616 . 34-002 . 1-053 . 3 : 576 . 851 . 4 8 (Buct. coli neupolitunum)

ISOLATION OF ANTIGENICALLY HOMOGENEOUS STRAINS O F BACT. COLI NEAPOLITANUM FROM SUMMER DIARRHCEA O F INFANTS

JOHN BRAY Prom nn Emergencg Medical Service Patl~ologicnl Lnborntory,

London, Sector V I

FEW investigations appear to have been made on the lactose-fermenting bacteria isolated from cases of the disease of infants variously known as summer diarrhea, cholera infantum or non-specific gastro-enteritis. The results of such an investigation carried out in parallel with a search for non-lactose-fermenters in these cases are here set out.

The investigation of the lactose-fermenting bacteria followed on the observation of Beavan (1944 and personal communication) that severe cases of diarrhoea often emit a characteristic seminal smell. Winter (1911-12) and Ornstein (1920-21) have commented on the strong semen- like odour given off by certain strains of dysentery bacilli, but dysentery strains are usually absent from typical cases of infantile diarrhea in this country (Topley and Wilson, 1936). It was considered at first that in our cases the smell might be due to the presence in the fseces of Proteus vulgaris (Metchnikoff, 1914 ; Bertrand, 1914 ; Costello and Lind, 1939), but on subculturing on nutrient agar the strains of Bact. coli isolated from cases, it became apparent that certain of these strains were responsible for the smell. It was noted that the strains in question, identified as Buct. coli neapolitanum, showed a delay in the fermentation of maltose. Further investigation showed that organisms with these characters were almost invariably present in cases of severe summer diarrhea but occurred very infrequently in the fseces of normal infants. It is with these bacteria that the present paper is chiefly concerned.

Cases investigated The investigation took place between March 1943 and April 1944.

There were 51 cases with 20 deaths. In 39 cases the disease was severe and during the summer of 1943 the mortality rate was 44 per cent. The clinical aspects of these cases will be described by Beavan else- where, but the usual features were vomiting, diarrhea and dehydration. The faxes of all cases were examined bacteriologically along with faxes

230

240 J . BRAY

from normal children. The ages of the patients ranged from less than one month to one year, the mean age being six months. The epidemic neonatal diarrhea described by Rice d al. (1937), Greenberg and Wronker (1938), Forbes and Olsen (1939),.Cron, Shutter and Lahmann (1940), Ormiston (1941) and Crowley et al. (1941) may not therefore be the same disease. Rice et al. expressly state that no cross infection took place from their cases to the older infants in the ward. As all our cases occurred in bottle-fed infants under one year of age, the controls were chosen from healthy infants of the same group.

Post-mortem findings

A post-mortem examination was made in 18 of the 20 fatal cases. The findings were meagre. The intestines were usually dilated and showed localised capillary engorgement but very little other evidence of inflammation. In fact the impression received was that the term gastro-enteritis is incorrect. A similar picture (hickshank, 1930) is seen in neonatal diarrhea. The stomach usually contained altered blood and this may have been related to the occurrence of the monilial esophagitis (Ludlam and Henderson, 1942) which was commonly found. Unfortunately this consideration was not thoroughly explored. The liver commonly showed marked fatty degeneration and in many cases this was the only striking feature. Inflammation of the middle ear (Dick et al., 1928) was exceptional. Some degree of bronchopneumonia, probably terminal, was usually present.

BACTERIOLOGICAL INVESTIGATIONS

Methods

All fecal samples were plated without previous dilution on ( a ) Leifson’s desoxycholate-citrate medium, (b) MacConkey’s agar and (c) 8 per cent. horse blood agar, and seeded into tet,rathionate broth containing 0.5 per cent. urea and coloured with British Drug Houses universal indicator. Excluding certain paracolon-like bacilli, which will be discussed later, P. morguni and Bact. sonnei were practically the only two types of non-lactose-fermenter which were isolated on the desoxycholate-citrate medium. Any non-lactose-fermenting organisms which appeared were investigated in the usual manner. On MacConkey’s agar colonies other than those of lactose-fermenters seldom appeared, even when Proteus was present in the feces. Three lactose-fermenting colonies from each plate were picked at random and transferred to peptone water, and the action of each culture on maltose then investigated.

The tetrathionate-urea broth was designed for the detection of urea-splitting strains and its efficiency in this respect had previously been thoroughly established. The indicator of the medium changes from green to blue (alkaline) on hydrolysis of the urea. For practical purposes the only organisms which bring about this change are members of the Proteus group.

The blood agar was used originally in the hope that i t might aid in the detection of strains exhibiting the characteristic smell. In the majority of the diarrhea cases and in many of the normals Proteus swarmed on the plate and the strong glue-like smell of this organism masked any other odour. When

RACT. COLI NEAPOLITANUM 24 1

this occurred the (blue) tetrathionate-urea broth was discarded. When swurming did not occur and the tetrathionate-urea broth indicated the preiqence of a urea-splitting strain the tube was subcultured and the presence of the non-swarming Proteus confirmed.

Late mnltoee fermentation. In order not to miss slight degrees of delay ill the fermentation of maltose the following procedure was adopted. One loopful of each overnight peptone water culture was inoculated into maltose tubes containing Andrade’s indicator. Control tubes were inoculated with rapid maltose-fermenters at the same time. The tubes were placed in the incubator for a few hours until the indicator in the control tubes began to tun1 pink ; they were all then withdrawn, left on the bench overnight, and the results rcud next morning.

Early in the invavtigatioii a11 the strains which had been tested for maltose ferniontation were plated thickly on nutrient agar, incubated overnight and tested for odour. Although only those strains which showed H.

delay in maltose fermentation exhibited a seminal srnell, there was considerable variation in the intensity of the smell produced by strains from different cases, and even by different colonies from the same case. -4fter testing cultures from 350 colonies this line of investigation wm abandoned as being unreliable.

An antiserum WHB prepared in the rabbit against a heat-killed suspension of a typical late maltose-fermenting odoriferous strain isolated from a severe case of diarrhwa. The titre of the serum against this strain was 1 : 16,000 and the agglutination was of the 0 type. All the late maltose-fermenting strains isolated were tested against this antiserum. Living unheated broth cultures grown overnight were used and tho results were road after incubation overnight at 56” C.

Of 20 maltose-fermenting strains taken at random from cultures of the fzcas of normal infants, the majority showed no reaction with tho antiserum diluted to 1 : 100 ; two strains agglutinated to 1 : 800 and one agglutinated to 1 : 2000. A further 50 strains were tasted by slide agglutination with negative results. Fifteen strains of non-rnaltose-fermentinfi Uact. coZi (referred to later) isolated from the control cases were tested with negative results at a serum dilution of 1 : 100. Fifteen maltose-fermenting strains of Buct. coli neupolitunu?n isolated from normal controls also failed to agglutinate at 1 : 100 serum dilution, as did five miscellaneous strains of paracolon bacilli. Of 4 strains of Buct. coli neapolitunum from the National Collection of Type Cultures one strain only (N.C.T.C. 4198), which exhibited delay in the fermentation of maltose, was agglutinated to titre. No reaction was obtained with the other three strains (N.C.T.C. 414, 660 and 4139), which all fermented maltose rapidly. In brief, no signifimnt reactions were obtained between the antiserum and strains of Bacl. coli other than those similar to N.C.T.C. 4198. It seems clear that these strains form an antigenically homo- gexieous group.

Testing for odour.

Prepctrution of untwerurn.

Specifwily of untwerum.

Dejinitwns

By Btict. coli ncupolilunum is meant the shcrose- and salicin-fermenting strains of the highly heterogeneous group of bacteria commonly clsssified as Bmt. wli. A description is given by Winslow el al. (1919).

The reactions of the strains encountered were similar to those given by Moltke (1927).

Organisms so classified produced acid and gas in glucose in 24 hours, but not in maltose, mannitol, lactose, sucrose or salicin ; they did not swarm at 2Z3 or 37” C. on blood agar (6 per cent. blood, 2A per cent. agar), did not liquefy gelatin, and produced indol.

Protewr vulgaris.

Proteua morgani.

JOUWN. OP PATH.-VOL. LVIl

242 J . BRAY

RESULTS

Controls

Fzcal samples from 100 control infants obtained during the March 1943-April 1944 period were examined in the same manner as the specimens from the diarrhea cases. These infants were hospitalised with the diarrhoea cases and can, therefore, be considered as contacts. Several of them subsequently developed the disease. The figures for normal infants in table I express the actual number of infants from whose faxes the different strains of bacteria were isolated. Bact. wli neapolitunum agglutinating to titre with the antiserum and biochemically identical with the strains recovered from the diarrhoea cases was isolated 4 times.

Further control specimens examined for late maltose-fermenters included 17 from normal non-contact infants from a welfare clinic and specimens from 80 adults and older children sent to the laboratory for investigation for various reasons. One strain was isolated from the first group and three from the second ; of the last three two came from children aged 2 years suffering from Sonne dysentery ; the other strain was from a child aged 5. These three children could be regarded as contacts.

Sixty more fzcal specimens from the same number of normal contact infants were then examined for sucrose- and salicin-fermenting strains of Bact. wli. Of the 8 strains isolated only one was an agglutinable late maltose-fermenter. In fma l samples from 12 normal breast-fed infants late maltose-fermentem were not found.

DiarrhQa m e s

The ewes are grouped into (a ) " Summer " cases, from March 1943 to October 1943 inclusive and those of April 1944 (the weather in March 1943 and April 1944 was exceptionally hot), and ( b ) " Winter " cases, from November 1943 t o March 1944 inclusive.

The percentage incidence of the different strains of organisms isolated from the 44 summer cases is shown in table I. Late maltose-fermenting Bact. coli neapolitunum was isolated from every case. Forty-two of these strains were agglutinable ; two were inagglutinable. As compared with the normal contacts, the frequency of P . morgani and the maltose+ strains of P. vulgaris was approxi- mately doubled ; the frequency of the maltose- strains of P. vulgaris approximately trebled ; while the frequency of Bact. wli neupolitunum rose from 4 to 100 per cent.-a twenty-five-fold increase. The paracolon bacilli and Bact. sonnei will be discussed separately.

These findings confirm the observations that have been made on the shift in frequency of P. morgani (Lewis, 1911-12), and of P. vulgaris (Metchnikoff, 1914) in cases of diarrhea as compared with normal infants. It was often found that each of the three

Summer cases.

BACT. COLI NEAPOLITANTJM 243

lactose-fermenting colonies picked from the MacConkey plate belonged to the same late maltose-fermenting type, suggesting that these organisms had largely replaced the normal Bact. coli. It is also

TABLE I

Percentage incidence of organim in the feces of normal infants and of the diarrhm mses

Organisms

Cases of diarrhcea

100 normal infants Summer (44 cases) and (51 cast

P. rnorgani . P . vulgaris maltose+ ,

P . vulgaris maltose - . Bact. sonnei . Paracolon bacilli . Pa. pyocyanea . Bact. coli neapolitanum .

15 (12)

30 (33) 6

0 4 1 4 I

32.0 (53) 11.0 84.0 (93.6) 6-8 4.5 4.5

100.0

32 10 82

8 4 4

92

The figures in brackets give the findings of Morgrtn and Ledingham (1908-09) for P. morgani during 1908 and of Metchnikoff (1914) for P. vulgarin dnring 1909-13.

noteworthy that in 13 of the diarrhea cases the faxes were examined before the acquisition of the infection in hospital and are included in the control series. From none of these cases was late maltose- fermenting Bact. coli neapolitunum found before the onset of diarrhea, but this organism was recovered from all after the disease had set in.

Winter cases. Seven cases were diagnosed as gastro-enteritis, with two deaths, during the months November to March. The bacteriological findings were irregular. Agglutinable Bact. coli neapolitanum was isolated from one case, Bact. sonnei from one case ; paracolon bacilli biochemically similar to the non-lactose-fermenting variants of Bact. coli neapolitanum (described later) but inagglutinable, were isolated twice. The frequency of the various coliforms found in all cases is shown in table I.

Characteristics of the late maltose fermenting strains

The biochemical characters of the late maltose-fermenting strains are given in table 11. Forty-two of the 44 strains from the cases of summer diarrhea were agglutinated to titre by the antiserum ; 2 strains were inagglutinable.

Biochemical variants. Towards the end of the March to October series attention was drawn to certain paracolon-like non-lactose- fermenters which appeared in 8 cases on the desoxycholate and MacConkey plates. Except for the action on lactose the fermentation and biochemical reactions, as shown in table 11, were identical with those of the late maltose-fermenting strains. All the strains isolated

244 J . BRA Y

- P

from the diarrhea cases were agglutinated to titre by the antiserum. These paracolon-like organisms are evidently non-lactose-fermenting

TABLE I1 Biochemical characters of strains of Bact. coli neapolitanum

from cases of summer diarrhm

SlA

SIA

AG

____ AG AG

AG - AG AG

- 45

- 10

2 -

Results were recorded after 4 days’ growth, except for maltose, when readings mere made after 18 hours’ growth.

V.-P. and M.R. tests made nftcr 7 days. AG = Acid and gas. SIA 1 Slight acidity.

For details see p. 241.

variants of the late maltose-fermenting Bact. co2i neapolituntsm. This was confirmed by the smell given off by these strains, by their absorption of the agglutinins from the antiserum, and by the fact that on subculture of one strain in lactose broth it rapidly acquired the ability to ferment this sugar. No red papillae, as with Bact. coli mutabile, were seen to form on the white colonies on MacConkey’s agar.

It seemed possible that, in view of the method adopted for detecting agglutinable strains of Bact. coli neapolitanum, some strains fermenting maltose rapidly might not be detected during the search for this organism in the normal controls. Towards the end of the investigation two such strains were found. One was isolated from a one-year-old child with diarrhea, the other from a healthy control. Both were detected by their marked seminal smell. Both were agglutinated to titre by the antiserum, but neither showed any delay in the fermentation of maltose. In view of this, 8 strains of sucrose- and salicin-fermenting Bact. coli were collected from 60 normal controls, as already mentioned, and tested for agglutination. None of the strains fermenting maltose was agglutinated. It seems therefore that maltose-fermenting agglutinable strains of Bact. coli neapolitanum are just as rare as those of the late maltose-fermenting variety in the faxes of normal children.

Non-maltose-j‘ermenting Bact. coli. Many strains of Bact. coli, like some strains of Bact. sonnei, have no fermentative action on maltose even after ten days. These strains are not to be confused with the late maltose-fermenting Bact. coli neapolitanum here described. Ten strains of non-maltose-fermenting Bact. coli were isolated from the 100 normal controls, but none from the summer

RACI’. COTJ NEAPOLITANUM 245

diarrhea cases. They occurred in 4 out of 7 winter cases, but this is of doubtful importance. There was no constancy in respect of motility or the fermentation of sucrose or salicin, but none of the 10 strains fermented both these sugars and none was agglutinated by the antiserum prepared against the late maltose-fermenting strain of Bact. coli neupolitanum.

Agglutinins in patients’ serum The titre of the agglutinins in the blood of 10 cases of diarrhea

for the autogenous strain of agglutinable Bact. wl i neapolitanum was not significant. The sera were taken from 6 caaes convalescent from gastro-enteritis and from 4 cases at autopsy. The titre in two normal controls tested against an agglutinable strain was 1 : 32 and 1 : 64. In the diarrhoea cases the titre was higher in only one caae (1 : 320). Hughes (1942-43) states that in pyelitis due to coliform infections the agglutinins against the autogenous strain do not reach a titre higher than 1 : 25, even in cases of long duration, and are absent in the majority of cases.

Origin of seminal smell The seminal smell already referred to is probably due to a derivative

of spermine (Rosenheim, 1924). Spermine is odourless, but a p p o l derivative produced on its oxidation (Dudley et al., 1926) has the characteristic smell. The smell is not detectable in cultures on Macankey’s agar except with the non-lactose-fermenting strains. The acidity of the medium appears to inhibit production of the odoriferous substance. Attempts to enhance the smell by the addition of various amino-acids to Capek-Dox agar medium were unsuccessful.

PreAence of dysentery strains in gastro-enteritis Bact. sonnei occurred 3 times in the 44 summer cases. This was

noteworthy, as cases of Sonne dysentery occurred sporadically among the older children during this period. In two cases the Sonne infection preceded the appearance of the late maltose-fermenters and in the other followed it. Where the Sonne infection occurred first there was said to be a sudden decline in the clinical condition coincident with the appearance of the Bact. coli neapolitanum. Where the Sonne infection followed, there wst9 said to be a sudden exacerbation of symptoms during convalescence. Cases of Sonne dysentery do not have the characteristic smell found in cases of summer diarrhea. Six strains of Bact. sonnei were tested for the smell in the usual manner and were found negative.

Occurrence of late mltose-fermenting strains in $ie,q

During 1943 flies were abundant in the gastro-enteritis wards ; 20 were cultured by being crushed in peptone water, which was then

246 J . BRAY

plated out. Besides Proteus vulgaris and P. morgani, the late maltose- fermenting Bact. coli neapoliitcbnum (agglutinable) was isolated twice. This evidently shows that not only had the flies access to the infants’ dejecta, but also that if the organism is transmissible these insects could act as vectors.

DISCUSSION

Whether or not Bact. coli neapolitanum of type 4988 is setiologically connected with summer diarrhoea of infants, neither P . vulgaris nor P. morgani seem likely to fit this role. Neither of these organisms is antigenically homogeneous. The low carrier rate of Bact. coli neapolitanum in normal infants supports the idea that its presence in the disease may be significant. Moreover, it is normally abundant in the intestinal tract of the higher animals, but not in man (Winslow et al.). If the late maltose-fermenting strains have any aetiological importance it does not appear that they act in the same way as organisms of the dysentery group in view of the absence of gross inflammation of the intestine. The changes in the liver may suggest the absorption of some toxin from the intestinal tract ; on the other hand these changes are more probably due to starvation (Dible,

No experiments have yet been carried out to determine whether the organism produces a soluble toxin. Crowley et al. in their in- vestigations on neonatal diarrhoea found no evidence of a soluble toxin by injecting fzecal filtrates into mice. Feeding experiments which we made with living broth cultures on small numbers of lactating mice, kittens and rabbits gave negative results. There is some evidence (Light and Hodes, 1943) that neonatal diarrhea is caused by a virus and it is possible, if summer diarrhoea and neonatal diarrhoea are aetiologically related, that Bact. coli neapolitanum may be associated with tt virus on the analogy of the association between the virus of hog cholera and Bact. cholera suis.

Nevertheless, it has yet to be shown that these organisms cause the diarrhoea and not that the diarrhoea enables the organisms to flourish. In view of the somewhat hit-or-miss methods of detecting them it may be objected that these strains may always be present in small numbers in normal fseces. This may well be true, and perhaps the best that can be said is that, by present methods, these strains of Bact. coli neapolitanum are very hard to find in normal feces but very easy to isolate in cases of summer diarrhoea.

1938-39).

SUMMARY

A particular serological type of Bact. coli neapolitanum has been recovered from 42 out of 44 cases of summer diarrhoea of infants. The characteristics of these strains are described and their possible aetiological connection with the disease is discussed.

BACT. COLI NEAPOLITANUM 247

My thanks are due to Sir Alexander Fleming for his interest in this in- vestigation, to Dr T. E. D. Beavan for his valuable co-operation, to Dr Doris Stone for reading the MS. and to Dr C. N. Iland and Dr Glynn-Jones for specimens. Thanks are also due particularly to Sister M. L. Chaplin for arranging for the supply of numerous specimens and to Mr J. F. Stevenson for technical assistance.

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