THE ISOLATION OF ATOXIC STRAINS OF BACILLUS TETANI IN EGYPT.

By W. LEONARD FORSYTH, m.b., d.p.h. (Lond.),

MAJOR, I.M.S. (Retd.),

Professor, and

A. F. I. ZANATY, m.b. (Cairo), Demonstrator.

(From the Department of Bacteriology, University of Egypt.)

While investigating the Bacillus welchi content of the stools of Egyptians we observed that a very large number of samples of faeces

grown in Robertson's bullock heart medium

produced terminal drumstick spore-bearing bacilli morphologically resembling Clostridium tetani. Accurate figures concerning the incidence of

clinical tetanus in Egypt are not available. For the past decade in Kasr el Ainy Hospital (800 beds; outpatient weekly attendance over 10,000) an average annual death rate of 7 from tetanus in 1,200 from all causes is estimated. In a country like Egypt where the fellah is a

true son of the soil this incidence would appear to be a low one. Tenbroeck and Bauer (1) found that 34 per

cent, of the stools of 78 individuals in Peking yielded Bacillus tetani, and in a subsequent paper (2) that individuals carrying the bacilli had an appreciable amount of antitoxin in their blood sera. Their impression was that clinical tetanus was relatively uncommon in that area. Bauer and Meyer (3) in California found

B. tetani in 24 per cent, of cases investigated, at the same time finding no atoxic strains. ln

California the incidence of the disease is fairly high. Fildes(4) isolated it in only 1 per cent- of cases examined, while Tulloch(5) found in 16 per cent, of cases drawn from a civile11

pjg i.?Agar stab culture. Fig. 1.?Agar stab culture.

Sept., 1930.] ATOXIC B. TETANI IN EGYPT: FORSYTH & ZANATY. 501

population. Kerrin(6) in Aberdeenshire exa-

mined 204 cases without finding B. tetani

although he states that 36 of these showed organisms morphologically suggesting B. tetani, but in no case was it obtained either by re-

peated plating out or by the method of Fildes. These workers have used different methods of

isolation, and it is possible this may have some- thing to do with the different findings. This

paper is concerned more in the description of a large number of strains of what appears to be B. tetani of the atoxic variety occurring in the faeces of the fellaheen. These strains have all been isolated by the

following technique:? A tube of Robertson's medium, pH distinct-

ly alkaline to litmus, is inoculated with a bean- sized sample of faeces from the patient in the ward. This is then heated between 75? and 80 ?C. for 20 minutes, corked, and incubated

aerobically for 7 days. Routine examination

by staining shows that four out of five cultures usually contain bacilli with spherical terminal spores resembling B. tetani. A few drops of this fluid are transferred by sterile pipette to the condensation water of a Fildes' slope and examined after 48 hours' incubation in a

Mcintosh and Fildes' jar (Wilson modification). Usually two or three will show the film and

filamentous edge diagnostic of B. tetani

j Fildes). If incubation be continued to the fourth day terminal drumsticks usually appear, from the filamentous edge another slope is

inoculated and also a melted Fildes' agar Petri dlsh. In the latter typical cotton-wool colonies appear and we have found it relatively easy to obtain these in pure culture. Colonies are

Picked and sown in Parke and Williams' broth and incubated anaerobically for 48 hours for

examination for motility and agglutination

Jcsts, and for longer periods up to 14 days for he development of toxin. As an alternative method for the isolation of

these bacilli in pure colony culture we have ?und the following most successful:? 1 c.c. of the Robertson fluid containing round

terminal spores is pipetted to a test tube con-

taining 1 c.e. of 1 per cent, phenol. This is

left overnight in the incubator and next morn- ing it is used to inoculate melted and poured Fildes' medium in a Petri dish. Incubation in a Fildes' jar, using aqueous vapour instead of hydrogen in the manner recently described by Herrington and Knaysi(7), results in the pro- duction of well isolated typical colonies. The use of aqueous vapour instead of hydrogen greatly simplifies matters, although the rate of

growth of the colonies by this method is slower; if the Petri dishes are wrapped in oil cloth or

gutta-percha tissue there is no fear of flooding the dishes. The characters of ten of these atoxic strains

isolated by the first described technique is as

follows:?

Morphology, cultivation and biochemistry.

1. Broth (Parke Williams).?Slight milky turbidity after 24 hours, motility sluggish though definite; the culture can be used for the purposes of agglutination and the fourth day growth for the presence of toxin; morpho- logically the bacilli are from 2ji upwards to filamentous forms, and have rounded ends; it is essentially a slender bacillus resembling B. Klebs-Ldfller in breadth. Gram-positive, with variation in depth of staining. No spores are seen at the end of 48 hours' incubation.

2. Gelatin stab.?Usually no growth at bcnch temperature 20? to 22 ?C. if the gelatin is uncovered with vaseline. In the jar at 37?C. growth occurs, but the gelatin is not usually liquefied until the 21st day. We find these atoxic strains liquefy gelatin less rapidly than toxic ones.

3. Glucose agar stab (or agar stab without glucose) (Fig. 1). At 72 hours at 37?C. shows no growth aerobically, but when such stabbed tubes arc subsequently transferred to the jar, typical textbook fir tree growth extends to the sides of the tube by the 4th day; slight gas formation may occur; some strains do not pro- duce growth along the central stab, but rather concentrate on the lateral growth without having an obvious central core.

Fig. 2.?Surface colonies. Fig. 2.?Surface colonies.

Pijr_ 3. Photomicrographs. Deep colonies. piK_ 3. Photomicrographs. Deep colonies.

502 THE INDIAN MEDICAL GAZETTE. [Sept., 1930.

4. Fildes' agar slope.?On the 4th day rods appear from 2ji upwards, length indeterminate, many showing terminal spherical spores; the

typical film with filamentous edge is apparent, but we have found this film at its best when in process of isolation B. tetani is in associa- tion with other anaerobes.

5. Fildes' agar plates.?(a) Surface colony irregularly filamentous (Fig. 2) spreading, translucent and very difficult to photograph. (b) Deep colony may or may not have a darker brown centre distinctly filamentous with fuzzy periphery (photomicrograph, Fig. 3).

6. Potato.?No growth after 5 days' incuba- tion.

7. Loffler's serum.?After 5 days there is definite though feeble digestion.

8. Coagulated egg mediuvi (Locke).?Glis- tening growth is evident after 48 hours, when early surface pitting becomes visible; continued incubation up to the 7th day results in almost complete liquefaction with collection of the fluid at the bottom of the tube, the back of the tube showing slight blackening; films from the fluid show typical drumstick bacilli; this

growth on egg is vigorous and characteristic. 9. Horse blood agar plates.?Diffuse surface

greyish translucent colonies with filamentous edge. No haemolysis around the colony. Hemolytic tests have also been done by Neil's method" and by Greig's method, using sheep's cells, with no resulting ha?molysis.

10. Cooked meat medium (Robertson).? Some turbidity indicating moderate growth after 48 hours; softening of meat at the top of the tube with a slight pinkish coloration; no

odour of putrefaction; odour of the jar contain- ing the culture suggests manure. No spolia- tion till the 4th day, if then; growth appears to have stopped by the 14th day and usually slight blackening is visible.

11. Carbohydrate jermentation.?No carbo-

hydrate is fermented with sufficient activity to cause any change in the indicator; in litmus milk, anaerobic conditions interfere with the in- terpretation of any biochemical change.

12. Toxicity.?1 c.c. of a 4th day broth (Parke Williams) culture injected intramuscu- larly fails to produce in guinea-pigs either local or generalised tetanus; thirty animals have been inoculated in this way with consecutively isolated strains with uniformly negative results.

13. Serology.?In the absence of type sera for which we have made application we cannot say a great deal about these atoxic strains. For group identification we have however

made use of two antitoxic sera in agglutination tests (a) Concentrated tetanus antitoxin [Bur- roughs "Wellcome (17896)] (8), which is made mainly with the toxin from a good toxin pro- ducing strain. It contains 1,000 U. S. A. units Per c.c., or 2,000 international units per c.c. (o) Tetanus antitoxin (Hoechst), containing 300 international units per c.c. Several strains

of 48 hours' broth culture have been put up against these sera, with the usual controls, using Dreyer's technique. Most of them agglutinate in dilutions varying from 1:50 to 1:500 with the Burroughs Wellcome serum but not with the Hoechst serum.

Using 48-hour broth cultures injected intra-

muscularly and intravenously we have succeed- ed with difficulty in immunising a rabbit, the serum of the animal giving an agglutination of 1:250 only with the homologous organism and other strains.

Conclusions.

The following are the conclusions in this pre- liminary communication.

(a) Atoxic B. tetani occur in the stools of

from 40 per cent, to 50 per cent, of Egyptian fellaheen.

(b) These strains show deviation from the

genuine toxin producer in such attributes as

ha?molysis, gelatin liquefaction, poor antigeni- city, and absence of toxicity as assessed by ordinary methods.

References.

Bauer and Meyer (1926). Journ. Inf. Disy 38; p. 296. Fildes (1925). Brit. Journ. Exptl. Path. Herrington and Knaysi (1930). Journ. Bad., Vol. 19 Kerrin (1928). Brit. Journ. Exptl. Path., April. Tenbroeck and Bauer (1922). Journ. Exptl. Med,.,

Vol. 36, 261. Tenbroeck and Bauer (1923). Ibid., Vol. 37, 479. Tulloch (1919). Journ. Roy. Army Med. Corps, 29;

Journ. Hyg. Carnb., 18, 103.