Battle Casualties in Korea: Studies of the Surgical Research Team Volume III
The Early Bacterial Flora of Wounds in the Korean Conflict
Lieutenant Colonel R. B. Lindberg, MSC, USA
Theodore F. Wetzler, B. S.
1st Lieutenant Joseph G. Strawitz, MC, USAR
Captain John M. Howard, MC, USAR
Introduction
A study of the bacterial contamination of battle wounds, as it occurred during the first 4 to 8 hours after injury, was made at the 46th Surgical Hospital on the east-central front in Korea during the last year of the Korean conflict. Cases were observed during August and September 1952, and from December through February 1953. Various reports in the literature regarding World War I and World War II, for the most part, included cases in which less recent wounds were considered.1-3 Under these circumstances, infection was far more likely to be well established at the time of culture than it was in the case of wounds observed in this study. The earlier reports showed that Clostridia were present in from 60 to 80 percent of the wounds within 1 to 4 days after injury. Most of these observations were made on soldiers who had not received intensive and early penicillin prophylaxis and therapy to the extent that it was employed in the Korean conflict.
The incidence of gas gangrene was lower in World War II than in World War I. In both conflicts, such infections were extremely rare in comparison with the extensive contamination of wounds by pathogenic and saprophytic Clostridia. In the Korean conflict, the exact incidence rate of gas gangrene and of anaerobic cellulitis was not ascertained, but it was evidently lower than in the previous wars. This improvement has been ascribed largely to the rapid evacuation of the wounded, prompt and thorough surgical débridement coupled with more effective shock therapy, and the extensive use of broad-spectrum antibiotics. It is important, however, that the degree and nature of bacterial contamination of these wounds be determined as a part of the basis for evaluating the role of wound infections in modern war surgery and for maintaining a perspective on the survival potentialities of
215
patients having Clostridia and receiving antibiotic prophylaxis soon after wounding. This report presents an analysis of the bacterial flora of wounds incurred by troops during the latter part of the Korean conflict and some observations on flora of the environment of these troops.
Materials and Methods
Sources
Of the two series of cases observed during 1952 and 1953, Series A was composed of 39 wounded patients who were studied during August and September 1952, and Series B consisted of cultures taken on 87 cases from November 1952 through February 1953. Specimens from these patients were cultured at. the time of arrival and débridement at the surgical hospital. The specimens represented mainly wound cultures taken 4 to 6 hours after injury, with follow-up cultures in some instances. The cases thus illustrate wound contamination and early proliferation of bacteria, rather than infection. Principal emphasis was placed on isolation and identification of Clostridia since these organisms, in addition to their classical role in gas gangrene, offer a potential source of toxins which may contribute to the development of secondary shock and to the kidney damage connected with posttraumatic anuria.
The speed of routine evacuation of patients limited most observations to initial débridement specimens in which typical infection had not yet appeared. Cases selected included principally arm and leg wounds.
In addition to tissues, the flora of the skin of patients and troops, of clothing worn in combat, and of the soil of the combat zone were studied to determine the relations of such organisms to the flora of contaminated wounds. Blood cultures were obtained in some cases of both series at the time of admission to the hospital. In Series A, 30 men arrived at the hospital in time to permit cultures to be obtained at an average interval of 4.5 hours following injury. However,13 percent of the patients arrived 6 or more hours following injury. In three patients, cultures were taken 1 or more days after wounding.
Technics of Culture
Blood cultures were obtained using evacuated rubber-stoppered bottles with reduced oxygen and increased CO2 tension over a trypticase soy broth. Skin cultures were taken by means of swabs moistened in broth, rubbed over the lateral aspect of the lower leg, and inoculated in chopped heart broth. Clothing samples were placed in dry, sterile vials. Wounds were sampled during débridement by collecting portions of tissue on sterile gauze pads as they were removed by the surgeon.
216
These samples were planted at once in a modified Robertson`s cooked meat medium in screw- capped tubes. This medium was found to maintain virtually all anaerobic and aerobic flora when held at room temperature for at least 3 months. The inoculated meat-broth tubes were incubated for 48 hours at 37o C. They were then transferred to blood-agar plates containing yeast extract. Inhibitors were added to media to suppress interfering aerobic flora. These included sodium azide (0.2 percent), chloral hydrate (0.2 percent), and at times sorbic acid (0.15 percent). Subcultures to thioglycollate broth containing sorbic acid and polymyxin B were made when necessary, with repeated platings to isolate pure cultures of anaerobes. Anaerobiasis was maintained in Brewer jars, with hydrogen introduced at 200 mm. pressure. Differentiating reactions with pure cultures were carried out in thioglycollate broth or semisolid medium.
Particular emphasis was placed on isolation of Cl. perfringens. Presumptive recognition of this species was made with the acrolein reaction.4
Classification of Clostridia
Clostridia were classified when possible according to the modified Spray classification as presented in Bergey`s Manual of Determinative Bacteriology, 6th edition.5 Incases where the characterization of strains in this system was incomplete or ambiguous, the classification of Reed and Orr 6 was employed in conjunction with the Bergey outline. Four principal categories of strains were differentiated as shown in Table 1. Aerobic flora were classified chiefly according to the scheme of Bergey`s Manual.
Table1. Categories of Clostridia
217
Results of Observations, Series A, Summer 1952
The two periods of observation, Series A and Series B, showed culture results which varied so markedly that it was desirable to present them separately. The major distinction between the two was a marked decrease in the relative proportion of Clostridia isolated in the winter in comparison with the number found in wounds during the summer.
Of the 39 patients studied in Series A, 33 had one or more tissue specimens collected at débridement. Six patients had blood cultures taken simultaneously; while six other patients had only blood cultures. In attempts to ascertain sources of contamination, 29 soil samples were collected from the combat zone, from hillsides and rice paddies. The causative agents of wounds, when known, were tabulated in Table 2. Included in this series were 21 cases of leg wounds of varying severity, 11 arm and shoulder injuries, and 7 head and neck or body injuries.
Anaerobic Flora of Tissue and Embedded Fragments at Débridement, Series A
Cultures were made of 94 tissue specimens from the 33 patients. Twenty-eight patients harbored Clostridia in tissues, with a total of 63 tissue samples which were positive. Five samples of cloth were removed from various wounds during débridement and Clostridia were isolated, although the tissues in one of these wounds did not reveal Clostridia. A shell fragment removed and cultured showed two species of Clostridia; and the same species were also isolated from adjacent tissues. Among these 33 patients, 84.8 percent showed positive cultures of Clostridia from one or more débrided tissues. There were 125 strains recovered, including 19 species. Clostridia were isolated in 67 percent of the tissue specimens. An average of 4 Clostridial species per patient was present in cases harboring those organisms; while 2 strains were isolated per tissue sample. The five pieces of embedded clothing showed 1.8 strains per sample, representing 9 strains and 5 species. One shell fragment from deep muscle was cultured and showed two species of Clostridia.
Clostridia Isolated in Blood Cultures, Series A
Collection of 15 blood cultures from12 patients showed Clostridia in 11 patients. Strains of Clostridia isolated totaled 19, including four species and, four aerobic organisms.
The bacterial flora of the Korean soil in the combat area was studied in both the summer and the winter series. No essential difference was noted in the flora of the soil in these two seasons.
The anaerobic flora of tissues and blood cultures in Series A is summarized in terms of total strains isolated (Table 3).
218
Table2. Causes of Wounds and Clostridia Present in 32 Casualties in Korea, Series A, August-
September 1952
219
Table3. Anaerobic Flora of Wounds and Blood, Series A, August-September 1952
Predominant among the 19 species isolated from tissue and associated samples were Cl. sporogenes, Cl. perfringens, Cl. bifermentans, Cl. multifermentans, and Cl. novyi. Toxigenic species(excluding Cl. tetani, which is not pertinent to the problem of wound infection) included Cl. perfringens, Cl. novyi, Cl. sordelli, and the rare Cl. histolyticum. The diverse Clostridial flora of these wounds was comparable to that reported in earlier wound studies.7 It is unusual to observe this number of cultures with all strains identifiable.
The relatively large number of patients from whom Clostridia were isolated from blood cultures was a complete surprise. Careful check on handling methods failed to reveal any obvious contaminating error in technic; and it is quite improbable that such exotic Clostridial flora should be introduced as contaminants. Blood cultures on 41 normal individuals, taken later as controls under identical circumstances as those in patients, showed no positive cultures. The predominance of Cl. sporogenes and the presence of Cl. novyi, as well as failure to isolate Cl. perfringens-the most common soil contaminant, would indicate that these results are valid as representing
220
a widespread, early bacteremia among wounded soldiers. All patients who showed positive blood cultures were seriously wounded and in shock upon arrival at the surgical hospital. In two
patients, blood cultures taken 2 or more days after wounding showed Clostridia. Five traumatic amputations and several severe open, comminuted fractures were included in this list of patients. Other observers have reported Clostridial bacteremia in septicabortion,8 and in wound cases.9
Distribution of Clostridia by Cases
The population of Clostridia when correlated with the individual cases in which strains were found gave amore significant picture of the species incidence. Out of 28 cases showing Clostridia in tissues, the number of cases harboring each species and the total number of positive tissues are listed in Table 4.
Table4. Incidence of Clostridia in Tissues of 28 Cases with Recent Wounds
In numerical order, Cl. perfringens (46.4 percent of the cases), Cl.. novyi (25 percent), and Cl. sordelli (10.7 percent) were the major pathogenic species observed. The most commonly encountered Clostridia were Cl. sporogenes (64.2 percent of the cases). Proteolytic species were preponderant among the isolates; there were 12species which could be so classified. Among the nonpathogenic species, Cl.
221
Sporogenes, Cl. bifermentans, Cl. multifermentans, and Cl. paraputrificum were of chief numerical importance. The proportion of tissues cultured which were positive for each species was relatively high. If positive cultures were obtained, they showed up to 50 percent of tissue samples for a given species.
Aerobic Flora of Wounds, Series A
A limited study of the aerobic flora of Series A. is summarized in Table 5, listing the species totals isolated from 90 tissue samples. The cultures, from the cooked meat broth used also for anaerobic enrichment, showed as high a proportion of aerobes as did direct aerobic procedures. The predominance of gram-negative bacilli and the infrequency of hemolytic streptococci was in marked contrast to the flora of older wounds in this conflict.
Table5. Aerobic Flora of Recent Wounds in Korea, Series A
Cases in which positive Clostridia cultures were obtained from tissues showed from one to nine species. The variation in number of Clostridia per patient in 27 instances is shown in Table 6.
Table6. Total Clostridia in Tissues in 27 Cases, Series A
222
Series B, Winter 1952-1953
The high incidence of Clostridia in battle wounds cultured shortly after injury, and the fact that such a limited series was sampled, prompted a more exhaustive study which was initiated in December 1952, and completed in February 1953. Eighty-seven cases were examined. Tissues were cultured in 75 of these cases, while blood cultures only were taken on the remaining 12. In marked contrast to the 93.9 percent of patients harboring Clostridia in wounds or in the blood soon after injury (Series A), only 43 of this series (57.3 percent) showed cultures positive for Clostridia. Aerobic flora remained ubiquitous, and all cases had contaminated wounds. The reduction in recovery rate in this series does not seem to have a rational basis, although several hypotheses can be offered. In an effort to correlate the wound flora with a probable source, cultures were taken of the soil of the combat zone, clothing of troops, and the skin surfaces of the soldiers tested.
Anaerobic Flora of Tissues, Series B
A total of 443 tissue specimens, including some embedded foreign bodies, were cultured from 75 patients. Of these cases, 43 (57.3 percent) harbored Clostridia in one or more wounds, in marked contrast to the 84.8 percent of cases with wound cultures positive for Clostridia in Series A. Eighty-four of the tissue samples cultured showed Clostridia (19 percent of the total). The positive series comprised 285 tissues, so that the rate of isolation of Clostridia was 29.4 percent of these specimens. There were 127 strains isolated, including 20 identified species and a group of unidentified strains. Out of the cases positive for Clostridia, an average of 2.9 strains per patient was obtained; while among tissue samples which were positive, there were 1.4 strains per sample. Thus, in terms of patients involved and the extent and intensity of tissue contamination, the anaerobic flora of this series of cases was greatly reduced from that observed during the previous summer (Series A).
Blood Cultures, Series B
Sixty blood cultures were obtained from 53 patients. Three individuals (5.6 percent) harbored Clostridia, in striking contrast to the 91 per cent with Clostridial bacteremia among the 12 patients in Series A. Four strains and three species were isolated.
Cause of Wound and Clostridia Present, Series B
The degree of wound contamination in relation to wounding agent was observed in this series in a manner similar to that. recorded in Series A. Results are summarized in Table 7.
233
Table7. Causes of Wound and Strains of Clostridia Present in 28 Patients, Series B, Winter 1952-1953
234
The earlier observation of a preponderance of contamination occurring with high-explosive and mortar-shell injuries, as contrasted to the effect of small-arms fire, was not borne out in this series. It is possible that the heavier clothing worn during the winter reduced the amount of contamination introduced at the time of wounding. The initial contamination of a wound is assumed to be imparted chiefly by the blast effect of the missile. No distinction associated with variation in this effect was apparent in this series. Rifle and machine-gun missiles occasioned the most severely contaminated wounds (55 percent), while shell and grenade injuries were positive in from 25.4 to 38.9 percent of the cases.
Total Number of Clostridial Strains Observed, Series B
The Clostridial flora, in terms of strains isolated from tissues and blood, is shown in Table 8.
The predominant species were not significantly different from those observed in the summer series. Cl. perfringens, Cl. sporogenes, and Cl. novyi were the predominant organisms. Cl. bifermentansap-
Table8. Anaerobic Flora of Wounds and Blood, Series B, December-February 1952-1953
225
peared less often, with Cl. sordelli being proportionately more common. In no other series did these closely related organisms occur with equal frequency. Cl. carnis was more common in the winter series than in the summer series. Cl. fallax and Cl. difficile were first encountered in Series B. The latter strain is extremely rare in war wounds, and it may not have been observed previously.10 A significantly large group of unidentifiable strains was observed. These strains included both toxigenic and non-toxigenic organisms, many of which were microaerophilic. Their taxonomic position could not be defined on the basis of the current classification.
The isolates from the blood, as regards Clostridia, were limited to three species and four strains. As pointed out previously, the wide discrepancy in these two series poses an unanswered question as to the actual over-all incidence of bacteremia with Clostridia in the period soon after injury.
Distribution of Clostridia in Cases, Series B
Table 9 gives a summary of the case incidence and frequency of occurrence in tissues of the various species of Clostridia in Series B. The two principal species, Cl. perfringens and Cl. sporo genes, were present in almost one-third of the cases; of the remainder, species of principal interest included Cl. novyi, Cl. lentoputrescens, Cl. paraputrificum, and Cl. carnis. Case incidence of Cl. novyi was significantly lower than in Series A. It was also distinctly below the level encountered in most studies of wounds in World War II in North Africa and Europe.3 The percentage of tissue specimens positive for each species was much lower than in Series A. This may reflect a decreased concentration of Clostridia in wounded tissue incurred under winter conditions.
Aerobic Flora of Wounds, Series B
A summary of the aerobes isolated from a representative set of 42 cases in Series B is shown in Table 10. The principal aerobes were streptococci (Group D), alpha hemolytic and nonhemolytic strains, staphylococci and Bacillus species. Corynebacteriuin strains were present in 50 percent of the cases. The numbers of Proteus strains and coliform bacilli were unexpectedly low, and Pseudomonas aeruginosa was found in only one case. These results are significantly at variance with the findings of Series A. Cultures during the summer rarely showed hemolytic streptococci or staphylococci, but organisms of the Enterobacteriaceae group were more common. Bacillus species were relatively common in both series. None of Group A streptococci were isolated, nor were they found in the remaining 45 cases cultured.
Table9. Incidence of Clostridial Species in Tissues of 43 Cases Showing Cultures Positive for
Clostridia, Series B
Table10. Incidence by Case of Aerobic Flora in 42 Cases Positive for Clostridia, Series B
227
Clostridial Flora of Skin, Clothing, and Soil
The bacterial flora of intact skin constituted an obvious potential source of wound contamination. Two sets of skin swabs were cultured, one from patients upon admission, the other from members of an infantry company returning from the battle line for shower baths. During the winter study, 66 samples were collected. Results of the two sets of cultures were pooled.
Seventeen samples of clothing, including fatigues, socks, and woolen underwear, were collected from patients on admission to the hospital.
During both Series A and Series B, a total of 50 samples of soil was collected. Specimens were obtained from rice paddies, hillsides, bunkers, and a variety of areas occupied by the line companies along an active, division front. Of course, Clostridia were most numerous in the soil; and all specimens showed strains if carefully handled to control interfering growth.
The distribution of strains isolated from these three environmental sources are shown in Table 11.
The Clostridia found in the three environmental areas (skin, clothing, and soil) presented data of interest when it was compared with the flora isolated in wounds shortly after injury. In proportions of the principal species, Cl. perfringens, Cl. sporogenes, and Cl. novyi isolated from the skin showed a close resemblance to the bacterial flora of wounds studied simultaneously. The relatively small number of samples and the relatively low isolate rate (46.9 percent of 66 samples which were positive) may explain the failure to obtain some species observed in wounds, including Cl. lentoputrescens and Cl. carnis. Among the clothing samples, 66 percent were positive for Clostridia. The preponderance of Cl. perfringens and the absence of various species observed in wounds, would indicate that Cl. perfringens may not be the major source contributing Clostridia to wounds. The absence of Cl. novyi, Cl. multifermentans, and Cl. lentoputrescens is noteworthy.
When the soil was considered, the preponderance of Cl. perfringens was of particular interest because it was not as prominent as it was in the flora of recent wounds. Cl. butyricum was much more common in soil than in wounds. Although any of the three sources could contribute to the flora of the wound, the data indicated that in terms of relative incidence, the strains isolated on the skin showed a ratio comparable to the principal species occurring in wounds. Therefore, strains found on the skin are always suspected as a source of contamination.
The total of 25 species recognized among these was greater than the total number of species found in any tissue series; and several species were not isolated from tissues, including Cl. subterminale, Cl. pasteurianum, and Cl. beijerinckii.
228
Table11. Clostridial Strains Isolated from Skin, Clothing, and Soil in Korea, 1952-1953
Discussion
Studies reported were unusual in observations on war wounds, particularly in their emphasis on cultures obtained at from 4 to 8 hours after injury and in the fact that most cases received from 300,000 to 600,000 units of penicillin intramuscularly shortly after injury. Almost all wounds were contaminated; but the incidence of Clostridia varied widely between wounds in Series A and in Series B. Of the 33 cases in Series A in which tissues were cultured, 84.8 percent har-
229
bored Clostridia, in contrast to 57.3 percent of the 75 cases in Series B. Of the cases in Series B diagnosed as Clostridial infection, only 29.4 percent of the biopsied tissues showed Clostridia. This was in contrast to (17 percent of all tissues which were positive in the summer series. A group of blood cultures in the summer series was positive for Clostridia in 91 percent of the cases, while only 5 percent. of the winter series showed Clostridia. This striking discrepancy has no valid explanation at present.
Blood cultures were more apt to be positive in the case of severe wounds with shock; and there was additional indication that the patients who failed to receive penicillin were more prone to bacterial invasion of the blood. Reports included little data on Clostridial bacteremia; and the proportion of cultures which was positive appeared to he unusually high.
The nature of the wounding agent was associated with marked differences in the degree of wound contamination in Series A, shell fragments being far more prone to produce a contaminated wound than rifle fire. In Series 13, however, there was little difference in degree of contamination in wounds due to grenades, mines, rifle or artillery fire.
The time lapse between wounding and débridement was much shorter during the last year of the Korean conflict than it was in previous wars. Apparently the mean time of 4.5 hours in the summer and 7.9 hours in the winter was within the period in which bacterial proliferation is present, but not sufficiently long to initiate the tissue defense against infection. Wounds were apt to be far more heavily contaminated when cultured one day after injury.
The primary emphasis in these observations was placed on the anaerobic flora. The aerobic flora was characteristic in Series A, showing a predominance of Enterobacteriaceae, Bacillus species, and nonhemolytic streptococci. In Series B, however, there were more staphylococci and far more hemolytic streptococci, chiefly Group D. By contrast, the various Enterobacteriaceae were less common than in Series A. Bacillus strains were prominent in both series. The failure to isolate even one strain of Group A streptococci was significant.
The anaerobic streptococci isolated were numerous enough to be of interest, but could not be studied effectively. They were grouped in the table with streptococci. Studies should be made of these organisms at some future time.
When environment was considered, the skin of patients showed a flora quantitatively similar to that of the wounds. Among anaerobes, Cl. perfringens was predominant in the samples from soil and clothing. Cl. multifermentans and Cl. bifermentans were more common
230
in the soil than in wounds. The latter two organisms, however, were a conspicuous part of the wound flora, despite some observations questioning their existence in wounds. The Clostridia in the soil were extremely varied; and 25 species were recognized among 243 strains, with several unclassified strains. All soil samples showed Clostridia. There was no significant difference between the species of anaerobes isolated from soil in rice paddies, lowlands, and hillsides. An unusual fact was the total absence of Cl.. tetani in soil; this species was readily isolated from tissues, and it was evident that the organism was extremely rare in the soil of Korea.
During the Korean conflict, fresh wounds were frequently contaminated with a diverse Clostridia. Pathogenic species were less often encountered, however, than in reports of World War II. The effect of time lapse on this factor was obvious. Wounds serious enough to require culture at a general hospital would logically be more apt to harbor Clostridia.
Summary
Two groups of patients. totaling 108, were observed for Clostridia of recently incurred wounds by culture of tissue samples at initial débridement within a few hours after injury.
Contamination by Clostridia was relatively frequent during the summer (84.8 percent of the cases studied), but less frequent during the winter (57.3 percent).
Cl. perfringens and Cl. sporogenes were the principal Clostridia isolated from wounds. Investigators observed a total of 21 species, including eight pathogenic types.
Clostridial bacteremia occurred in a total of 14 cases (19.4 percent) of the 71 cases on which blood cultures were obtained. The incidence of this bacteremia varied with the two series, but it was far higher in the summer series than in the winter series.
In the summer series, aerobic flora was predominantly Enterobacteriaceae, while the majority of the organisms present in the winter series were streptococci and staphylococci. Bacillus species were prominent in both series.
Soil cultures showed a predominance of Cl. perfringens, in contrast to skin swabs from which the most frequently recovered strain was Cl. sporogenes. In the distribution of Clostridial species, clothing samples were similar to those in the soil, while flora, on the skin was more like that of the wounds.
231
References
1. Weinberg, M., and Seguin, P.: La Gangrene Gazeuse. Masson, Paris, 1918.
2. Coupal, J. F.: "Pathology of Gas Gangrene Following War Wounds," The United States Army in the World War, Vol. XII., pp. 407-567. Medical Department, United States Army, Washington, D. C., 1929.
3. Smith, L. D.: Clostridia in Gas Gangrene. Bact. Rev. 13: 233, 1949.
4. Humphreys, F. B.: Formation of Acrolein from Glycerol by B. welchii. J. Inf. Dis. 35: 282, 1924.
5. Breed, R. S.; Murray, E. G. D.; and Hitchens, A. P.: Bergey`s Manual of Determinative Bacteriology, 6th edition. Williams and Wilkins Company, Baltimore, 1948.
6. Reed, G. B,. and Orr, J. H.: Rapid Identification of Gas Gangrene Anaerobes. War Medicine, 1: 493, 1941.
7. Rustigian, R., and Cipriani, A.: The Bacteriology of Open Wounds. J. A. M. A., 133: 224, 1947.
8. Butler, H. M.: Further Bacteriological Studies of Severe Clostridium welchii Infection Fol1owing Abortion. J. Obst. Gynec., British Empire, 50: 105, 1943.
9. Stock, A. H.: Clostridia in Gas Gangrene and Local Anaerobic Infections During the Italian Campaign. J. Bact. 54: 169, 1947
10. MacLennan, John: Personal communication, 1954.
