576. 851 . 4 (Bac. coli) : 546. 3 2 2 . 44

THE INHIBITORY ACTION OF POTASSIUM TELLURITE ON COLIFORM BACTERIA

ALEXANDER FLEMING and M. Y. YOUNG From the Laboratory of the Inoculation Department,

St Mary's Hospital

POTASSIUM tellurite has been shown to have a remarkably selective antibacterial action in that a concentration of 1 : 1,000,000 or less will inhibit some species, whereas others will grow freely in a concentration of 1 : 1000. Among the sensitive species are the coliform bacilli and, as it was found that different strains vary considerably in their resistance, it was thought worth while to test a large number to see whether anything useful would emerge.

METHODS Dilutions of a stock solution of 1 per cent. potassium tellurite were

added to agar or broth t o make concentrationr of tellurite of 1 : 10,000- 1 : 4,000,000.

When tellurite broth was used, this was made up in 1 C . C . quantities and the tubes were implanted with one drop (about 30 c.mm.) of a 1 : 200 dilution of a 24-hour broth culture. With tellurite agar the plates were dried off and loopfuls of a 1 : 200 dilution of a 24-hor. broth culture were planted in spots on tho surface so that 20 or more cultures were grown on the same plate. Cultures were incubated at 37" C. and were examined daily.

It may be said herr that the results obtained were essentially the same whcther the cultures were grown in tellurite broth or on tellurite agar.

RESULTS. In table I (p. 30) is given a typical result of testing a batch of

strains of B. coli taken at random. It will be seen that most of the strains are extremely sensitive,

being completely inhibited by tellurite in a concentration of 1 : 2,000,000 ; one, however, was resistant and grew well in a con- centration of 1 : 10,000. For comparison one strain of B. typhosus was used which was inhibited even by the weakest tellurite.

Table I1 summarises the results obtained with 302 cultures of various coliform bacilli. They have been arbitrarily divided into three groups-a very sensitive group which would not grow in a concentration greater than 1 : 1,000,000, a resistant group which grew in I : 50,000, and a partially sensitive group occupying an intermediate position.

25)

30

z:ii;:

A . FLEMING AND M . Y . YOUNG

TABLE I Inhibitory effect of potassium tellurite on the growth of a

number of strains of B. coli

Very sensitive

(growth only in 1 : 1 x108

or less)

__-

Strain no.

11 19 1 1

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 I 6

5. typhosuc

0 3 0 0

Control

+++ +++ +++ +++ +++ +++ +++ +++ +++ +++ +++ + -t + +++ +++ + + 4- +++ +++

1 : 4x106

+ + + + + + + + + +++ +++ +++ +++ +++ +++ f - t + -

Concentration of telluritc

1 : 2 x 1 0 8

Out of 302 cultures of coliform bacilli of various kinds 236 or 78 per cent. were extremely sensitive and 35 or 12 per cent. insensitive. If we exclude B. proteus, B. pyocyaneus and B. cloaca we are left with 271 strains of which 86 per cent. are very sensitive and only 9 per cent. resistant (table 11). Some of the results obtained call for comment.

TABLE I1 Growth of bacteria in media containing p o k w i u m tellurite

Organism

R. t y p h o m ,

B. paratyphosus A . . ,, B . .

Other Salmonellm . . B. fcecalis alkaligenes . B. dispar . B. dysenterice Shiga .

,, Flexner . ,, Sonne .

Unclassified non-lactose fermenters . B. coli . .

B. pyocyancus . B.proteus . B. morgani . B. cloacae .

7 5 5

27 5 3 6

7 5 5

27 5 3

0 0 0 0 0 0 1 2 1 2 I

11 7 0 0

Resistant (growth in 1 : B x 104- 1 : 1 x 10')

0 0 0 0 0 0 0 0 0 3

21

0 9 1 1

GROWTH INHIBITION BY TELLURITE 31

Category

B. typhosus. Other Salmonellze. These were all very sensitive but not

quite so sensitive as B. typhosus. A series of 27 different Xalmonellce from the National Collection were all inhibited by the same con- centration of tellurite (1 : 2,000,000).

Four strains out of 24 were classed as partially sensitive but of these, three were inhibited by concentrations greater than 1 : 500,000, so there was really little difference between them and the majority of the cultures which fell into the very sensitive class.

B. pyocyaneus. All eleven strains grew well in 1 : 250,000 and poorly or not at all in 1 : 100,000.

B. proteus. This group varied enormously, some being very sensitive and many resistant. The fact that many strains of this organism are resistant is in accordance with the common observation that B. proteus may appear in the special tellurite media prepared for the isolation of B. diphtherice and containing a high concentration of tellurite.

B. coli. Of these cultures 17 were from the National Collection and 141 were taken at random from hospital patients. The vast majority (82 per cent.) were very sensitive to tellurite, but 2 1 strains were resistant. Table I11 shows in more detail the variations which occur in this group.

These were all extremely sensitive.

B. dysenteria.

TABLE I11

Masimiirn concentration of tellurite in which

growth occurrrd R o . of strains

Growth of B. coli in media containing potassium tellurite

Resistant . . .. 1 : 50,000 3 1 : 20,000 3 1 : 10,000 15 or greater

I I -I_______ < I : 4,000,000 13

1 : 4,000,000 1 : 2,000,000

Very sensitive .

I I 40 1 1 : 1,000,000

Partially sensitive . 1 : 500,000 3 1 : 250,000 1 : 100,000

It will be seen that some 10 per cent. of the strains tested would grow in the presence of 1 : 10,000 tellurite while about the same number were inhibited by as little as 1 : 4,000,000. It will also be noted that the number of partially sensitive strains is relatively small.

32 A . FLEMING A N D M . Y . YOUNG

Resistant strains of B. coli and B. proteus Attention was paid to the resistant strains of B. coli as it was

thought that they might form one homogeneous group. The sugar fermentations of all the B. coli strains were tested but no difference was found between the very sensitive and the resistant strains. Rabbits were immunised with vaccines of sensitive and insensitive strains and produced good agglutinins to the homologous strains, but as regards the other cultures each serum agglutinated only a few, and these were irregularly distributed among the sensitive and resistant groups. The question of sensitiveness or otherwise to tellurite, therefore, did not seem to be correlated with biochemical activity or antigenic specificity.

Similar investigations were made with sensitive and resistant strains of B. proteus with exactly the same results.

Absorption of tellurite by the growing bacilli An interesting observation was made when investigating the

In the inhibitory power of tellurite incorporated in agar plates.

Influence of a resistant strain on adjacent implants. 48 hours’ growth of coliform bacilli on 1 : 100,000 tellurite agar.

resistant strain ; the others are sensitive. Dotted lines indicate where implants were made ; dark shading indicates growth.

No. 6 is a

method employed, a considerable number of different strains were grown in spots on the same culture plate. It was noticed that

GROWTH INHIBITION BY TELLURITE 33

Control

+++ f++ +++ +++

when a tellurite-resistant strain grew luxuriantly on the lower concentrations of tellurite the other strains in the neighbourhood began to grow after an interval of perhaps 48 hours (fig.).

This was confirmed in another experiment in which about one- third of a plate containing 1 : 100,000 tellurite was thickly planted with a resistant strain of B. coli and allowed to grow for 48 hours. Then three sensitive strains were planted in streaks across the plate at right angles to the resistant strain. After another 24 hours' incubation there was growth of the sensitive strains for a distance of 16 mm. from the resistant strain, but none further away. These sensitive strains would not normally grow on a greater concentration of tellurite than 1 : 1,000,000, so it is clear that the resistant strain had in 48 hours absorbed or rendered inert at least 90 per cent. of the tellurite over a distance of 16 mm.

1 : 1 x los 1 : 5 x lom 1 : 2 x lo4 1 : 1 x l o 4 1 : 6700 -________-_____

- - - -

+++ i- +++ +++ ++ - +++ +++ +++ +++ - +++ +++ +++ +++ +++

Acclimatisation of B. coli to tellurite Some experiments were made to see whether by passage in

tellurite broth the resistance of B. coli to the inhibitory action of the chemical could be enhanced. A series of tubes of tellurite broth was inoculated with B. coli and incubated for 24 hours. A

TABLE IV Adaptation of B. coli to growth in tellurite

Control ~~

Original culture + + + E X I : ~ X ~ O ~ +++ EX 1:25x104 +++ Ex 1 : 1x10s +++ ~ ~ 1 : 5 ~ 1 0 4 t +

Inoculum

1 : 1 x loe

+ + + +++ ... ... ...

Original culture E Z 1 :j.x105

+ +++ +++ .I.

...

___ Original culture EX 1 : 5 x 1 0 4 EX 1 : 2 x 1 0 4 EX 1 : 1x104

- ++ +++ +++ ++

~

Concentration of tcllurite

Control 1 1 :2x106 1 1 : 5 x 1 0 6 i 1:25x10' 1 1 : l x 1 0 3 ~ _ _ _

I I 1 I

~

I 1 I

+ I 1 : 5000

- - -

+++ 1 : 5 x 1 0 5 1:25x106 I 1 : 1 xi05 1 1 : 5 xi04 1 : B x 104

fresh series of tubes was then inoculated from the tube containing the strongest concentration of tellurite in which growth was visible. This was repeated on successive days. It will be seen from table IV

JOURN. OF PATH.-VOL. 1.1 a

34 A . FLEMING AND M . Y . Y O U N G

FBces “M ” 1 : 5000-1 : 800,000 1 : 2,000,000 1 : 4,000,000

Control

that the bacteria could easily be educated to grow in broth containing tellurite in concentrations far greater than would have completely inhibited the original culture.

Streptococci Streptococci+minute colonies of B. coZi Same: B. coli colonies larger, but still smaller than

About 3 colonies of B. coli to 1 of streptococcus streptococci

Uses of tellurite in practical bacteriology Potassium tellurite has long been used in many different

forniulze for the isolation of the diphtheria bacillus. It is especially useful for this purpose as the diphtheroid bacilli are among the most resistant to its inhibitory powers.

One of us (Fleming, 1932) has pointed out the value of tellurite for the isolation of Gram-positive cocci from faxes or other infected material. Relatively little attention has been paid to the isolation of streptococci or staphylococci from faxes, and public health bacteriologists have been more concerned with the isolation of the non-lactose-fermenting coliform bacteria from the normal B. coli in sewage and excreta, for which purpose tellurite is useless, as all the common bacteria concerned with epidemics are extremely sensitive to its inhibitory action.

Hospital bacteriologists on the other hand frequently have to investigate the intestinal contents of individual patients with no regard to public health but in respect only of the patient’s condition, and it is in such cases that tellurite is of the greatest value. By its use, in about 90 per cent. of cases B. coli can be completely eliminated and the streptococci, staphylococci, diphtheroid bacilli, etc., can be isolated with the greatest ease. Table V shows the results obtained when two samples of fzeces were plated on agar containing tellurite in various concentrations.

TABLE V Result of plating human feces on tellurite agar

Faces “N ” 1 : 5000-1 : 800,000 1 : 2,000,000

Concentration of tcllurite j

4 or 5 colonies of streptococci Same+B. coli colonies resembling small colonies of

Nature of growth

1 : 4,000,000 Control

- streptococci

B. coli colonies still smaller than normal Well grown B. coli : no streptococci visible

In the case of faxes “ M ” it will be seen that €3. coli was completely eliminated on the plates containing tellurite in con-

GROWTH INHIBITION BY TELLURITE 35

centrations from 1 : 5000 to 1 : 800,000, but streptococci grew freely on all these plates. Even when the tellurite was as weak as 1 : 4,000,000 the B. eoli colonies which appeared were smaller than the streptococcal colonies.

Faxes " N " is interesting, as in this specimen the streptococci were very scanty, so that in the control plate they were completely obscured by the profuse growth of B. coli. On the plates containing tellurite in concentrations of 1 : 800,000 or greater the few strepto- ccicci present grew out and there was no difficulty in their isolation.

In all the experiments detailed in this paper the tellurite has been incorporated in the medium, but in practice tellurite can be used even more simply. The faxes or other material is spread in the usual way on the sorface of blood agar or other suitable medium and then with a capillary pipettc two drops (about 60 c.mm.) of a 1 : 1000 solution of tellurite are placed on the plate and are spread over one half of the surface. This gives a plate of which one-half is an ordinary culture and the other a selective culture in which the coliform bacilli arc eliminated.

A simple method such as this might be very useful in the investigation uf septic wounds in which large nnmbors of coliform organisms are present.

SUMMARY The Salmonella and dysentery groups of bacteria are very

sensitive to the inhibitory action of potassium tellurite, B. pyocyaneus is moderately sensitive, while many strains of B. proteus as well as B. morqani and B. cloacce are resistant.

B. coli strains are usually very sensitive but 10 per cent. of them are very resistant.

Resistant and sensitive strains of B. coli or B. proteus do not differ in their fermentative or antigenic characters.

Bacteria can be readily acclimatised to tellurite. The use of tellurite to facilitate the isolation of Gram-positive

cocci from fzces is indicated. We wish to acknowlodge our gratitude to the officials of the National

Collection of Type Cultures for many strains of coliform bacilli.

REFERENCE

FLEMING, A. . . . . . 1932. This Journal, xxxv, 831.