containing streptomycin have not been used for bacteriologic ex11. Paine, T . F., and Finland, Maxw ell, Strepto mycin-sensitive, -dependent, and -resistant Bacteria. Science 1 0 7 :143 (February 6) 1948. 12. M iller, C . P., Developm ent of Bacterial Resist ance to Antibiotics. J .A .M .A . 135:749 (Novem ber 22) 1947*
62
The Journal of the American Denial Association
aminations of patients. Hall and Spink,13 however, have described a strain of Brucella abortus that acquired resistance to streptomycin during treatment and grew better in the presence of the com pound. It is likely that if and when de pendent mutants produce infection, the administration of streptomycin may not only fail therapeutically but actually pro duce an enhancement of the infection. Mechanism of A cq uired Resistance
Mutants naturally resistant to strepto mycin occur much more frequently than mutants naturally resistant to penicillin. This probably accounts for the fact that bacteria acquire resistance to streptomy cin much more frequently and more rap idly than to penicillin. Furthermore, nat urally resistant mutants occur more fre quently in some bacterial species than in others. For example, staphylococci as a group show more mutants naturally re sistant to penicillin and acquire resist ance to the compound both in vitro and in vivo much more readily and frequently than most other bacterial species. Al though Treponema pallidum is highly susceptible to penicillin, Tung and Fra zier14 were unable to increase the re sistance of the Reiter strain in vitro. Kolmer and Rule 15 observed no detect able increase of resistance of the virulent Nichols-Hough strain after three trans fers through rabbits treated with sub curative doses of penicillin. Gezon16 has observed that beta hemolytic strep tococci of group B acquire resistance to 13. H all. W . H., and Spink, W . W., In V itro Sen sitivity of Brucella to Streptomycin: Development of Resistance D u rin g Streptomycin Treatment. Proc. Soc. Exper. Biol. & M ed . 64:403 (A p ril) 1947. 14. Tung, T., and Frazier, C. N., Penicillin Sensi tivity an d M o rp h olo gy of the Reiter Strain of Trepo nema Pallidum After Cultivation in M e d ia Containing Penicillin. A m . J . S y p k .t Gonor. & Ven. Dis. 30:5205 (M a y ) 1946. 15. Kolm er, J. A., and Rule, A . M ., Acquired Resistance of Treponem a Pallidum to Penicillin. Proc. Soc. Exper. Biol. & M ed. 63:240 (Novem ber) 1946. 16. Gezon, H . M ., Antibiotic Studies on Beta H em o lytic Streptococci. I. Penicillin Resistance Acquired by G rou p A Organism s. Proc. Soc. Exper. Biol & M ed. 67 :2 08 (February) 1948.
penicillin in vitro much more frequently than streptococci of groups A and C. The exact mechanism of acquired re sistance of bacteria to antibiotics is still unknown. According to Demerec17’ 18 the mechanism consists in the elimination of susceptible micro-organisms, leaving nat urally resistant mutants which undergo further enhancement of resistance upon repeated exposures to the compound. Demerec has interpreted this as being due to the summation of the effects of several independent genetic factors for resistance which undergo consecutive mutation. In other words, according to this hypothesis, acquired resistance consists of the selec tion of naturally resistant mutants which undergo a further increase of resistance through “training” in vitro or in vivo. From this point of view the mechanism is to be interpreted as essentially one of bacterial genetics. Certainly considerable evidence has accumulated in support of the theory,19’ 20 although it must be ad mitted that it is difficult to determine whether or not only mutants are in volved. Some investigators maintain that this selective process is not as important as the physiologic response of bacteria to antibiotic compounds.21 A m in o -A cid s
As previously stated, Gale and his col leagues9’ 10 concluded that penicillin im pairs the ability of staphylococci and other gram-positive micro-organisms to assimilate certain free amino acids. In 17. Demerec, M ., Production of Staphylococcus Strains Resistant to Va rious Concentrations of Peni cillin. Proc. N at. Acad. Sc. 3 1 :16 (January) 1945. 18. Demerec, M ., Genetic Aspects of Changes in Staphylococcus Aureus Producing Strains Resistant to Various Concentrations of Penicillin. A n n . Missouri Botan. Garden 3 2 :131 (M a rc h ) 1945. 19. Kle in , M . , and Kiram elm an, L. J., T h e Role of Spontaneous Variants in the Acquisition of Strepto m ycin Resistance by the Shigellae. /. B a d . 52:471 (October) 1946. 20. Alexander, H . E . } and Leidy, G., M o d e of Action of Streptomycin on Type b H . Influenzae. I. O rigin of Resistant Organism s. J. Exper. M ed. 85:329 (A p ril) * 94721. Seligman, E., and Wassermann, M ., Induced Resistance to Streptomycin. /. Im m unol. 5 7 :351 (D e cember) 1947.
Kolmer
their opinion acquired resistance is due to a reduced dependence on assimilation with an acquired ability to synthesize glutamic acid and other amino acids from ammonia and glucose in the pres ence of thiamine. In other words, accord ing to their observations, acquired resist ance consists in the selection of mutants that are less dependent on the process of assimilation of free amino acids until, eventually, all micro-organisms are sup pressed except those that grow by syn thesis instead of by assimilation of these substances. Since gram-positive bacteria usually become gram-negative upon acquiring a high degree of resistance in vitro, these investigators have logically concluded that the capacity for assimila tion of free amino acids is associated with the presence of the gram-positive com plex in the cell wall. Whether or not this mechanism is in volved in acquired resistance of meningo cocci, gonococci, and other gram-nega tive micro-organisms to penicillin or of acquired resistance of gram-positive and gram-negative bacteria to streptomycin cannot be stated. Nevertheless, the results indicate that the mechanism of acquired resistance of micro-organisms to anti biotics may be similar in principle to that involved in acquired resistance to the sulfonamide compounds. Woods22 has shown that sulfonamides compete with bacteria for para-aminobenzoic acid which they require in enzyme systems necessary for their survival and growth. Since susceptible micro-organisms are regarded as unable to synthesize paraaminobenzoic acid, this competition re sults in their crippling or complete de struction. Upon repeated exposure to sulfonamide compounds in vitro or in vivo, however, the bacteria may acquire the ability to synthesize sufficient amounts of para-aminobenzoic acid to meet their requirements, which results in acquired
J.A.D.A., Vol. 38, January 1949 . . . 63
resistance. Fildes23 has suggested that a rational approach to chemotherapeutic research consists in the endeavor to alter the structure of a known essential metab olite of bacterial cells in such a way that the resulting compound may block an enzyme system of which the metabolite normally occupies an important func tional part. Penicillinase
As is now well known, many different gram-positive and gram-negative micro organisms produce penicillinase, which undoubtedly accounts, at least partly, for their natural resistance to penicillin in vitro and in vivo. But micro-organisms may be naturally resistant to penicillin without the detectable production of this inhibiting agent.24 Furthermore, it does not appear that staphylococci which acquire resistance to penicillin in vitro owe this resistance to an acquired ability to produce penicillinase, although this may be true of some strains of staphylo cocci acquiring resistance in vivo.2* At all events it is practically certain that natural and acquired resistance of bac teria to penicillin is not due solely to the production of penicillinase. Furthermore, bacteria apparently do not produce a similar inhibitor for streptomycin.21’ 26 C h an ge s A c co m p a n yin g A cquired Resistance
When bacteria acquire resistance to penicillin or streptomycin in vivo during treatment they are not likely to show de tectable changes in morphology or in tinctorial or biochemical properties. Con
23. Fildes, P., A R ational Approach to Research in Chemotherapy. Lancet 1:955 (M a y 25) 1940. 24. Bondi, Amedeo, Jr., and Dietz, Catherine, R e lationship of Penicillinase to the Action of Penicillin. Proc. Soc. E xper. Biol. & M ed. 56:135 (June) 1944. 25. Spink, W . W ., and Ferris, V ., Penicillin I n hibitor F ro m Staphylococci W h ich H a s Developed R e sistance to Penicillin in the H u m a n Body. Proc. Soc. Exper. Biol. & M ed. 5 9 :188 (June) 194526. Graessle, O . E., and Frost, B. M ., Induced in 22. Woods, D . D ., T h e Relation of A m in o Acids V itro Resistance of Staphylococci to Streptomycin and Penicillin. Proc. Soc. E xper. Biol. & M ed. 6 3 :171 to the M echanism of the Action o f Sulfonamide. Brit. (October) 1946. J . Exper. Path. 2 1 :7 4 (A p ril) 1940.
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64
sequently, resistance acquired in vivo is not usually detectable in the course of routine bacteriologic examinations al though it is sometimes suspected because of slower growth and the production of smaller colonies. But pronounced resist ance acquired in vitro through “training” frequently results in such marked pleo morphic and tinctorial changes that staphylococci assume coccobacillary or diphtheroid forms and gram-positive micro-organisms become gram-negative. Pronounced physiologic or biochemical changes may also occur; for instance, reduced growth and smaller colonies in culture media, reduction or loss of ability to grow anaerobically, and reduction or loss in the capacity for producing pig ments or hemolysins or for fermenting sugars, reducing nitrates or producing hydrogen sulfide. Staphylococci that be come resistant in vitro may acquire the ability to synthesize nicotinic acid .21’ 26' 31 Curiously enough, however, bacteria that have undergone these profound changes while acquiring resistance gradually re turn to their original state when culti vated in antibiotic-free broth media .28’ 32 Induced Susceptibility
Even more surprising is the recent re port of Voureka 33 that some strains of penicillin-resistant staphylococci and streptococci were readily reversed to a susceptible state by growing them in mixed culture with other penicillin-sus 27. Silver, H . K ., and Kem pe, C . H ., Resistance to Streptomycin: A Study of the M echanism of Its Development. /. Im m u n o l. 57:263 (Novem ber) 1948. 28. Klim e k, J. W .; Cavallito, G. J., and Bailey, J. H ., Induced Resistance of Staphylococcus Aureus to V a rious Antibiotics. /. Bact. 55:139 (February) 1948. 29. Bellamy, W . D ., and K lim e k, J. W ., Som e Properties of Penicillin-resistant Staphylococci. /. B act . 55:153 (February) 1948. 30. Bellamy, W . D ., and Klim ek, J. W ., T h e Rela tion Between Induced Resistance to Penicillin and O xygen Utilization. J. Bact. 5 5 :i4 7 (February) 1948.
31. Dufrenoy, J ., and Pratt, R ., Cytochemical Mech anism of Penicillin Action. III. Effect on Reaction to the Gram Stain. J . Bact. 54:283 (September) 1947« 32. Bailey, J. H ., and Cavallito, C . J., T h e R e versal of A ntibiotic Action. J. Bact. 55:175 (Febru ary) 1948. 33. Voureka, A ., Sensitisation of Penicillin-resistant Bacteria. Lancet 1:62 (January 10) 1948.
ceptible or penicillin-resistant micro-or ganisms. Indeed, it is stated that this re versal can be induced if the bacterial association occurs in the refrigerator, where growth is suspended, or by using autolysates of sensitizing micro-organ isms. Voureka offered no explanation of this phenomenon—whether, for exam ple, the sensitizing agent adsorbs the re sistant micro-organisms, or whether sen sitized staphylococci lose their capacity to produce penicillinase— but her work certainly opens up a new and interesting field in the problem of both natural and acquired resistance. This transference of susceptibility to penicillin from one micro-organism to another apparently has been confirmed by Winner,34 who has reported that the susceptibility of penicillin-resistant staphylococcus was increased about two hundred times by growing it for four hours in contact with a susceptible strep tococcus. Barber,35 however, has pointed out that since many penicillin-sensitive staphylococci kept in the laboratory do not breed true, such claims are unreliable if based on the testing of single colonies. By studying 50 colonies from each of the two cultures used by Voureka, Barber was unable to find any significant in crease in the number of colonies rendered more susceptible to penicillin through exposure to the Milne strain of strepto coccus for “sensitizing” purposes. Under the circumstances Barber has expressed the opinion that the results reported by Voureka and Winner were due merely to spontaneous mutation. O f further interest in this connection is whether or not bacteria with an ac quired resistance to either penicillin or streptomycin also display acquired re sistance to the other drug. Graessle and Frost26 have reported that one of two strains of penicillin-resistant staphylo34. W inner, H . I., Quantitative Sensitisation of a Penicillin-resistant Staphylococcus. Lancet 1:674 (M a y 1) 1948. 35. Barber, M a ry, Sensitisation of Penicillin-resistant Staphylococci. Lancet 1:730 (M a y 8) 1948.
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Kolmer
cocci showed increased resistance to streptomycin, but that strains of strepto mycin-resistant micro-organisms showed no cross resistance to penicillin. Klimek and his associates,28 however, have ob served this cross resistance. Sullivan and his colleagues36 found that a strain of Staphylococcus aureus with acquired resistance to streptothricin was more resistant to streptomycin than the parent culture. Meleney and John son37 have reported that aerobic and anaerobic cocci resistant to penicillin were susceptible to bacitracin and organ isms resistant to bacitracin were suscep tible to penicillin. Unfortunately, and of great impor tance in relation to treatment as well as to public health and epidemiology, patho genic micro-organisms acquiring resist ance to penicillin or streptomycin in vivo or in vitro usually retain their virulence. Thus all but one of seven strains of men ingococci with acquired resistance to penicillin were found to be virulent for mice.38 Tubercle bacilli with acquired resistance to streptomycin have also been found virulent for mice and guinea pigs.39’ 40 Salmonella, with acquired re sistance to streptomycin in vitro were stated, however, to have lost their viru lence for animals.26 Clinical Aspects
How the dangers of acquired resistance to antibiotic compounds are to be over come is still conjectural, especially in the 36. Sullivan, Marguerite» and others, Reciprocal Sen sitivities of Staphylococcus Aureus to Streptomycin, Streptothricin and Penicillin. Science 104:397 (O cto ber 25) 1946. 37. Meleney, F. L., and Johnson, Balbina, Bacitracin Therapy. J .A .M .A , 133:675 (M a rc h 8) 1947. 38. M iller, C . P., and Bohnhoff, Marjorie, Studies on Action of Penicillin. V . Virulence of Penicillin Resistant Strains of Meningococcus. Proc. Soc. E xper. Biol. & M ed. 60:356 (December) 1945. 39. Youm ans, G. P., and W illiston, E . H ., Effect of Streptomycin on Experimental Infections Produced in M ic e with Streptomycin Resistant Strains of M . Tuberculosis var. H om inis. Proc. Soc. Exper. Biol. & M ed. 6 3 :131 (October) 1946. 40. Karlson, A . G.; Feldman, W . H ., and Hinshaw, H . C., Persistence^ of Resistance of Tubercle^ Bacilli to Streptomycin D u rin g Passage T h ro u gh Guinea Pigs. Proc. Soc. Exper . Biol. & M e d . 6 4 :6 (January) 1947.
case of streptomycin. Fortunately the lit erature contains few reports presenting convincing evidence of resistance to peni cillin acquired by bacteria during treat ment. Resistance is usually acquired slowly and the dosages ordinarily pre scribed are sufficient for the destruction of naturally resistant variants. Miller,12 discussing infections that apparently be come resistant to penicillin during treat ment, said: In such instances, one must be sure that the infection is being maintained by the same strain which initiated it, for secondary infec tion with a penicillin-resistant micro-organism often accounts for failure of the drug to con trol an infection rather than the actual devel opment of penicillin resistance.
Whether or not bacteria, including oral spirochetes, may acquire resistance in the course of topical treatment with peni cillin cannot be stated, as this phase of the problem has not been sufficiently in vestigated. Available data, however, indi cate that bacteria may acquire resistance to penicillin through topical treatment with the compound. Under the circum stances I believe that topical solutions should contain at least 1,000 to 2,000 units per cubic centimeter and ointments at least 500 to 2,000 units per gram for dental use, and that applications should be frequent enough not only for prompt therapeutic results but for complete de struction of any naturally resistant mu tants that may be present. As previously stated, tests for suscep tibility of micro-organisms to penicillin or streptomycin should be capable of detecting the presence of any naturally resistant mutants in order that dosage and intervals of administration of these compounds may be so arranged as to bring about their complete destruction as promptly as possible. The danger of inadequate dosage over long periods has been demonstrated clinically and like wise by Silver and Kempe 27 who found
66
that Escherichia coli and Aerobacter aerogenes acquired resistance to strepto mycin when exposed to subinhibitory concentrations of the compound in vitro. Miller and Bohnhoff,41 however, have stated that gonococci exposed to bacterio static concentrations of penicillin in vitro did not acquire resistance. Furthermore, Eagle42 has reported that certain micro organisms are killed more rapidly in vitro at low concentrations of penicillin than at high concentrations. Nevertheless there is no escape from the conclusion that in antibiotic therapy it is always advisable to secure complete destruction of the infecting micro-organisms as promptly as possible. This is particularly important in streptomycin therapy. The problem of acquired resistance is far more difficult and complex in the antibiotic treatment of chronic infections than of acute infections because of the need for prolonged therapy. Bigger43 has suggested that since bacteria are espe cially likely to be killed while they are multiplying, intermittent treatment might be more effective than continuous treatment. That is, it might be well to alternate periods of effective blood con centrations and penicillin-free intervals, since resting micro-organisms ( “persisters” ) would resume multiplication in the penicillin-free intervals and thereby be come more vulnerable to succeeding in jections of the compound. 41. M ille r, G. P., and Bohnhoff, M arjorie, Studies on the Action of Penicillin. IV . Development of Peni cillin Resistance by Gonococcus. Proc. Soc . Exper. Biol. & M ed. 60:354 (Decem ber) 1945. 42. Eagle, Harry, A Paradoxical Zone Phenomenon in the Bactericidal Action of Penicillin in Vitro. Science 107:4 4 (January 9) 1948. 43. Bigger, J. W ., Treatment of Staphylococcal I n fections W ith Penicillin. Lancet 2:497 (October 14) 1944.
The Journal of the American Dental Association
C om b ination Therapy
One promising approach to the prob lem of acquired resistance is synergistic . and additive chemotherapy, commonly designated as combination therapy. Ex tensive experimental data and some clin ical data indicate that treatment of infec tions with such combinations as penicillin and sulfonamides, streptomycin and sulfonamides, streptomycin and sulfones (for tuberculosis), penicillin and strep tomycin, penicillin and oxophenarsine hydrochloride (Mapharsen) (for syphi lis) and penicillin and bismuth (for syph ilis) may be more effective than treat ment with single compounds and may reduce the chances of acquired resistance by materially shortening the duration of treatment.44'48 Whether or not penicillinsensitive micro-organisms may render penicillin-resistant micro-organisms sensi tive in vivo, as reported by Voureka and Winner to occur sometimes in vitro, is not known at the present time, but such action appears doubtful. If the observa tions of Voureka and Winner are con firmed, however, the incidence of ac quired resistance of micro-organisms in mixed infections may be thereby reduced. 44. Eagle, H ., M agnuson. H . J., and Fleischman, R., T h e Synergistic Action of Penicillin and M apharsen (Oxophenarsine Hydrochloride) in the Treatm ent of Experimental Syphilis. J. Ven. Dis. Inform . 2 7 :3 (January) 1946. 45. Kolm er, J. A., and Rule, A . ' M ., Studies on Penicillin in the Treatm ent of Experimental Syphilis of Rabbits. I I . T h e Synergistic or Additive A ctivity of M apharsen (Oxyphenarsine H ydrochloride) and Po tassium Bism uth Tartrate. Arch. D erm at. & Syph. 5 6 : 179 (August) 1947 . 46. Kolm er, J ; A., and Rule, A . M ., T h e Synergistic or A dditive A ctivity of Chemotherapeutic Compounds. A m . J . M . Sc. 215:136 (February) 1948. 47. Kolm er, J. A., T h e Present Status of Synergistic and Additive Chemotherapy. Texas State /. M ed. 4 4 : 81 (June) 1948. 48. Kle in , M ., and Kim m elm an, L . J., T h e C orre lation Between the Inhibition of D ru g Resistance and Synergism in Streptomycin and Penicillin. /. B a d . 5 4 : 363 (September) 1947.
A — An average of one out of eight Americans received hospital care in 1947, the American Hospital Association reports. Eighteen million were admitted to the nation’s 6,173 hospitals. T en years ago the figure was 9,221,517. Average stay in hospital was eight days compared to 9.1 in 1946 .— Insurance E conom ics Surveys, Septem ber 1948. Hospitalization Averages.