OBSERVATIONS ON THE PATHOLOGY AND BACTERIOLOGY O F GAS GANGRENE.l

By KENNETH TAYLOR, M.A., M.D., Pathologist, The Bobert Walton Goelet Research Fund (H6yital Compldmentaire, Ris-Orangis, 8. & 0.1.

(PLATES XXVI1.-XXX.)

CLINICAL CASES AND EXPERIMENTAL INFECTIONS.

THE following informal paper on the work of this laboratory is published in the hope that some of the results may lead to a clearer understanding of the problems presented by the cases of infection with the 6. aerogenes capulatus among the wounded.

CLINICAL MATERIAL.

The study of the pathology of “human” gas infections covers fifteen autopsies of clinical gas gangrene in which this infection was evidently the cause of death, and eleven biopsies (eight arms and three legs).

Seven of the fifteen cases coming to autopsy gave a pure culture of the 6. aerogenes capsnlatus from the heart blood ; in one other case the same bacillus was associated with streptococcus. Blood cultures taken shortly before the death of these patients were sterile in every case. The other six autopsies gave positive cultures of the bacillus from the wounds or subcutaneous tissues. In nine cases the autopsies were performed within five hours of death, and in four cases within one hour, three of the latter showing the bacillus a t that time in the heart blood. In only one case was the bacillus obtained from the blood of a living patient, although many cultures have been taken within an hour of death. The one exception was in a case of gas gangrene of the thigh, which ultimately recovered. In this case the bacillus was obtained twice in culture from blood taken from the arm vein shortly after an operation for drainage of the thigh ; it was prob- ably a post-operative and temporary invasion of the blood by way of the vessels cut in the treatment of the thigh, and the blood proved sterile forty-eight hours later. Invasion of the blood by this organism

I Received January 26, 1916.

OBSER V'A TIONS ON GAS GANGRENE. 385

during life is apparently rare, although the fact that one case of gangrene of the shoulder developed a metastasis in the buttock before death tends to show that such invasions may occur. I n two cases the bacilli have been found at autopsy in the liver and spleen-probably the result of a terminal invasion. In the guinea-pig, however, judging from the changes in the internal organs on autopsy immediately after death, ante-mortem septicsmia seems to be usual.

The internal organs from nearly all the cases coming to autopsy have been examined microscopically, as well as the skin, subcutaneous tissues, and muscle from both autopsies and biopsies; in all of the fifteen autopsies and eleven biopsies the symptom of gaseous disten- sion of the muscle has been present, and in all the B. aerogenes capsulatus has been obtained by culture or found in microscopical sections of the tissues. I n no case where the autopsy was performed within two hours of death were macroscopic gas excavations found in the internal organs.

EXPERIMENTAL MATERIAL.

The animals used in the experimental infections were guinea-pigs and occasionally rabbits. For inoculation 48-hour broth cultures of the B. aerogenes capsulatus were most successful. Eleven strains, isolated from different clinical cases, were employed. The methods of inoculation were subcutaneous, intraperitoneal, and intramuscular, the latter proving the most reliable. The dosage was varied to suit the purposes, but a reliable lethal dose for ordinary work appeared to be 1 C.C. or less from a 48-hour culture for all strains but one, although for certain strains 0.1 C.C. was uniformly fatal.

In nearly all cases the animals were autopsied at once or iced and autopsied within a few hours. Specimens of muscle and of some of the internal organs were preserved in formalin and subsequently examined microscopically. At the time of autopsy smears were made, usually from the subcutaneous tissues, peritoneum, and from the heart blood. Aerobic and anaerobic cultures from the heart blood were taken as a routine. The Gram stain was employed for the examination of smears, and hEmatoxylin and eosin, or a combination of haxnatoxylin, eosin, and Gram, for the microscopical sections.

Culture from the heart blood taken in thirty-four autopsied animals has given positive growth of the B. aerogenes eapsulatus twenty- five times. In the other nine cases, in which culture from the heart blood did not develop this organism, it was obtained from cultures taken from the peritoneum.

ISOLATION OF BACILLUS.

The following method has been found of service in isolating the gas bacillus from wounds :-

386

Six or more slender test tubes are filled with about 2 c.c. each of a dextrose broth medium, heated in a water-bath t o dispel the air, cooled suddenly and placed in series. The first tube is inoculated from the pus, and the whole set placed in the incubator until bubbles of gas begin to form in the inoculated tube. As soon as this occuis, one drop of the culture is transferred to the second tube in the rack ; the incubation is then continued and the tubes inoculated from each other at intervals of about half an hour. As the bacillus rapidly outgrows most of the organisms commonly present in pus, the last tube gives, not infrequently, a pure culture of the bacillus, which may be verified if necessary by transferring to solid media (Plate XXIX. Fig. 10).

INCIDENCE OF THE BACILLUS IN WOUNDS.

During the past four months 216 wounds have been examined bacteriologically, smears and aerobic and anaerobic cultures being made in each case. Of the 216, 1.30 (or 6 1 per cent.) have shown the presence of a gas-producing organism resembling morphologically the B. aerogenes capsulatus. The cases received during this period were for the most part old ones, the examinations being made a week or more after the date of injury. In the fresh cases the incidence of the gas bacillus reached as high as 75 per cent.

Of the 1 3 0 cases in which the gas bacillus was found, clinical symptoms of gas infection were present in forty-five (or 35 per cent.). In all cases in which the symptoms pointed to gaseous infection, the bacilli were found in culture from the wounds, and usually in the neighbouring muscle when examined microscopically.

No difference in the incidence of the bacillus between the spring and summer months has been noticed, nor between the different localities from which the patients have come.

No characteristic bacterial flora has been found in the wounds infected with the gas bacillus.

VIRULENCE OF GAS-FORMING BACILLI.

Eleven strains of anaerobic gas-forming bacilli, isolated from separate clinical cases, were identified as B. aerogcnes capsulatus by Drs. George Benet and L. B. Rogers of the Harvard Unit a t the American Ambulance, Neuilly, Paris, and their virulence tested in this laboratory. Ten of these proved fatal to guinea-pigs on intramuscular inoculation of 1 C.C. or Iess of a 48-hour broth culture. The other strain failed to kill in any amount tried ; there was, however, some uncertainty as to the identity of this organism, as it did not show all the characteristic features of the B. aerogenes capsulatus in culture.

I n testing the virulence of these various strains, fifty-two guinea- pigs were inoculated ; of these forty-four died and eight recovered.

OBSERVATIONS O N GAS GANGRENE. 381

Of the eight that recovered, three had received intraperitoneal in- jections, three subcutaneous injections, and two intramuscular injections. These last two were inoculated with the strain referred to as being of uncertain identity. The majority of the inoculations were intra- muscular, and, with the exception of these two, all proved fatal. The average duration of survival of forty-two animals after inoculation was twenty-four hours.

No relation has been found between the virulence of the organism as manifested by clinical symptoms in the patient and its ability to kill a guinea-pig. The pathological conditions produced in the animals have been similar with all the strains, no matter what the type of the cliuical case. Thus, those strains isolated from the cases in which cedeina was the marked feature, produced gas readily when injected into the muscle of the thighs of guinea-pigs.

IMMUNE REACTIONS.

Agglutinin experiments with the serum from a number of clinical cases, and with the serum from inoculated laboratory animals, have been performed both during the active stage of the disease and after recovery. So far it has been impossible to demonstrate the presence of agglutinins, or of any bactericidal properties in the blood in any case. Occasionally active phagocytosis by leucocytes in the sub- cutaneous tissues and peritoneum of laboratory animals has been seen, but never in the pus from infected wounds.

In order to determine whether or not immunity to the infection had been acquired by the laboratory animals which had recovered from active infections with sublethal doses of various strains of the bacillus, twelve guinea-pigs which had previously suffered from the disease were inoculated along with three fresh animals for control- All received a dosage of the B. aerogenes capsulatus just under that invariably fatal. Only one of the three control animals and two of the previously treated animals survived. It seemed evident that no active immunisation had occurred in this set of animals.

MACROSCOPICAL PATHOLOGY.

Localisation+-Nearly all the cases developing clinical gaseous gangrene had received injuries to the thigh or the arm associated with open fracture of the bone. Of the twenty-six cases examined, sixteen showed fracture of the femur, six fracture of the humerus, and one fracture of the ilium.

Muscles.-The B. aerogenes capsulatus is essentially a muscle feeder (Plate XXVIII. Fig. 7)) and no cases of subcutaneous infection without previous muscle invasion have been seen among the patients. The pathology is, therefore, chiefly limited to muscle destruction.

3aa KENNETH TAYLOR.

Great swelling, gas in the muscle, and subcutaneous cedema were always present. The evident tension of the muscle tissues was demonstrated by the bulging of the muscle substance, through wounds in the muscle sheaths, with a tendency towards eversion of the muscle. The colour of the muscle was usually dark red where exposed, but pale, dull pink and dry in thb deeper portions. The groups of fibres were frequently widely separated from each other by air spaces {Plate XXVII. Fig. 1). The muscle substance frequently crepitated and on section cut crisply, somewhat like lung tissue. Specimens free from fat usually floated in water ; in fact, in several cases the entire arm or leg floated when immersed. There was usually a small amount of thin, hEmorrhagic pus along the muscle sheaths. Gas generally escaped freely from the wound and from the more remote portions of the muscle when the muscle sheath was incised. The gas had a heavy, sweetish odour and would often explode with a snap when a lighted match was applied.

Vascular system.-The blood at autopsy was, as a rule, fluid, rather light in colour, and the larger veins and heart frequently contained a considerable amount of gas. Pinkish staining of the intinia of the cavities of the heart and the larger arteries was usual. Thrombosis of important blood vessels was found in four of the twenty-six cases.

Skin.-Subcutaneous cedema was usually associated with the muscular distension ; and incision of the skin allowed a free flow of pink-stained serum. In nine cases subcutaneous crepitation, due to the escape of gas into the edematous subcutaneous tissues, was present. The same cases showed discoloration of the skin, due to the subcutaneous hzemorrhage, and characteristic blebs containing thick hemorrhagic fluid. The four symptoms of subcutaneous crepitation, cedema, discoloration of the skin and blebs were iisually associated in the same cases, and represent conditions which we have grouped under what we term the " second stage " of the infection.

Liver,--No characteristic macroscopical changes in the liver could be demonstrated in the fifteen cases examined. In guinea-pigs, however, macroscopic vacuoles were frequently found in the livers of animals autopsied immediately after death, and the presence of the bacilli readily demonstrated by microscopical examination of the lesions.

Spleen.---No characteristic changes in the spleen could be demon- strated. It seldom showed the characters expected in septic conditions. I n guinea-pigs macroscopic cavities and abscesses harbouring the B. aerogenes capsulatus were not infrequent.

Kidney.-The kidneys were usually rather large, the capsules stripping readily, and the parenchyma was frequently pink-stained and bulging from the cut surface.

The other internal organs did not show any consistent characters which could be attributed to the gaseous infection.

OBSERVATIONS O N GAS GANGRENE. 389

MICROSCOPICAL PATHOLOGY.

From the examination of the tissues obtained from autopsy and operation in the hospital, and from experiments on animals, the microscopical picture showed the following characters :-

Distribution of bacteria.-In the microscopical sections of muscle the B. aerogenes capsulatzu was usually found in great numbers. It was universally Gram-positive except when forming spores. The latter generally failed to retain the Gram stain, and were sometimes rather obscure even when numerous. This variation in the staining reaction may account for the occasional statement that the bacillus rarely forms spores in tissue. Nearly half of the specimens we have seen have shown spores in some of the tissues examined. Phagocytosis has not been observed except in the animal experiments, and in one case in an infection of the knee-joint. The bacteria were usually most numerous in the muscle and along the muscle sheaths. They were distributed diffusely between the individual fibres with, frequently, larger masses between the smaller fibre bundles. They were rarely found within the fibre itself. They were occasionally present in the muscle layers of the walls of arteries in the neighbourhood of the infection. Less frequently the bacillus has been found in the blebs of the skin, in the subcutaneous tissues, or in the deeper layers of the epidermis. In the latter situation their presence appeared to be associated with the incipient formation of blebs (Plate XXIX. Fig. 11). In one case the bacillus was found in the spleen ; in another, in the liver and lung ; and in a third, in the cerebro-spinal fluid at autopsy. I n one case it was found in pure culture in free blood in the.abdomina1 cavity ; in another, in the pleural exudate in a case of empyema ; and in a third, in the exudate from a knee-joint. In the animal experi- ments, however, the bacilli were frequently found in the spleen and liver in addition to the muscle and subcutaneous tissue.

Muscle.-Specimens of muscle examined from clinical cases and animal experiments have shown very similar characteristics. I n the early stages of the infection or in regions somewhat remote from the chief infective processes, microscopical examination has shown a marked swelling of the individual fibres. They have usually been crowded together, obliterating most of the intervening space. Asso- ciated with this condition, vacuolation of the nuclei and some separation and disorganisation of the fibrill= could be noted. The swollen fibres also stained irregularly (Plate XXVIIT. Fig. 5).

The specimens from a more advanced stage of the infection showed the individual fibres separated widely from each other (Plate XXVII. Fig. 2, and Plate XXVIII. Fig. 4)) as compared with normal muscle (Plate XXVII. Fig. 3). Between them was frequently seen an accumulation of eosinophilic exudate, often interrupted by air spaces and containing muscle ddbris, many bacteria, and a small number of

390 KENNETH TAYLOR.

yolymorpho-nuclear leucocytes. The fibres themselves frequently showed rupture of the fibre sheaths with solution or disintegration of the fibre itself.

Other sections from a more advanced process showed complete dissolution of the muscle fibres, which were merged into a more or less homogeneous mass of eosinophilic ddbris showing numerous air spaces and diffusely infiltrated with the bacilli (Plate XXVIII. Fig. 6). The polymorpho-nuclear infiltration appeared t o increase rapidly and become quite marked in the older lesions.

Muscle sheath.-The sheaths of the muscle groups and the con- nective-tissue partitions between the smaller bundles of the fibres usually showed a marked cellular exudate and numerous bacteria. The connective-tissue elements did not seem to be especially affected by the process. Areas of muscle adjacent to the sheath frequently showed the early stages of degeneration described above without the presence of bacilli within the muscle.

Blood vessels.-Blood vessels in the affected areas have usually shown more or less characteristic changes, beginning with a swelling of the muscle fibres of the arterial wall and frequently the deposit of a brownish pigment. Eyosion or sloughing of the intima was of frequent occurrence. The walls of vessels more extensively involved showed loosening of the supporting tissue elements and further dis- integration of the muscle layer (Plate XXIX. Fig. 9) with a condition frequently resulting which permitted of the escape of cells from the blood into the perivascular tissues. It is this process that allows of the hieniorrhage into the subcutaneous tissues which is responsible for the discoloration of the skin. As the process continues, complete disintegration of the entire wall of the vessels results (Plate XXX. Fig. 12).

Skin.-In the majority of cases the skin showed a condition of subcutaneous cedema with swelling of the connective-tissue fibres and, frequently, a swelling and vacuolar degeneration of the basal cells of the epidermis. Haeniorrhage following the pathological changes in the subcutaneous blood vessels was frequently a prominent factor throughout the subcutaneous connective tissues and fat (Plate XXX. Fig. 13). Finely granular deposits of blood pigments were also occasionally seen. The limits of the subcutaneous hzmorrhage corresponded to the areas of discoloration of the skin. Occasionally infiltration of the basal membrane and invasion of the epidermis by the bacilli were remarked. In these cases collections of bacilli and serum could be found between the deeper cells, resulting in a separation of the more superficial layers and incipient bleb formation.

The subcutaneous fat has always appeared normal except for occasional infiltration with red blood cells.

Liver.-In most cases dying from the infection and in most of the experimental infections the liver has shown an abnormal picture.

OBSEX VATIONS ON GAS GANGRENE. 391

Marked congestion and engorgement of the central veins have usually been present. The parenchyma (especially the central portions of the liver cords) frequently stained irregularly, and in many cases a diffuse solution of the cytoplasm appeared to have taken place, often leaving large portions of the lobules with a meshwork or skeleton of connective tissue in which very little parenchyma persisted. In two of the cases coming to autopsy and in several of the experimental infections, microscopical cavities (Plate XXIX. Fig. 8) could be found, about the margins of which the B. aerogenes eupsulatus could be seen. These specimens showed no leucocytic reaction except a slight perivascular infiltration of polymorpho-nuclear cells.

Spleert.--Sections from the spleens of autopsied cases and experi- mental animals showed a condition of passive congestion. In two specimens from guinea-pigs, definite abscesses were present with the bacillus in the necrotic tissue.

Lung.-In one case coming to autopsy and in several of the specimens from experimental infections, the bacilli were found in local areas of broncho-pneumonia with early abscess formation.

Kidneg.--The usual picture, both in the natural and experimental infections, was that of an early acute parenchymatous nephritis such as may frequently be seen resulting from extensive burns or other acute intoxications.

Adrenuk-The adrenal bodies have usually shown a marked con- gestion and occasionally bzmorrhage into the medulla. In one case the bacilli were found in the medullary portion, and marked degenera- tion of the parenchyma was present.

DIAGNOSIS.

Muscle distension has been a consistent feature in all the clinical cases coming to autopsy or amputation.

In some obscure cases the deep muscles only have been involved and the tension has not been obvious on the surface. Frequently i t has been possible to demonstrate the gas in the deep muscles by diagnostic aspiration, using a 5 C.C. or 1 0 C.C. glass syringe and a long needle. One or 2 C.C. of water is drawn up into the barrel of the syringe and the air expelled. The needle is then inserted into the deep muscles and the plunger withdrawn. If gas is present about the needle-point, bubbles appear in the syringe. Care must be taken to assure tight joints between the needle and syringe. This method has been practised with success in the wards, and may occasionally be of service.

About one-third of the cases have shown the generally associated symptoms of subcutaneous crepitation, blebs, and discoloration of the skin.

The area of subcutaneous crepitus has borne no consistent relation

392 KENNETU TAYLOR.

to the region of the muscle involvement, but was usually only a sign that escape of the gas from the muscle had occurred. Subcutaneous crepitus over the foot has been seen from a focus limited to the upper part of the thigh. Cultures taken from the distant emphysematous areas have usually shown no bacilli.

Subcutaneous cedema was usually considered a sign of venous obstruction, often due to intramuscular pressure. Cultures from these areas were usually sterile.

The B. aerogenes capsulatus could always be demonstrated in the wound by anaerobic cultural methods.

Gas escaping from the wound was, with rare exceptions, positive evidence of the activity of the bacillus.

Glassi$eation of the Stages i n the Pathology of the Infection.

After watching the cases aud examining specimens bacteriologically and pathologically, i t would seem tha,t the course of many of the infections by the gas bacillus might perhaps be divided into more or less definite stages. It is suggested that the fatal cases pass through the following phases :-

Phase ~.-DORMANT STAGE-Localised infection of the wound. Gas bubbles may he present, and smears and cultures show the presence of the B. aerogenes capsulatus.

Phase 2.-STAGE OF ACUTE GASEOUS DISTENSION.-The result of obstruction to the escape of the gas generated locally in the wound. This obstruction appears to be the factor determining the development of a malignant from a comparatively innocent infection. During this stage are seen the swelling, subcutaneous edema and discoloration of the skin. The death of the affected muscles results from the anpmia due to the intramuscular pressure of the gas and from the exotoxin of the bacillus. Pernianent vascular obstruction, such as thrombosis, may also occur a t this stage. The focus of gas formation and other hacterial activity may still be limited to the region of the wound.

Phase 3.-ExrLos1v~ STAGE.-This is characterised by the rapid extension of the swelling associated with subcutaneous crepitus, and is probably the result of the rupture of the muscle sheath and the escape of the gas contained within it into the loose and usually edematous subcutaneous tissue. The rapid expansion of the gas probabIy aids in the dissemination of the bacteria.

Phase 4.-sTAGE OF SYSTEMIC INTOXICATION.-This is probably due to the absorption of the highly toxic by-products of the autolysis of the muscle killed during Phases 2 and 3, augmented by the absorp- tion of the exotoxin produced by the bacilli.

The rate of production of the gas and the high pressure i t map induce are discussed in a previous paper on “ Factors responsible for Gaseous Gangrene,” Lancet, London, 1916, vol. i. p. 123.

OBSERVATIONS O N GAS GANGRENE. 393

Phase ST STAGE OF SEPTICAM1A.-The terminal invasion of the blood by the bacilli, when it happens, is probably a very late pheno- menon occurring approximately at the time of death.

The first phase, or the stage of local infection in the wound with the free escape of a small amount of gas, represents a common type seen in the fresh wounds. The great majority of cases do not pass beyond this stage. When, however, for any reason, the free escape of the gas becomes interfered with, the second phase, with its clinical picture of gas gangrene, develops rapidly and passes frequently within a few hours into the third stage. If sufficient drainage be established at this point for the escape of the gas, and too large a mass of muscle has not been destroyed, recovery is usual; otherwise the patient passes shortly into the stage of intoxication and dies within a few hours-frequently before the fifth or septiczmic stage is reached.

The possibiIity of gas embolism cannot be excluded in all cases, but seems improbable as a cause of death.

In connection with this paper it is my pleasure to acknowledge the laboratory's financial assistance from Mr. Robert Walton Goelet, which has made the work possible, the interest and valuable ctiticism of Dr. Joseph A. Blake, and the active co-operation of Dr. Bertram H. Buxton and my assistants, Miss Mary Davies and Miss Nathalie Bach.

DESCRIPTION OF PLATES XXVI1.-XXX.

PLATE XXVII. FIG. 1.-Specimens of gluteus maximus muscle hardened in formalin and sectioned.

( b ) Section of muscle showing gaseous infiltra- (Note that the subcutaneous tissues appear approximately normal.)

(Note separation Subcutaneous tissues show hemorrhage into the subcutaneous fat.}

FIG. 2.-Specimen of extensor muscle from thigh of guinea-pig inoculated thirty hours Leg hardened entire

(Note separation of muscle fibres and accumulation of

(a) Section of normal muscle. tion. ( c ) Similar specimen showing effect of gaseous distension. of fibres. (d) Cross section of (c). (About natural size.)

previonsly with the B. aerogenes capstdatw of Welch. and sectioned later. serum between them.) ( x 25.)

FIG. 3. -Normal muscle from the same region for oomparison with Fig. 2. ( x 25.)

PLATE XXVIII.

FIG. 4.-Specinien of extensor muscle of thigh showing separation and various stages of

FIG. 5.-Longitudinal section of specimen similar t o those shown in Fig. 4. (Rote basket- Same may be seen in Fig. 4 cut trans-

(Note the fragments of degenerated muscle

(Oil immersion lens.)

degeneration of individual fibres.

like skeleton of degenerated fibres. versely.) ( x 150.)

FIG. 6.-Muscle debris with gas bubbles. fibres persisting.

FIG. 7.-The gas bacillus feeding on an individual muscle fibre. ( x 350.)

( x 50.)

This dGbris was packed with the gas bacillus.) ( x 50.)

394 OBSERVATIONS ON GAS GANGRENE.

PLATE XXIX. FIG. %-Liver of guinea-pig dying from inoculation with the gas bacillus. (Picture shows

gas excavations frequently found in the organs of animals but not seen in the internal organs of any of the patients dying from gas gangrene on whom autopsies were performed shortly after death.) ( x 25.)

FIG. g.-Port.ion of wall of a branch of the femoral artery from a case of gas gangrene infection of the thigh. (Note the beginning degeneration of the muscle layer as shown by the disintegration of the fibres and the shrinkage of their nuclei.) ( x 360.)

Frc. 10.-Typical culture of the B. aerogeizes cq~,srsZatus of Welch. (Oil immersion.)

FIG. 11.-Section of skin from thigh showing a smallnest of the gas bacilliin the epithelial

( x 800.)

layer. (This illustrates the early stage of bleb formatiou.) ( x 250.)

PLATE XXX. FIG. 12.-Thrombosis of a small artery in the thigh with complete degeneration of the

It is surrounded by muscle showing extensive degeneration muscular wall. due to infection with the gas bacillus.

the subcutaneous fat. associated with the infection.)

( x 50.)

FIG. 13.-Section of skin from a discoloured area showing cedema and hemorrhage into (Cause of the discoloration of the skin frequently

( x 25.)

JOURNAL OF PATHOLOGY.--VQL. XX.

Fro. 1.

F I G . 2.

PLATE XXVII.

VK. 3.

JOURNAL OF PATHOLOOY.-V~L. XX.

V I G . 4 .

F I G . 6.

PLATE XXVIII.

F I G . 5 .

FIG. 7.

JOURNAL OF P A T ~ O L O G Y . - ~ O L . xg.

F I G . 8.

F I G . 10.

WIG. 9.

Fro. 11.

JOURNAL OF PATHOLORY.-VOL. XX. PLATE XXX.