Surgical Laboratory Surveys of Clinical Material
Sensitivities of Ten Species of Clostridia to Penicillin,
Aureomycin, Terramycin, and Chloramphenicol:
A Study of Battle Wounds in Korea*
Major Arthur Newton, MSC, USA
1st Lieutenant Joseph G. Strawitz, MC, USAR
Lieutenant Colonel Robert B. Lindberg, MSC, USA
Captain John M. Howard, MC, USAR
Lieutenant Colonel Curtis P. Artz, MC, USA
Battle casualties in the Korean conflict suffered less from wound infections than casualties in any previous war. During the last half of the war, gas gangrene was a rarity; death from gas gangrene was almost unknown.1 Success in preventing severe, clostridial infections was probably due to the repair of arterial injuries, early and adequate débridement of wounds, and early and consistent use of antibiotics. It was demonstrated in Korea,2 as had been shown in earlier wars,3 4 that clostridia were present in approximately 46 percent of wounds at the time of primary débridement.
The average time-lag from injury to arrival at the 46th Surgical Hospital (Mobile Army) was only 3.5 hours on the eastern front during the last 18 months of the war; the subsequent time-lag before reaching the operating room being only 1.0 to 1.5 hours.
Thus, in the latter half of the war, short evacuation times and small casualty loads permitted optimal surgical care and resulted in a low incidence of severe clostridial infections.
In earlier wars, mass casualties such as occurred at times in Korea, or which might occur with atomic warfare and civilian disasters, would not permit the early or individualized attention given to the soldiers during the period of this study. For these reasons, as well as for the improvement of the care of soldiers in Korea, a re-evaluation of the antibiotic program in the combat zone was undertaken. Throughout the latter part of the war, penicillin (300,000 to 600,000 units) was administered intramuscularly as the casualty reached a battalion aid station. Following subsequent surgery, casualties
*previously published in Surgery 37: 392, 1955.
routinely received penicillin (300,000 units) every 12 hours. Those casualties having intraperitoneal injuries, arterial injuries, or intrathoracic injuries, also received 0.5 gin. of streptomycin every 12 hours. Aureomycin was available, but it was not used very often.
Material and Methods
In a previous study, 11 wounds inflicted during the Korean conflict were examined bacteriologically throughout the first week of healing.5 This was done by culture of tissue excised from the open wound at the time of primary débridement and every 2 days thereafter during the ensuing week. Cultures obtained included 56 clostridial isolates and numerous aerobic organisms.
In this study, the sensitivities of the clostridial isolates to penicillin, Aureomycin, Terramycin, and chloramphenicol are presented and correlated with human serum levels of these antibiotics. 6- 8
Isolates were grown for 18 hours at 37o C. in tubes of thioglycollate broth. One ml. of this culture was transferred to a flask containing 100 ml. of thioglycollate broth. This mixture served as seed broth. The antibiotics were prepared in thioglycollate broth in such a manner that the mixture of 2 ml. of the antibiotic solutions and 2 ml. of the seed broth gave the following concentrations of antibiotics in units/mi or ugms/ml.
Penicillin: .025, .05, 0.1, 0.2,0.4, 0.8, 1.6, 3.2, 6.4 units/ml.
Aureomycin: .0125, 0.25, .05, 0.1,0.4, 0.8, 1.2, 1.8, 3.2 µm/ml.
Terramycin: .025, .05 0.1, 0.2,0.4, 0.8, 1.2, 1.6, 3.2, 6.4 µgm/ml.
Chloramphenicol: 0.5, 1, 2, 4. 6,8, 12, 16, 20, 32 µgm/ml.
The tubes were incubated at 37o C. for 20 hours. At this time, the tube of lowest concentration showing no visible growth was read as containing the concentration of antibiotic which caused complete inhibitions of growth. A control tube containing 2 ml. of seed broth and 2 ml. of thioglycollate broth was used for each organism tested; and it was incubated for the same length of time and at the same temperature
Table 1 shows that sensitivities of the 56 clostridial isolates to the antibiotics, penicillin, Aureomycin, Terramycin, and chlorampenicol, follow normal distribution curves. The range of inhibition for penicillin is 0.2 to 6.4 units/ml; Aureomycin, 0.0125 to 0.8 µgm/ml; Terramycin, 0.55 to 3.2 µgm/ml; and chloramphenicol,1.0 to 32.0 µgm/ml. Those dilutions of the antibiotic which did not inhibit were omitted.
Figure 1 shows human serum concentrations following the administration of from 300,000 to 600,000 units of procaine penicillin G, and correlates these levels with the number of clostridial isolates inhibited by increasing concentrations of penicillin.6 A comparison of serum
Table1. Distribution of Sensitivities of Clostridial isolates to Four Antibiotics
FIGURE1. Sensitivity of the Clostridial Isolates to Penicillin. The percentages in the column on the far right are the results of an earlier study of battle wounds at the time of primary débridement.2 The dotted line represents the maximal concentrations of the drug.
levels in Figure 1 with concentrations of penicillin necessary for inhibition demonstrates that approximately 80 percent of the clostridial isolates are inhibited by the highest serum level reached after the administration of 600,000 units. Since this level is maintained for a short period only, the percentage of organisms falling outside of the range of inhibition would probably become greater when the entire absorption curve is considered.
Following the administration of 300,000units of procaine penicillin, 46 percent of the organisms (as indicated by the in vitro assay) would be inhibited by the maximal concentration found in the serum. The sensitivities in this study correspond closely with the results of
Table2. Inhibition of Clostridial Isolates by Various Antibiotics
a previous study of 126 clostridial isolates from many battle wounds at the time of the initial débridement.2 According to the previous study, 83 percent and 58 percent of the clostridia would be inhibited by the maximal serum concentration following the two dosages (Table 2).
Following the oral administration of 0.5 gm. of Aureomycin, the serum concentration ranged between 1.5 and 1.7 µgm/ml for 6 hours (Fig. 2) 7 This concentration proved inhibitory, in vitro, to 100 percent of the 56 clostridial isolates in this study and to 97 percent of the 126 isolates in an earlier study.
Following the oral administration of 0.5 gm. of Terramycin, the serum concentration ranged around 1.2 µgm/ml between 1 and 4 hours after ingestion (Fig. 3). 7 As indicated by the in vitro studies, this concentration was inhibitory to 98 percent of the 56 clostridial isolates in the study, and to 84 percent of the 176 clostridial isolates studied previously.
Following the oral administration of 0.5 gm. of chloramphenicol, the serum concentration rises to 6 µmg/ml after 3 hours. The concentration then gradually decreases (Fig. 4) 8 This maximal concentration, in vitro, inhibited the growth of 66 percent of the 56 isolates in this study; this finding confirmed the earlier study. After the administration of 1.0 gm. of chloramphenicol, the serum concentration rises to 12 µgm/ml, a concentration sufficient to inhibit 78 percent of the organisms in this study and 87 percent in the earlier studies.
FIGURE 2. Sensitivity of the Clostridia1 Isolates to Aureomycin. This figure, constructed as was Figure 1, shows that in the current study all 56 clostridial isolates were inhibited by the therapeutic concentration of aureomycin.
Antibiotic therapy has not supplanted and probably never will supplant débridement in the fundamental role of preventing or treating infections of war wounds. As an adjunct to débridement, all four of the antibiotics included in this study appear to be beneficial in preventing or minimizing clostridial infections.9-15
FIGURE3. Sensitivity of the Clostridial Isolates to Terramycin.
Within the limitations of the invitro sensitivity method, Aureomycin or Terramycin (of the drugs and dosages studied) would appear the drugs of choice in preventing or treating clostridial wound infections.
Experimental studies conducted in this laboratory have shown that concentrations of most antibiotics in the muscle are less than concentrations found in the serum.16 If a time should come again when-as a compromise-antibiotic therapy must be used as a bulwark to support delayed surgery of mass casualties, increased dosages of these drugs should be recommended.
FIGURE4. Sensitivity of the Clostridial Isolates to Chloramphenicol(Chloromycetin).
In vitro sensitivity studies of the flora of battle wounds in Korea suggest that Aureomycin and Terramycin are more effective than penicillin and chloramphenicol in inhibiting the growth of clostridia.
1. Howard, J. M., and Inui, F. K.: Clostridial Myositis-Gas Gangrene: Observations of Battle Casualties in Korea. Surgery, 36: 1115, 1954. (Chapter 15 of this volume.)
2. Lindberg, R. B.; Wetzler, T.F.; Marshall, J. D.; Newton, A.; Strawitz, J.G.; and Howard, J. M.: The Bacterial Flora of Battle Wounds at the Time of Primary Débridement: A Study of the Korean Battle Casualty. Ann. Surg. 141: 369, 1955. (Chapter 18 of this volume.)
3. Stoddard, J. L.: The Occurrence of Significance of B. welchii in Certain Wounds. J. A. M. A. 71: 1400, 1918.
4. MacLennan, J. D.: Anaerobic Infections of War Wounds in the Middle East. Lancet, 245: 123, 1943.
5. Strawitz, J. G.; Wetzler, T.F.; Marshall, J. D.; Lindberg, B. B.; Howard, J. M.; and Artz, C. P.: The Bacterial Flora of Healing Wounds: A Study of the Korean Battle Casualty. Surgery, 37: 400, 1955. (Chapter 19 of this volume.)
6. Romansky, Monroe J., George Washington University School of Medicine, Washington, D. C. Personal communication.
7. Welch, H.; Hendricks, F.; Price, C.; Randall, W.: Comparative Studies on Terramycin and Aureomyein. J. Am. Pharm. Assn: 39: 185, 1950.
8. Burnell, J. M., and Kirby, W.M. H.: Serum Concentration on Chloramphenicol Following Intravenous and Intramuscular Administration. J. Lab. & Clin. Med. 38: 234, 1951.
9. Smith, L. D.: Clostridia in Gas Gangrene. Bact. Rev. 13: 233, 1949.
10. Ulio, J. A., and Marshall, G.C.: 1945 Notes on Care of Battle Casualties. War Department Technical Bulletin, T. B. Med. 147, War Medicine, pp.234-248.
11. Hac, L. R.: Experimental C. welchii Infection. IV. Penicillin Therapy. J. Infec. Dis. 74: 164, 1944.
12. Taylor, W. I., and Novak, M.V.: Prophylaxis of Experimental Gas Gangrene in Mice. J. Bact. 61: 571, 1951.
13. Altemeier, W. A.; Furste, W.L.; and Culbertson, W. B.: Chemotherapy In Gas Gangrene. Arch. Surg. 55:668, 1947.
14. Sandusky, W. R.; Keeble, C.F.; Wharton, W. P.; and Taylor, B. N.: An Evaluation of Aureomycin and Chloromycetin In Experimental Clostridium welchii Infection. Surgery, 28:632, 1950.
15. Altemeier, W. A.; McMurrin, J. A.; and Alt, L. F.: Chloromycetin and Aureomycin in Experimental Gas Gangrene. Surgery, 28: 621, 1950.
16. Marshall, J. D., and Strawitz, J. G.: A Comparison of Antibiotic Levels in Serum and Muscle of Rabbits.(To be published.)