576 . 8 . 077 . 34 : 576 . 851 . 48 (Bact. coli var. neapolitunum)

SLIDE AGGLUTINATION OF B A C T E R I U M COLI VAR. NEAPOLITAN UM IN SUMMER DIARRHCEA

JOHN BRAY and T. E. D. BEAVAN From Hillingdon Hospital, Uxbridge

INVESTIGATIONS made during 1943 (Bray, 1945) showed that a particular serological type of Bact. coli was recoverable from 95 per cent. of cases of summer diarrhea or non-specific gastro-enteritis of infants. Originally these strains were detected by taking advantage of the delay which, under certain conditions, they showed in the fermentation of maltose. I n the present investigation, carried out during 1945, the method of slide agglutination was used for the detection of these strains, as the earlier method of isolation proved unreliable. The results suggest that this method might be of practical value for the laboratory confirmation of the clinical diagnosis of a particular type of gastro-enteritis.

Clinical material

During 1945, 40 cases were diagnosed as gastro-enteritis in infants of a mean age of 5 months, the cases being spread over the period February to October. Faecal samples from these and from 80 non- diarrheal controls from the same wards were examined, the cases selected being from bottle-fed infants under one year of age.

In three-quarters (28) of the diarrheal cases the disease was of a severe type, i.e. requiring parenteral fluids, and there were 11 deaths (28 per cent.). The characteristic smell noted by Beavan (1944) was obviously present, although not especially sought for, in 17 cases. In nearly half the cases (18) i t was considered that the disease had been acquired during the infant’s stay in hospital.

Post-mortem jindings

Six autopsies were performed and the findings were character- istically meagre. In 2 cases there was marked monilial msophagitis. There was macroscopic evidence of fatty changes in the liver in 4. In 3 cases partially digested blood was present in the stomach, but in none were there significant changes in the mucosa of the intestines. Gdema of the brain wm noted in 4 cases. Agglutinable strains of Bact. coli var. neapolitanum were isolated from all these cases.

J . PATE. BACT.-VOL. LX 395

396 J . BRAY AND T. E. D. BEAVAN

1943

1945

Methods

Faecal samples from each case were plated on desoxycholate-citrate agar and MacConkey’s agar. The former was examined for non-lactose fermenters with the usual lack of significant findings, except in one case where Bact. sonnei was isolated. From the MacConkey’s agar a single pink colony w a picked and tested on a slide against the antiserum which had been used in the 1943 investigations (Bray, 1945) and which had been preserved in the refrigerator. In the selection of a colony, one which was transparent t o transmitted light and not obviously rough or mucoid was taken. Otherwise, apart from choosing one that was well isolated, the colony was taken at random. Before use the serum was diluted with saline 1 in 10 and the h a 1 dilution on the slide was approxim- ately 1 in 400. In spite of this there were few doubtful reactions. Agglutination was, in the positive cases, usually immediate and unequivocal and of the granular kind, like that given by an 0 strain.

The identity of all agglutinating strains was later confirmed by tube agglutina- tion and the testing of fermentative reactions against sucrose, salicin and maltose, and by motility tests. After picking the colony the MacConkey plate was laid aside on the bench and examined from time t o time for the growth of Monilia. It had been previously noted that an easily recognisable growth of Monilia appeared in about 4 days in the form of discrete granular white domes on the site of the thickest inoculum, where the coliform growth was confluent.

-

50 95.0 30

40 87.5 28

RESULTS Table I shows the number of isolations of Bact. coli var. neapolit-

anum resulting from the investigations of 1943 and 1945.

TABLE I Isolations of Bact. coli var. neapolitanum (B.C.N.) f rom cases of

gastro-enteritis and f rom controls

I I Qastro-enteritis I

Isolation of Mortality (per cent.) (per cent.)

Year 1 1 No. ofcases 1 B.C.N. I Controls I

Isolation of

(per cent.) KO. of controls B.C.N.

100 I 4 1

In the gastro-enteritis samples the slide agglutination was positive in 35 cases. Later tests showed these strains of Bact. coli to be of the same type as those reported in the earlier communication (Bray). In the controls, agglutination was positive 5 times, 3 of the strains being of the same type as those from the diarrheal cases, the other 2 of a different type as judged by fermentation and motility tests ; in other words there were 2 false-positive agglutinations. Considering its dilution of 1 in 400, therefore, the serum seemed to show consider- able’ discrimination.

With regard to the Monilia, these yeasts grew in 15 of the diarrhaeal cases (37 per cent.) and in 18 (22-5 per cent.) of the controls.

SLIDE AGGLUTINATION OF COLI VAR. NEAPOLITANUM 397

Although Monilia is found rather frequently in the mouths of normal adults (14 per cent., Todd, 1937) i t seems that there is a close connection between the presence of Monilia in the oral cavity (Anderson, Sage and Spaulding, 1944) and perhaps in the faxes (Ludlam and Henderson, 1942) and the existence of clinical thrush in new-born infants. It is likely that our figures represent a high incidence of thrush infection in the wards, although we have no clinical data to confirm this. The point to note is that there is little difference in the incidence of Monilia in the faxes of the diarrheal and non-diarrheal cases.

Identity of strains

It has been noted that in isolating the strains the slide agglutinations were done with the antiserum prepared in 1943. During 1946 a further antiserum was prepared in the rabbit against one of the strains (G.R.) isolated in 1945. Thirteen of the 1945 strains, which had been preserved in nutrient broth at room temperature, were tested against it by the tube-agglutination method. The results were that 10 of the strains were agglutinated to titre, 2 had become rough, and 1 strain which in the meantime had lost its agglutinability to the 1943 serum also failed to react with the 1946 serum. A strain (Lindsay) which had been isolated in 1943 and preserved was also agglutinated, to titre, by the 1946 serum. While the type of agglutina- tion on the slide was granular, in the tubes it was semi-flocculent and showed some adherence to the sides in the lower part of the tube.

Identity of the 1943 and 1945 strains was further supported by a small number of absorption tests which were carried out in the manner described by Mackie and McCartney (1948). The 1943 and 1946 sera were each absorbed with a strain of the homologous and hetero- logous organism and then re-tested for agglutination against the same 1943 and 1945 strains. It was found in each case that the agglutinins had been removed from the sera. These results are shown in tables 11-IV.

The various serological tests may thus be summarised : (1) Serum 1943 tested against Lindsay (1943) in order to determine

titre. Approximately 1 in 8000. (2) 38 strains (1945) isolated by slide agglutination with the aid

of 1943 serum (results confirmed by tube agglutination). (3) Serum 1946 prepared against G.R. (1945). Titre 1 in 16,000.

This serum was prepared in a rabbit of the same strain and the same dosage of bacterial suspension was used as in the preparation of the 1943 serum.

(4) 13 strains of 1945 origin tested by the tube-agglutination method against serum 1946.

(5) Lindsay (1943) shown to agglutinate to titre with 1946 serum. (6) Serum 1943 absorbed with Lindsay (1943) and G.R. (1945)

and re-tested against these strains. The agglutinins for both strains

10 strains were agglutinated to titre.

398 J . BRAY A N D T . E. D. B E A V A N

were shown to have been completely absorbed by each. absorption of the 1946 serum gave identical results.

Similar

TABLE I1 Absorption of 1943 serum by strain G.R. (1945) and

strain Lindsay (1943)

Lindsay G.R. suspension suspension . . *

Lindsay G.R. suspension suspension . .

I 1 i n j 1 i n I l i n 1 l i n I 1 i n 1 1 i n 1000 2000 4000 8000 16,000 32,000 i

Final dilution

- j : l : l : l ' ~ - . . .

Serum absorbed with Lindsay

- - - . - - i - ~ + l - ~ - Serum absorbed with G.R.

Unabsorbed serum

G.R. suspension . Lindsay suspension . .

Serum absorbed with Lindsay

- -

G.R. suspension . - 1 -

TABLE I11

Absorption of 1946 serum with the homologous strain G.R. and strain Lindsay (1943)

1 1 i n 1 1 i n I 1 i n 1 1 i n 1 1 i n I 2000 4000 8000 16,000 32,000 Final dilution

I Unabsorbed serum

- - G.R. suspension . +

Serum absorbed with G.R.

G.R. suspension Lindsay suspension

(7) Two other 1945 strains (Fisher and Alyott) were used to absorb the 1946 serum. On re-testing the serum against these two strains agglutinins were found to have been removed as in (6). Unfortunately

S L I D E AGGLUTINATION OF COLI V A R . NEAPOLITANUM 399

' 1 i n 1 1 i n 1 1 i n I 1 i n 1 1 i n I 1 i n 500 1000 2000 4000 8000 16,00(

we have no record that this absorbed serum also failed to agglutinate strain G.R., so that the identity of these two strains with G.R., though probable, is not proved.

TABLE I V

Absorption of 1946 serum with strains Fisher (1945) and Alyott (1945)

...

Final dilution

+ + + + + T r + + + + + - + + + + Tr

Fisher unboiled suspension . Alyott unboiled suspension . Fisher boiled suspension . Alyott boiled suspension . + + + + + T r

Fisher suspension . Alyott suspension .

1

/ + I T r

I 1 Fisher suspension 1 Alyott suspension

Serum unabsorbed

+ + + + Serum absorbed with Fisher

, Serum absorbed with Alyott

Tr -

In these experiments broth cultures grown at 22" C. overnight and diluted with normal saline were used. The water-bath was kept at 56" C. and the results read after overnight incubation.

Nature of antigens

Little work has been done to elucidate the nature of the agglutinogens present in these bacteria. Broth cultures of G.R., Fisher and Alyott were boiled for ten minutes in the water-bath and tested against the 1946 serum. No difference was found in titre whether using the boiled or unboiled cultures. This indicated that H agglutinogens were not responsible for the agglutination. On the other hand on heating the serum to 70" C. for 10 minutes, a t which temperature 0 agglutinins are labile, the titre was not shown to be altered when tested against a single strain.

Alpha agglutinins of Stamp and Xtone (1943-44)

These agglutinins are present in the sera of some rabbits, possibly as the result of infection with paracolon bacilli, and their presence has been found to lead to false positive reactions in diagnostic slide- agglutination tests. It was considered possible that these agglutinins might also be present in our sera. An alpha-agglutinating serum

400 J . B R A Y A N D T . E. D. BEAVAN

and also an appropriate bacterial strain were procured and a number of slide agglutinations carried out. No evidence was found of the presence of agglutinins of this type in the antisera we had prepared or of the corresponding agglutinogens in the Bact. coli var. neapolit- anum strains.

Action on carbohydrates

A characteristic of the strains isolated in 1943 was that of late maltose fermentation under the conditions of the test as defined in the earlier communication. This, in contrast, was not a property of the strains isolated in 1945. Only one of the 13 strains tested against maltose showed any delay in fermenting this carbohydrate. It is clear, therefore, that this is not a characteristic which can be relied on in the primary isolation of these strains.

With regard to sucrose and salicin the findings were similar to those of 1943. With both these substances fermentation was demon- strated if the tubes were kept in the incubator for 4 days. All the strains, like those of 1943, were non-motile.

DISCUSSION

Considering the multiplicity of antigenic types of Bact. coli normally found in the gut of the same and different persons (Boivin, Corre and Lehoult, 1942 ; Wallick and Stuart, 1943), and considering also that positive slide agglutinations were got so easily from the diarrheal cases by the picking of a single colony, it is strongly suggested that, in certain cases of gastro-enteritis, the coliform flora of the gut becomes replaced by a single type of these bacteria-a type which is found but rarely in normal children.

As different types of Bact. coli appear very alike on a MacConkey plate, the alteration from the normal is not apparent until the bacteria are investigated serologically.

Thus one is tempted to postulate that there exists a type of gastro-enteritis in bottle-fed infants which has a high mortality rate and an association with warmer weather, and is marked clinically in many cases by a seminal smell and bacteriologically by a change in the Bact. coli flora of the gut.

This type of gastro-enteritis might be separated on the one hand from infections due to Bact. sonnei, which appear to have a greater incidence in older children and are marked clinically by the presence of blood and pus in the faxes, and on the other from the cases of gastro-enteritis, including neonatal diarrhea, which are distinguished by no special features, clinical or bacteriological.

What setiological connection, if indeed any, Bact. coli var. neapolit- anum has to this suggested group of cases remains, in the absence of the demonstration, for example, of an enterotoxin, entirely sub judice.

SLIDE AGGLUTINATION OF COLI VAR. NEAPOLITANUM 401

SUMMARY

The technique of slide agglutination has been applied to the detection of a serologically homogeneous type of Bact. coli var. neapolit- anum in cases of summer diarrhea.

Results were positive in 87.5 per cent. of the cases, and, if 2 false positive results are excluded, in only 4 per cent. of the controls.

The frequency of Monilia in the faxes of these cases was also investigated by culture.

To Mr John Stevenson, for his valuable technical assktance, our thanks.

REFERENCES

ANDERSON, NINA A., SAGE, DOROTHY N., AND SPAULDING, E. H.

BEAVAN, T. E. D. . , . . . B o r n , A., CORRE, L., AND

BRAY, J. . . . . . . . LUDLAM, G. B., AND HENDERSON,

MACKIE, T. J., AND MCCARTNEY,

STAMP (LORD), AND STONE, DORIS

TODD, RAMONA L. . . . . . WALLICE, H., AND STUART, C. A.

LEHOULT, Y .

J. L.

J. E.

M.

1944. Amer. J . Dis. Child., lxvii, 450.

1944. Lancet, i, 568. 1942.

1945. This Journal, lvii, 239. 1942. Lancet, i, 64.

Cmpte rend. SOC. Biol., cxxxvi, 257.

1948. Handbook of practical bacteriology, 8th ed., Edinburgh p. 243.

1943-44. J . Hyg., Camb., xliii, 266.

1937. Amer. J . Hyg., xxv, 212. 1943. J . Bact., xlv, 121.

POSTSCRIPT

Since this communication went to press, our attention has been drawn to a paper by Giles and Sangster (1948) in which it is stated that two types of Bact. coli were recovered in an epidemic of infantile gastro-enteritis occurring in 1947 at Aberdeen. One of these types, which was identical biochemically and serologically with strains Lindsay (1943) and G.R. (1945), was recovered in 94-7 per cent. of cases. Both the Aberdeen strains were sensitive to strepto- mycin in witro (Giles, 1948).

REFERENCES

GILES, C. . . . . . . . 1948. Lancet, ii, 706. GILES, C., AND SANGSTER, G. . . 1948. J . Hyg., Camb., xlvi, 1.