616. 126-002-022. 7 A CASE O F ENDOCARDITIS DUE TO A DIPH-

THEROID BACILLUS STRUCTURALLY AND CULTURALLY RESEMBLING THE DIPHTHERIA BACILLUS.

JAMES SUTHERLAND and RUPERT A. WILLIS.

Prom the Baker Medical Research Iwtitute, Alfred Hospital, Melbourne, Australia.

(mTE XXXII.)

HOWARD (1893) reported a case of acute ulcerative mitral and aortic endocarditis due to B. diphtherice, which organism he obtained in pure culture from the mitral valve, the lungs, liver, spleen and kidneys. There was nothing in the clinical history of the patient to indicate a preceding attack of diphtheria. Howard and others who studied the bacillus were unable to detect any morphological or cultural difference between it and B. diphtherice. The only point of difference was that it failed to kill guinea-pigs and rabbits.

Roosen-Runge (1903) described a fatal case of tricuspid and aortic endocarditis due to an organism structurally and culturally identical with the diphtheria bacillus. This organism was obtained during life both fiom blood cultures and from the discharge from an empyema cavity; and at autopsy the same organism was identified microscopically in the endocardial vegetations. As in Howard’s case, there was no clear history of preceding diphtheria, and the bacillus was non-virulent for guinea-pigs.

There are several records of cases of endocarditis in which diphtheroid bacilli have been cultivated from the blood stream or from the endocardial vegetations, but in which cocci have also been present (Tow and Wechsler, 1932). In these caaes the ztiological role of the diphtheroid organism is of course uncertain .

The case here described is very similar to those reported by Howard and Roosen-Runge. The diphtheroid bacillus was cultivated during life fiom the blood aa well as from the vegeta- tions on the heart valves post mortem. No other organism was cultivated or seen in histological sections.

127

128 J . SUTHERLAND AND R. A . WILLIS

CASE REPORT.

Clinical history.

C. R., a male of 18, was admitted to the Alfred Hospital on 4th September 1935 with a provisional diagnosis of infective endocarditis. He stated that he had had rheumatic fever and diphtheria during childhood but no serious illnesses since. On 14th August, i.e. three weeks before admission, he had a sudden severe rigor lasting about fifteen minutes, accompanied by headache and backache. The following day he felt weak and sick, and he was examined by his private doctor who found no physical abnormality except a temperature of 104' F. and who regarded the illness &s influenza. The pyrexia continued, but gradually declined until on 19th August it was 100" F. with pulse 72. On the two following days the patient was afebrile, though the general aching continued ; but when seen again by his doctor on 24th August his temperature was 104" with pulse 90 and he was delirious. There were still no abnormalities on general physical examination. He was transferred to a local hospital for observation. O n the night of 25th August he had a rigor with rise of temperature to 105' F. On the following day swinging pyrexia continued, his leucocyte count was 7500 per c.mm. and he suddenly developed severe colicky pain in the lower abdomen. This con- tinued on the 26th, but there was no indication for surgical intervention. The Widal test gave a negative result and the leucocyte count was 12,500. On the 27th the abdominal pain was better, but another rigor occurred and there were rigors also on each afternoon or evening of the subsequent days up to his admission to the Alfred Hospital on 4th September.

Examination on admission showed temperature 99", a regular, soft pulse of 78, respiration rate 18. The only abnormalities disclosed by general examination were slight tenderness in the right renal angle, hyperactivity of all deep reflexes, some linear hamorrhages beneath the nails and some doubtful petechie on the back.

During the remainder of the patient's life (nearly four weeks) he had an irregularly swinging temperature ranging between 97' and 104", pulse 78 to 130 per minute. He had several rigors. The main changes noted on physical examination during his illness were as follows. On 19th September the Grst cardiac sound was roughened and the aortic second sound accentuated. On 25th September grunting respiration appeared, and on the 26th, blood-stained sputum and dullness and diminished breath sounds over the axillary area and base of the left lung, but without adventitiae. Examination of the heart now revealed a loud systolic bruit. Death occurred on 30th September.

The results of special investigations during the illness were as follows. Blood culture on 6th September yielded a pure growth of a diphtheroid bacillus resembling the Klebs-Loeffler bacillus, and the same organism was isolated on several subsequent occasions (vide infra). Cultures made on inspissated serum from a throat swab, a nbsal swab and from the sputum on 16th to 21st Sep- tember contained no diphtheria bacilli, and a culture made from the centrifuged deposit from the urine on 25th September was also negative. The leucocyte count on 4th September was 10,000 and on 18th September 18,000 per c.mm. Albuminuria appeared first on 18th September and persisted until death.

ENDOCARDITIS DUE TO A DIPHTHEROID 129

In addition to treatment along general lines, the patient was given diph-

13th September, 20,000 units intravenously and 30,000 intramuscularly. 16th ,, 30,000 units intravenously. 17th ,, 20,000 ,) ,, daily. 18th ,, 20,000 )) I , ,, 19th ,, 20,000 77 7 7 7,

21st ,, 20,000 ,, J 2

27th ), 8,000 7 7 97

28th 7 7 16,000 ,) 77

29th ,) 8,000 97 7,

theria antitoxin as follows :-

The serum administration appeared to have little or no influence on the symptoms and course of the illness.

Post-mortem $dings. A post-mortem examination was performed on October 1st.

The lungs appeared normal. The heart showed pronounced general enlargement and weighed 14 oz. ; the myocardium was flabby and the chambers were dilated, A large mass of grey friable vegetations adhered to both cusps of the mitral valve and projected upwards into the left atrium. There was some recent destruction of the cusps but no indications that they had been the seat of previous disease. The remainder of the endocardium was normal. The liver was large (90 oz.) but appeared normal. The spleen weighed 19 oz. and contained several large mottled orange and yellow infarcts ; the remainder of the splenic pulp was red and soft. The right kidney weighed 10 oz. and presented many confluent firm yellowish areas of infarction, mainly in its upper half, the largest nearly 5 cm. in long diameter. The remainder of the renal tissue was pale and its pattern somewhat obscured. The left kidney, 12 oz., was pale and had a poor pattern ; it contained only one or two tiny areas of infarction. The adrenuls, alimentary tract, pancreas, pelvic organs, testes, lymph glands and brain all appeared substantially normal. The thyroid was slightly enlarged and firm and showed general colloid accumulation.

Histological Jindings. The cardiac vegetutim consisted of masses of unorganised

fibrin mingled with packed masses of Gram-positive bacilli morpho- logically similar to those cultivated from the blood during life. These bacillary masses formed fully half of the bulk of the vegeta- tions (fig. l). The cardiac muscle fibres were well preserved and showed no obvious degenerative changes.

Spleen. The infarcted areas consisted of granular amorphous material scattered through which were crystals and granular masses of blood pigment. At their periphery the splenic tissue was congested and pigmented and showed some degree of fibrosis. JO-. OF PAW.-VOL. XUII. N

130 J . SUTHERLAND A N D R. A . WILLIS

Gram-stained sections revealed occasional colonies of diphtheroid bacilli in the infarcted areas but none could be found in the healthy parts of the splenic pulp.

Kidneys. The infarcted areas consisted partly of diffusely staining hyaline material in which the ghost-like outlines of tubular arrangement could be detected, and partly of masses of granular amorphous material, some of which stained darkly with hsema- toxylin, suggesting early calcification. The renal tissue throughout exhibited pronounced idammatory changes, i .e. dilatation of vessels, and collections of lymphocytes, polymorphonuclear cells, monocytes and plasma cells. The glomeruli showed increased cellularity and they and their pericapsular tissues were studded with leucocytes. The convoluted and straight tubules were for the most part well preserved but there was patchy granular degeneration of the epithelium in places. The lumina of many of the tubules contained large numbers of leucocytes, most of which were of the polymorphonuclear variety and were laden with granules of brown pigment. The connective tissue stroma of the kidney appeared edematous, but fibroblastic proliferation was slight or absent, except a t the margins of the infarcts where there were conspicuous zones of young active fibrous tissue. In Gram- stained sections no bacilli could be found in either the infarcts or the renal tissue.

The liver showed general vascular engorgement and the portal tracts contained small collections of lymphocytes and a few poly- morphonuclear cells. The liver cells appeared healthy and free from degenerative changes. No bacilli could be discovered in Gram -s t ained sections.

The adrenals showed small groups of lymphocytes and mono- cytes scattered through both cortex and medulla, the parenchyma cells of which, however, appeared healthy.

The thyroid was the seat of slight colloid goitrous changes with some general increase in fibrous stroma.

Bacteriological Jindings.

The first blood culture, made on 6/9/35, resulted in the growth of a Gram-positive diphtheroid in four different culture media. At first it was thought that it might have been only a skin con- taminant. On 11/9/35 and 12/9/35 three series of blood cultures were made, special precautions being taken to avoid skin con- tamination. All media in each series again gave pure cultures of the same Gram-positive diphtheroid. An estimate of the number of bacteria present in the patient’s blood was made on 12/9/35 by plating 0.2 C.C. of blood on agar and counting the colonies that developed. In one plate 22 colonies appeared and

ENDOCARDITIS DUE T O A DIPHTHEROID I31

in the other 23, equivalent to 110 and 115 respectively per C.C.

of blood, a significant number of viable bacteria in the blood. It was concluded that the diphtheroid was actually in the blood stream and not simply a skin contaminant. On 13/9/35, 16/9/35 and 24/9/35 further samples of blood for bacterial counts were taken just prior to intravenous administration of diphtheria anti- toxin. The presence of 104, 360 and 1570 colonies per c . ~ . of blood respectively was demonstrated. A sample on 19/9/35 failed to give any growth from 0.2 C.C. of blood. This finding is difEcult to account for, since it was the only occasion when a negative result was obtained.

On 1/10/35 cultures from the heart vegetations, spleen, kidney and liver were made post w t e m . Innumerable colonies of Gram- positive diphtheroids were cultivated from the vegetations on the heart but only a few odd colonies from the spleen, kidney and liver. Fig. 1 shows how the vegetations were packed with Gram- positive diphtheroids in colony-like masses. No other organisms were found.

Identiificution of the GramTositive diphtheroid cultivated. Five cultures of the diphtheroid, four from the first four blood

cultures and the fifth from the heart vegetations post w t e m , were studied in parallel and found to be similar.

Morphology. Gram-stained direct smears of the blood cultures showed a Gram-positive diphtheroid, not particularly suggestive of C. diphtherice. Subcultures made in Loeffler’s inspissated serum, however, revealed marked similarity to C . diphtherice when stained by Loeffler’s methylene blue or Mallory’s stain (figs. 2 and 3). Some cultures on inspissated serum showed typical metachromatic and some polychromatic staining, while others showed marked involution, some individuals being small, almost coccal, and others in the same culture large and bulbous. The cultures have con- sistently preserved their diphtheroid morphology and have never been suggestive of streptococci.

Cultural characters. Growth on agar was poor after the first day’s incubation at 37” C. but increased considerably on the second day’s incubation. The addition of serum or blood resulted in quicker and much more luxuriant growth. No pigment was produced and gelatin was not liquefied. In broth the usual type of growth for diphtheroids was found, viz., a granular deposit, almost clear supernatant fluid and slight pellicle formation after some days’ incubation.

Fermentation reactions. Glucose, fructose, galactose, maltose, and glycerol were fermented, without gas. Lactose, mannite and sucrose were unchanged. These reactions agree with those of C. diphtherice, virulent or avirulent forms (see table). The sugar

132 J . SUTHERLAND AND R. A . WILLIS

*Virulent G. diphtherim *Non-virulent C. diph-

*Group V diphtheroids therim

(Berrett, 1924-25) Endocarditisstrain .

media used were of two varieties, ( a ) ordinary peptone water sugar medium enriched by the addition of 10 per cent. horse serum and ( b ) Hiss's serum water medium. Sterilisation of both sugar media

+ + + - - + + + + + - + + + - + + + -

-

- -

+ -

TABLE.

Pemntation reactions compared.

1 Qlucose. Maltose. 1 "2:;- !Suerose./lactare.( Dextrin.

I- I- I- /-/-I-

From " Diphtheria " (Medical Reaearch Council), 1923, p. 411.

was effected by atration through Seitz filters to avoid heat hydrolysis of the sugars, and the serum was heated separately at 60" C. for half an hour to destroy any sugar-splitting enzymes that might be present (Tenbroeck, 1920). The indicator used was brom-cresol purple. Hiss's serum media were not clotted even after a week's incubation at 37" C.

Barratt (1924-25) classified diphtheroids in a number of groups, group V being of particular interest, since its members give sugar reactions similar to those by C. diphtheria? except that dextrin is not fermented. Barratt, however, points out the unreliable nature of commercial samples of dextrin in respect of its fermentation properties and queries whether the strains of group V should be regarded as diphtheroids or as non-virulent strains of C. diphtheria?. Our strain fermented dextrin regularly, and on other characters, growth on agar, etc., would be classed as an avirulent C . diphtheria? rather than a type V diphtheroid of Barratt.

Toxin production. Attempts to produce a toxin in Hartley's diphtheria toxin broth proved unsuccessful. Doses of 2 C.C. of the broth culture after four and ten days' incubation, given sub- cutaneously to guinea-pigs, produced no ill effects.

Virulence test. Whole one - day - old cultures on inspissated serum were emulsified in 2 C.C. of normal saline solution and injected subcutaneously into guinea-pigs. The animals showed no ill effects.

From these bacteriological findings we conclude that the organism is a non-toxigenic strain of C . diphtherim. Save a history of an attack of diphtheria many years previously, we have no evidence as to the original source of the infection. In this respect our case resembles those of Howard and Roosen-Runge. Whether the blood stream infection was simply a leakage of bacteria from

JOURNAL OF PATliOLOUY-VOL. XLI11. PLATE XXXII

ENDOCARDITIS DUE TO A DIPHTHEROID

Fro. 1.-Low power view of a section of the cardiac vegetations stained by Gram’s met,hod, showing the dense colony-like masses of Grain-positive bacteria mingled with blood corpuscles and fibrin clot. x 50.

FIG. 2.--Sniear from a sub- culture on inspissated serum stained by Loeffler’s methy- lene blue. x 1000.

FIG. S.-Similar smear stained by Mallory’s method. x 1000.

ENDOCARDITIS DUE T O A DIPHTHEROID 133

the heart vegetations or an actual growth of bacteria in the blood stream we cannot say. Unfortunately the desirability of testing the patient's blood serum for diphtheria antitoxin was overlooked in the urgency of initiating antitoxin treatment. However, in view of the non-toxigenic nature of the organism and the previous history of diphtheria, it appears doubtful whether this would have been of much help.

Remarh on avirulent C . diphtheria Until some method is discovered whereby avirulent non-

toxigenic C. diphtherice strains may be made to produce toxin neutrahable by diphtheria antitoxin, there will persist doubts as t o whether these so-called avirulent strains, especially if not known to have been associated with diphtheria, are in fact related or not to the virulent form.

Arkwright (1910) stated that " The evidence for the transforma- tion of true non-virulent B. diphtherice into virulent B. diphtheria? or the reverse is scanty and unsatisfactory. I have tried very many methods, but have completely failed to change the one type into the other. Occasionally, however, a strain on being tested has proved non-virulent, whereas on a former occasion the same strain has been virulent. When this has occurred, the change has not been referable to any special treatment or external condition, and has been permanent."

Neufeld on " Variability of bacteria " given in the De Lamar Lectures, 1926-27, quotes Bernhardt and Paneth (1913) as having " injected diphtheria bacilli intravenously into guinea-pigs and rabbits, and on killing the animals a dayor two afterwards, succeeded in recovering from their organs non-toxic diphtheroid bacilli. They observed the same transformation in vitro when they inoculated diphtheria bacilli into undiluted serum of human beings or guinea pigs."

Neufeld also quotes more recent work by Levinthal (1926), who used single cell cultures of virulent diphtheria bacilli. Neufeld goes on to state, " We may assume that a like transformation of diphtheria bacilli into atoxic diphtheroids regularly takes place in human beings, and that the final issue of the diphtherial infection depends not only upon the antitoxic content of the blood, but also upon the transforming power of the individual. Patients who are not able to transform the virulent into atoxic bacilli may partly on this account succumb to the infection. The cases of diphtheria in which typical toxic bacilli appeared in the urine nearly always ended fatally, whereas the urine of patients afterwards recovering from the disease often contains atoxic diphtheroids which we may presume to be modified diphtheria bacilli. . . . Killian succeeded in imitating more closely the process of human infection. He

JOumL 01 p ~ . - v o L . XTnI. N 2

134 J. SUTHERLAND AND R. A. WILLIS

brought diphtheria bacilli into the mouths of guinea-pigs and mice, and was able to cultivate atoxic diphtheroids some hours later from the cervical glands of the animals. We may assume that the bacilli undergo the same degeneration, not only when passing through the mucous membranes, but also when growing on their surfaces. In this way, most of the toxic bacilli present on the tonsils at the height of the disease may be disposed of.”

Our case, like Howard’s and Roosen-Runge’s, shows that avirulent C. diphthericz! may establish themselves selectively in the patient’s endocardium and produce fatal results without toxin production.

Diphtheroids in blood cultures. It is the experience of most workers occasionally to find

diphtheroids present in blood-inoculated culture media, such diphtheroids usually being considered as contaminants from the patient’s skin in collecting the sample of blood by vein puncture. Diphtheroids and Staphylococcus albus are the most common skin contaminants met with. As the present case and others quoted later show, however, diphtheroids may be more than mere con- taminants from the skin, and this possibility should always be considered.

Thompson (1932) studied a large series of blood cultures with a view to correlating the presence of diphtheroids in the blood culture with the type of case, and to testing the hypothesis advanced by Koch and Mellon that diphtheroids are found in the blood stream of persons convalescent from streptococcal infections. One thousand and seventy-nine blood cultures were made in 730 cases and 33 cultures of diphtheroids were obtained in 30 cases, an incidence of 3 per cent. Diphtheroids were obtained twice in the same case on two different occasions. Thompson, however, found it impossible to* correlate the finding of diphtheroids in blood culture with the clinical diagnosis. He concluded that diphtheroids appear to have the same sqpficance in blood cultures as the saprophytic cocci.

Kessel and Romanoff (1930) cultivated a Hofmann type of diphtheroid from the blood of a cme that had been operated on for an anal infection. A diphtheroid and a coliform bacillus were cultivated from the pus from the operation site. Later, meningitis developed and the diphtheroid organism was isolated from the cerebro-spinal fluid. This case recovered. The blood and cerebro- spinal infections were considered to have had their origin in the anal infection.

sumnzary. (1) A fatal case of ulcerative endocarditis due to an avirulent

C. diphtherice type of organism is described.

ENDOCARDITIS DUE TO A DIPHTHEROID 136

(2) The organism was cultivated in pure culture repeatedly from the blood during life and from the vegetations on the heart valves post mortem.

(3) It is emphasised that, although diphtheroids in blood cultures are usually considered contaminants from the skin, the possibility of their having clinical significance should not be overlooked.

(4) The significance of diphtheroids in blood cultures is discussed.

We desire to acknowledge our indebtedness to Dr M. D. Silberberg for permission t o publish the case, to Miss H. M. Butler for supplementary counts of bacteria in the blood and for post-mortem cultures, and to Mr Reg. Prosser for cutting the sections.

REFERENCES.

ARKWRIOHT, J. A. . . . . Diphtheria carriers, Brit. Med. J . , 1910, ii. 1608.

BARRATT, M. M. . . . . A study of C . diphtherice and other members of the genus Corynebacteriurn with special reference to fermentative activity, J . Hyg., 1924-25, xxiii. 241.

BERNHARDT AND PANETH . . Ueber die Variabilitiit des Diphtherie- bazillus, Cbl. Bakt., Abt. I. Ref., 1913, Ivii. Beiheft, 83* (quoted by Neufeld).

HOWARD, W. T., Jr. . . . Acute ulcerative endocarditis due to the bacillus diphtheriae, Johns Hopkim H o q . Bull., 1893, iv. 32.

General infection with a diphtheroid bacillus complicated by diphtheroid meningitis, J . A m r . Med. Assoc., 1930, xciv. 1647.

KILLIAN, H. . . . . . . Uber die Urnwandlung pathogenen Bak- terien beim Durchtritt durch die Schleim- haut der Verdauungswege, 2. Hyg., 1924, cii. 262 (quoted by Neufeld).

LEVINTHAL, W. . . . . . Studien an Diphtheriebacillen, Ibid., 1926, cvi. 679 (quoted by Neufeld).

NEUFELD, F. . . . . . Variability of bacteria, Johns Hopkim Univ. School Hyg., De Lamar Lectures, 1926-27 (1928), p. 13.

ROOSEN-RUNQE . . . . . Ein Fall von Diphtheriebazillensepsis, Munch. med. w8chr., 1903, I. 1252.

TENBROECK, C. . . . . . Effects of enzymes in serum on carbo- hydrates and their relation to bacterio- logical technique, J . Exper. Med., 1920, xxxii. 346.

THOMPSON, L. . . . . . Occurrence of diphtheroids in blood cultures, J . Infect. Dis., 1932, 1. 69.

Tow, A., AND WECHSLER, H. F. Diphtheroid bacillus as the cause of acute endocarditis, A m r . J . D k . Child., 1932, xliv. 156.

KESSEL, L., AND ROMANOFP, A.