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Bacterium alkalescens in the stools of normal infants
B A C T E R I U M A L K A L E S C E N S I N T H E S T O O L S OF NORMAL INFANTS MAn.s~ ANN
L. SNYDE~, PH.D. A~BO~,
MICH.
E R I A L studies of the fecal flora of twenty-three n o r m a l infants showed three babies to have consistently large numbers of dysentery organisms in their stools. These strains were at first identified as Bact. dysenteriae (Flexner), but later studies showed them to be Bart. alkalescens, a species closely related to the F l e x n e r t y p e but to which Andrewes 1 ascribed doubtful pathogenicity. Following Shiga's description iI~ 1898 of the org"a~lism associated with an epidemic of dysentery in J a p a n , 2 several types of dysentery bacilli were isolated and classified in a short time. Their relation to the summer diarrhea of infants was quickly sought, and extensive investigas were made. The failure to recover these or similar forms from the stools of normal infants was recorded by Wollstein, 3 Da.vison, 4 and Soule and Heyma.n, ~ but D u r a l 6 sta~ed that he found the Flexner type in the stools of normal bottle-fed infants. Collins 7 also obtained "Bacillus p,aradysen.tericte" in one of a series of ten normal infants, a 2-yearold child who had no previous history of diarrhea.. However, she could not find arty of these organisms in a duplicate series. The presence of dysentery bacilli in the stools of infants led Veeder, Kilduffe, and D e n n y s in 1912 to the conclusion that the appearance of a few dysentery bacilli was of little significance and even in a. ease of colitis did not mean t h a t these germs were the cause of this condition. Specifically, the presence of Bact. alkalescens in the stools of normal infants has not, as f a r as the author is aware, been reported. Its occurrence has for the most p a r t been associated with abnormal conditions of the u r i n a r y tract as recorded by Well, 9 Popoff and Spanswiek, ~~ Smith and Fraser, 1~ Welch and Miekl@ 2 M u r r a y and Pike, ~3 Starkey, ~4 3/[aekenzie mid Rather, x~ Snyder and I-Ianner, ~6 mad Neter27-~9 I n these instances Bact. alkcdescens was usually isolated from the feces. However, its presence in the blood stream was observed by Smith and Fraser as well as by Starkey. Welch and Mickle found Bact. alkalescens in the stools of several university students d u r i n g an acute outbreak of dysentery which could be tra.eed to a food handler who was a carrier. Neter also mentioned the isolation of Bact. a lkalescens from the stool of a healthy person. Because of these references dealing with the presence of Bact. alt~alescens in normal and pathologic intestinal and u r i n a r y tracts, it seemed of interest to report the following 9bservations.
S
T h i s w o r k w a s d o n e a s C h i l d R e s e a r c h F e l l o w in t h e D e p a r t m e n t o f t l a e t e r i o l o g y a n d P u b l i c H e a l t h a n d C h i l d I ~ e s e a r e h Co,uneil, U n i v e r s i t y of C o l o r a d o S c h o o l o f Medicine, Denver. 341
342
T H E JO.UI%NAL 0F PEDIATlgICS MATERIAL
This s t u d y of Bact. alkalescens represents only a small portion of the work done on the feces of twenty-three infants under 1 y e a r of age who were a p a r t of a group of one hundred children being" followed f r o m birth to adolescence b y the staff of the Child Research Council. In addition to the extensive routine q u a r t e r l y examination given these children, stools were obtained biweekly f r o m the breast-fed infants and monthly f r o m the babies who were weaned. With the exception of one baby there was no acute gastroenteritis recorded. All of the infants were n o r m a l in d e v e l o p m e n t as d e t e r m i n e d by e x h a u s t i v e clinical study. The isolation of a d y s e n t e r y bacillus in the one case of gastroenteritis cast suspicion upon it as the etiological agent. I I o w e v e r , similar organisms were also f o u n d in the stools of two n o r m a l infants. No other a p p a r e n t l y a b n o r m a l species was observed. B r i e f histories of the three babies are as follows: CASE ] . - - A b a b y girl, 6 weeks old at t h e t i m e of the f i r s t - e x a m i n a t i o n , h a d r e c e n t l y b e e n t r a n s p o r t e d f r o m I d a h o to Denver, a n d a f t e r h e r a r r i v a l s t a r t e d to p a s s six to e i g h t w a t e r y g r e e n i s h stools a d a y which con%ained m u c u s b u t n o blood. Recovery w i t h o u t m e d i c a t i o n was complete w i t h i n a f o r t n i g h t . N o m o r e t r o u b l e w a s r e c o r d e d during, t h e p e r i o d of o b s e r v a t i o n , w h i c h w a s u p to 1 y e a r of age. A l t h o u g h t h e b a b y w a s b r e a s t fed~ t h e r e w a s t h e p o s s i b i l i t y of i n f e c t i o n b y w a t e r during the journey. C~sE 2.--A_ mule child, 6 m o n t h s old a n d a l r e a d y w e a n e d b e f o r e his first examination~ h a d no previous record o f d i a r r h e a or d y s e n t e r y . D u r i n g t h e n e x t six month.s t h e r e w a s l i k e w i s e no o c c u r r e n c e of g a s t r o i n t e s t i n a l l r o u b l e . CASE 3 . - - T h e first stool w a s o b t a i n e d f r o m t h l s h e a l t h y b r e a s t - f e d male i n f a n t w h e n he w a s ~ weeks old. E x c e p t f o r a s l i g h t d i a r r h e a a~ 5 ~ m o n t h s w h e n t h e first t e e t h erupted, no a b n o r m a l i n t e s t i n a l condition w a s noted.
The stools of the b r e a s t - f e d infants were c h a r a c t e r i s t i c a l l y deep yellow in color, semisolid in texture, and acid in reaction. A f t e r weaning, the usual shift to the d a r k e r stools w i t h fecal odor was observed. Microscopically, slide smears of the n u r s l i n g s ' feces were observed to be ahnost u n i f o r m l y composed of slender or slightly c u r v e d g r a m positive rods, some of which showed bifid forms. These organisms were assumed to be Lactobacillus bifidus. To obtain members of the colon-typhoid-dysentery group, suspensions of the specimens were made in normal saline of which a loopful was streaked over the surface of an eosin-methylene-blue a g a r plate. Instead of the ordinarily expected metallic colonies of Bact. coli or the mueoid growth of Bact. aerogenes, it was extremely interesting to find numerous smooth fiat grey or white colonies which even replaced the coliform organisms, as in Case 3 when it was the f o u r t h month before Bact. coli appeared. These organisms were repeatedly isolated in Cases I and 3 up to 6 months of age, a f t e r which they a p p e a r e d less frequently. F o r the
SNYDER:
BACT. ALXAL,ESCENS IN STOOLS OF NOR,NIAL INFANTS
343
other members of the fecal flora in these babies the author's thesis ~~ may be consulted. To show the regularity of the presence of Bact. alkalescens the results of the periodic examinations are given in Table I. TABLE I FtlEQUENCY
OF
ISOLATION
OF
Ba,6t.
A~]6c~le8(~e~8
IN
TKE
~FECES
OF
THREE
BABIES
UNDEI~ ONE YEArr OF A~E WEEKS CASE
~1 GI s lo 1~]14 16117118119
a (7404) *Serial
+ numbers
+
+
+
+
for laboratory
+
p,~,124128 a2136140 44:14:8
+
cultures
o
of hospital
+1
Ol +
material.
LABORATORY STUDIES
Preliminary work showed these suspicious forms on rosin-methyleneblue agar to be short nonmotile rods that did not ferment laetose or liquefy gelatin. Fermentation of mannite tentatively classified the groups as strains of Bact. dysenteriae (Flexner). However, extended cultural studies revealed the constant production of acid in dulcitol which identified the strains as B a c t alkalescens (Andrewes * and Bergey~*). Acid was not produeed in laetose in two weeks, which reaction differentiated these strains from the mutating Sonne and Dispar types. The cultural reactions were repeated at the Hygienic Laboratory and compared with two strains of BacL alkalescens sent by Professor E. D. G. Murray, of McGill University, one of which was tile u r i n a r y strain (49226) isolated by Starkey and the other a subculture of Andrewes, original BacL alkalescens. The urinary strain (7172) of Snyder and Harmer was also included. The cultural reactions of these strains and Bact. dysenteriae (Plexner) are given in Table II. T A B L E II CULTURAL ]~EACTIONS OF ~TI~AINS OF BfbOt. AlkaIesceus COIIPAt~:D W I T H Bact. Dysenteriae (~LEXNEF~)
Case 1 (7204) Case 2 (7166) Case 3 (7404:) 7172 49226
Bact. a~lca~esce~s Bact. dyseq~teriae (Ftexner)
3
~
A
A
~
22A A A A
a
A A A
I
o
Ao A A A
AIPA
A A A
0
o?
oA A ~ I
0 0 0 0
A A 0
5~
O
~ 0
A A A A A
2 A O
O O O O O O O
0 0
o
+ + +
o+ O
+ +
Alk. Alk. Alk. Alk. Alk. A]k. Alk.
Table I I shows the uniform fermentation with acid but no gas of glucose, levulose, galactose, maltose, xylose, mannitol, and dulcitol by the
344
THE JOUR.NAL OIO PEDIATRICS
strains Of Bact. alkalescens, whereas B,ct. dysentcriae (Flexner) did not produce acid in xylose or du]citok The reactions with mannitol and dulcitol are in accord with the excellent differential chart given by Andrewes and I n m a n } ~ Milk was also t u r n e d alkaline more r a p i d l y with the Bact. cd/calescens than with the F l e x n e r type. Another characteristic of Bact. aZka~escens as pointed out by Andrewes is its susceptibility to agglutination b y acid after the method of Michaelis. 2~ This feature is in contrast to the inagglutinability of dysentery bacilli by these acid concentrations (Michaelis2~). All the present strains of Baet. alkalescens could be shown to be completely flocculated in buffers of p H 3.2-2.4. The Flexner strain was not agglutinated by respective hydrogen ion concentrations. Serologieally, Andrewes found Bact. alkalescens to stimulate antibody formation with difficulty. ]in his monograph with Inlnan, however, he noted that good agglutination could be obtained by incubation at 55 ~ C. for t w e n t y to twenty-four hours. H e felt that this delayed reaction by Bact. alkalescens and lactose fermenters (B. ambiguous) was so striking as to be regarded of special significance. Also, a common antigen with the Flexner type, especially at 55 ~ C., was present. Well, Smith and F r a s e r , Popoff- and Spanswick, Welch and Miekle, a n d Neter were likewise able to obtain not only specific antibodies but also showed a common antigen with Flexner which could be absorbed. P r e l i m i n a r y tests with the strains at time of isolation showed 7204 and 7404 to agglutinate with a known Flexner antiserum but not with Shiga, Park, Hiss Y, and Sonne antisera. Strain 7166 was not agglutinated b y the Flexner antiserum. The reactions of ttnese strains with an antiserum p r e p a r e d against Strain 7404 are listed in Table I I I . TAtTlE IIi[ CZOSS-AGGLUTJ[NATION TESTS OI,' STRAINS OF Bac t . At]~cdesce'Y~s ISOLATED 1~I%0.~ IN?~ANTS WITII AN 2AkNTISEP~USI .D]%EPARED AGAINST ONE OF TIIE FECAL STI~AINS, 7404 SEI~U EI D I L U T I O N S
Cont. 7204 7166 7404
. ++§247247247
++++
q-+++
+4-++
(horn.) Flexner
/++++1++++1++++1++++1++++/++++/
-t-+l
-/
-
H e r e again the relation of Strain 7404 to the F l e x n e r type is shown, but there was no macroscopic agglutination with the other two strains. A f t e r these tests the cultures were placed on agar slants and sealed for more t h a n a year, when they were opened for additional study. Routine transfers were made f r o m one- to three-month intervals. All strains w h e n first plated on nutrient agar showed the colonies to be mostly smooth with some colonies showing slightly wrinkled edges i n forty-eight
SNYDEI%:
BACT.
ALKALESCENS
IN
STOOLS
OF
NOI'~i\{AL I N F A N T S
345
to seventy-two hours. Recently the Andrewes and 7166, 7404, 49226 strains were found to be completely dissociated into the R colony form. P r e p a r a t i o n of antisera against the Flexner type and Strains 7172, 7204, 7166, and 7404 was done with heat-killed saline suspensions as antigens which were given subcutaneously in rabbits in 2.0 c.e. doses ten times at four-day intervals, and the rabbits were bled for serum a few days a f t e r the last injection. Macroscopic tests showed no agglutination of the strains of B a c t . a l k a l e s c e n s by the Flexner antiserum. Strain 7172 was not flocculated by its own or the other antiscra, but the Andrewes, 7166, 7204, and 7404 strains and the Flexner type agglutinated up to a ] :40 serum dilution, and occasionally partial clumping as high as 1:320 by antisera 7204, 7166, and 7404 was observed. Antisera p r e p a r e d against the Andrewes and 49226 strains showed a similar lack of agglut, inins. The tests on the whole were v e r y inconclusive. Since plain broth cultures flocculated somewhat better than saline suspensions, new rabbits were inoculated with forty-eight-hour broth cultures of the Andrewes and 7204, 7172, 7166, 7404 strains, which were given either subcutaneously (2 e.c.) at four-day intervals or three weekly series of daily intravenous doses (0.5 to 2 e.c.) with a week's interval between the series. The agglutination reactions with these new antisera were not significantly different f r o m the preceding ones. Failing satisfactory agglutination tests, recourse was made to the complement fixation test as recommended by Starkey. Portions of 7204, 7172, Andrewes and Plexner antisera were heated to 56 ~ C. for t h i r t y minutes and mixed with saline suspensions diluted to a point determined not to be anticomplementary. I n the presence of complement and a hemolytic system the results are recorded in Table I V for a 1:20 sermn dilution. TABLE COMP],E~ENT BACTEI~IAL S Y S T E S I :
IV
FIXA!PlON T E S T S W I T I I
~O~(Y~6T$'g, Tg& fl,~/~Cd~68(J6'[b3
A N T I G E N 0.2 C,C. SALINE S U S P E N S I O N
SEr~U~t 0.2 c.c. (]-20) CO~PnE~ENT 0.2 C.C. (1-20) ItEI~,IOLYTIC SYSTE.~{: INCUBATION:
AIV[BOCEPTOR 0.2 C.C. ( 1 - 2 0 0 ) F~.B.C. ( S I I E E P ) 0.5 C.C. 2 PEN CENT S U S P E N S I O N BACTEI{IAL SYSTENI INCUBATED I N A WATEI% BATH 30 M I N U T E S AT 37~ ~IES~OLYTIC SYSTE~,I ADDED AND INCUBATED E01~ AN ADDITIONAL 3 0 1V[INUTES
9
7172 7204
o
o
o
o
Baet. alkalcscens Bact. dysenter4ae
o
o
o
o
(Flexner) C : Complete -hemolysis. O ---- N o h e m o l y s i s .
o o~
59
cc ce
c c
cc
346
T t I N , J O U R N A L OF P E D I A T R I C S
Serum dilutions of 1:5 and 1:10 gave the same results; namely, that there were apparently specific antibodies produced by the Bact. alkalescens strains which did not fix complement in the presence of Flexner antigen. DISCUSSION
The interesting feature in this study was tile isolation of Bact. alkalescens, a dysentery bacillus, from the stools of three normal infants in only one of whom occurred a marked gastroenteritis. In this instance it seemed probable that these organisms were the cause of the illness, since there were no other pathogenic forms demonstrated. Their presence was not chance finding since they were repeatedly obtained and in one ease were the only gram-negative aerobic nonsporulating rods in the stools up to the f o u r t h month, when a few coliform bacteria appeared. Except for the possibility in Case 3 in which a similar organism was found in the father's stool, the source of infection could not be determined. These findings support the slight pathogenicity of Bact. alkalescens as reported in the literature. However, the chance of increased ~irulenee through transmission remains, and thus a public health problem is raised. On a cultural basis the fermentation of dulcite seems to be the chief differential criterion and for routine diagnostic purposes should serve to separate Bact. alkalescens from the Flexner, Hiss Y, Park, and Strong varieties. Serologically, however, the problem, is complicated by the inability of some strains either to stimulate the production of antibodies or to agglutinate with known serum. Neter (personal communication) thinks there are three types of agglutination response with this species: first, a high titer agglutination; second, a delayed though high titer agglutination; and third, sera with little or no ability to agglutinate. A hig'h titer serum (2567) prepared by Neter failed to agglutinate any of the present Bact. alkalescens strains, but it did agglutinate the Flexner strain to a serum dilution of 1:1280, and its homologous antigen 1. :5,120. Complement fixation tests with this serum showed Neisser-Weehsbur~ types of antigen-antibody combination with the Andrewes and 7404 strains in sermn dilutions of 1:80 and 1:160 but not at 1:20 and 1:40. Strains' 7166, 7172, 49226, and 7404 did not fix complement, but the Flexner strain absorbed the complement in a sermn dilution of 1:160. Recent observations on these strains showed all but 720r to be completely or nearly completely dissociated into the rough phase on the basis of colony morphology. The Flexner type remained smooth. Undoubtedly this change explains some of the difficulties and divergent results encountered in the serological tests, but considering the inability to produce antibodies even with freshly isolated smooth strains, it would appear that bacterial variation was not the whole story. In an exchange of strains and antisera of Bact. a~l~alescens with Dr. Neter, both of us obtained little or no agglutination with the present strains as antigens and his high titer Bact. alkalescens (2567) antiserum as the antibody. "However,
SNYDEP~:
BACT. ALKALiESCENS IN STOOLS O.P NOIgl~AL INFANTS
347
Neter pointed out, as Andrewes had already indicated, that extensive agglutination took place at 55 ~ C. in forty-eight hours with nil strains except 7172. This delayed reaction could be confirmed. F u r t h e r studies are being made to determine other variables besides temperature acting u p o n the antigen-antibody reactions of Bact. alkalescens. At present it would seem that while strains of Bact. alkalescens exist that are capable of producing high titer antisera, the strains u n d e r study do not possess this power. As a result of low and irregular agglutination response, no absorption tests were made; only the gross relationship of the strains of Bact. alkalescens to each other and the Flexner type was indicated. SUMMARY
Strains of dysentery bacilli were frequently isolated from the stools of three infants under one year of age. 0 n l y one of these babies had any history of gastrointestinal trouble. The identity of these organisms with a subculture of the original strain of Bact. alkalescens was shown by culturaI tests. The serologic picture was complicated by the inability of the strains to stimulate the production of agg]utinins or other antibodies so that adequate titers for absorption tests could be made. Nevertheless, tile strains seemed to have an antigen in common which was also shared by the Flexner type in the agglutination test but not in the complement fixation reaction. REFERENCES J. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Andrewes, F. W.: Lancet 1: 560, 1918. Shiga, K.: Centralbl. f. Bakt. 23: 599j ]898. Wollstein, IV[.: J. 1VI. Research 10: 11, 1903. Davison, W. C.: Medicine 1: 389, 1922. Soule, M. H , and Heyman, A.: J. Lab. & Clin. Mud. 36: 281, 1933. Duval~ E.: Studies from Rockefeller Inst. 2: 121, 1904, Collins, /(. R.: J. Infect. Dis. 2: 620, 1905. Veeder, 9. S., Kilduff% R., and Denny, O. T.: Am. J. Dis. Child. 4: 75, 1912. Well, A . J . : Centralbl. f. Bakt. Orig. I, 112: 376~ 1928. Popoff, N. W., and Spanswick, M. P.: ,l. Lab. & Clin. Med. 16: 437~ 1931. Smith and Fraser, A. M.'. J. Path. & Baet. 31: 511, 1928. Weleh~ I-I., and Mickle, :F. L.: Am. J. Pub. ]-Iealth 2~: 219, ]93&. Murray, M. F., and Pike, :R. 3/I.: Clin. Misc. iVlary I. Bassett Hospital 1: 69, 1934. Starkey, D. I-I.: Canad. 1Vf. A. J. 31: 42, 1934. ~ackenzie, D. W., and ]~atner, ~ . : J. Urol. 31: 671, 1934. Snyder, M. L , and l:Ianne% J.: J. Infect. Dis. 60: 51, 1937. Neter, E.: J. Infect. Dis. 61: 338, 1937. Neter~ E., a~d Rappo]e, F.: Arch. Path. 25: 298, 1938. Neter, E.: J. Urol. 39: 727~ 1938. Snyder, 1V[.L.: A S~udy of the Normal Fecal Flora of I n f a n t s Under One Year of Age, :1935~ Univ. of Colorado Library, Boulder, Colo.
2]. ]3ergey, D. H.: ~anual of Determluative ]3aeteriology, ed. ~, Baltimore, 1934, Williams and Wilkins Company. 22. Andrewes, F. W., and Inman, A. C.: Special Rep. Med. Res. Committee, No. 42, 1919. 23. 3/[iehae]is, L.: 2~. Miehaelis, L.:
Deutsche meal. Wehnsehr. 37: 969, 1911. Deutsche reed. Wehnsehr. 43: lgO6, 19;I.7.
HYGIENE LABORAT01~Y~ "[J'NIVEr~SITY OF MICHIGAN"
L. SNYDE~, PH.D. A~BO~,
MICH.
E R I A L studies of the fecal flora of twenty-three n o r m a l infants showed three babies to have consistently large numbers of dysentery organisms in their stools. These strains were at first identified as Bact. dysenteriae (Flexner), but later studies showed them to be Bart. alkalescens, a species closely related to the F l e x n e r t y p e but to which Andrewes 1 ascribed doubtful pathogenicity. Following Shiga's description iI~ 1898 of the org"a~lism associated with an epidemic of dysentery in J a p a n , 2 several types of dysentery bacilli were isolated and classified in a short time. Their relation to the summer diarrhea of infants was quickly sought, and extensive investigas were made. The failure to recover these or similar forms from the stools of normal infants was recorded by Wollstein, 3 Da.vison, 4 and Soule and Heyma.n, ~ but D u r a l 6 sta~ed that he found the Flexner type in the stools of normal bottle-fed infants. Collins 7 also obtained "Bacillus p,aradysen.tericte" in one of a series of ten normal infants, a 2-yearold child who had no previous history of diarrhea.. However, she could not find arty of these organisms in a duplicate series. The presence of dysentery bacilli in the stools of infants led Veeder, Kilduffe, and D e n n y s in 1912 to the conclusion that the appearance of a few dysentery bacilli was of little significance and even in a. ease of colitis did not mean t h a t these germs were the cause of this condition. Specifically, the presence of Bact. alkalescens in the stools of normal infants has not, as f a r as the author is aware, been reported. Its occurrence has for the most p a r t been associated with abnormal conditions of the u r i n a r y tract as recorded by Well, 9 Popoff and Spanswiek, ~~ Smith and Fraser, 1~ Welch and Miekl@ 2 M u r r a y and Pike, ~3 Starkey, ~4 3/[aekenzie mid Rather, x~ Snyder and I-Ianner, ~6 mad Neter27-~9 I n these instances Bact. alkcdescens was usually isolated from the feces. However, its presence in the blood stream was observed by Smith and Fraser as well as by Starkey. Welch and Mickle found Bact. alkalescens in the stools of several university students d u r i n g an acute outbreak of dysentery which could be tra.eed to a food handler who was a carrier. Neter also mentioned the isolation of Bact. a lkalescens from the stool of a healthy person. Because of these references dealing with the presence of Bact. alt~alescens in normal and pathologic intestinal and u r i n a r y tracts, it seemed of interest to report the following 9bservations.
S
T h i s w o r k w a s d o n e a s C h i l d R e s e a r c h F e l l o w in t h e D e p a r t m e n t o f t l a e t e r i o l o g y a n d P u b l i c H e a l t h a n d C h i l d I ~ e s e a r e h Co,uneil, U n i v e r s i t y of C o l o r a d o S c h o o l o f Medicine, Denver. 341
342
T H E JO.UI%NAL 0F PEDIATlgICS MATERIAL
This s t u d y of Bact. alkalescens represents only a small portion of the work done on the feces of twenty-three infants under 1 y e a r of age who were a p a r t of a group of one hundred children being" followed f r o m birth to adolescence b y the staff of the Child Research Council. In addition to the extensive routine q u a r t e r l y examination given these children, stools were obtained biweekly f r o m the breast-fed infants and monthly f r o m the babies who were weaned. With the exception of one baby there was no acute gastroenteritis recorded. All of the infants were n o r m a l in d e v e l o p m e n t as d e t e r m i n e d by e x h a u s t i v e clinical study. The isolation of a d y s e n t e r y bacillus in the one case of gastroenteritis cast suspicion upon it as the etiological agent. I I o w e v e r , similar organisms were also f o u n d in the stools of two n o r m a l infants. No other a p p a r e n t l y a b n o r m a l species was observed. B r i e f histories of the three babies are as follows: CASE ] . - - A b a b y girl, 6 weeks old at t h e t i m e of the f i r s t - e x a m i n a t i o n , h a d r e c e n t l y b e e n t r a n s p o r t e d f r o m I d a h o to Denver, a n d a f t e r h e r a r r i v a l s t a r t e d to p a s s six to e i g h t w a t e r y g r e e n i s h stools a d a y which con%ained m u c u s b u t n o blood. Recovery w i t h o u t m e d i c a t i o n was complete w i t h i n a f o r t n i g h t . N o m o r e t r o u b l e w a s r e c o r d e d during, t h e p e r i o d of o b s e r v a t i o n , w h i c h w a s u p to 1 y e a r of age. A l t h o u g h t h e b a b y w a s b r e a s t fed~ t h e r e w a s t h e p o s s i b i l i t y of i n f e c t i o n b y w a t e r during the journey. C~sE 2.--A_ mule child, 6 m o n t h s old a n d a l r e a d y w e a n e d b e f o r e his first examination~ h a d no previous record o f d i a r r h e a or d y s e n t e r y . D u r i n g t h e n e x t six month.s t h e r e w a s l i k e w i s e no o c c u r r e n c e of g a s t r o i n t e s t i n a l l r o u b l e . CASE 3 . - - T h e first stool w a s o b t a i n e d f r o m t h l s h e a l t h y b r e a s t - f e d male i n f a n t w h e n he w a s ~ weeks old. E x c e p t f o r a s l i g h t d i a r r h e a a~ 5 ~ m o n t h s w h e n t h e first t e e t h erupted, no a b n o r m a l i n t e s t i n a l condition w a s noted.
The stools of the b r e a s t - f e d infants were c h a r a c t e r i s t i c a l l y deep yellow in color, semisolid in texture, and acid in reaction. A f t e r weaning, the usual shift to the d a r k e r stools w i t h fecal odor was observed. Microscopically, slide smears of the n u r s l i n g s ' feces were observed to be ahnost u n i f o r m l y composed of slender or slightly c u r v e d g r a m positive rods, some of which showed bifid forms. These organisms were assumed to be Lactobacillus bifidus. To obtain members of the colon-typhoid-dysentery group, suspensions of the specimens were made in normal saline of which a loopful was streaked over the surface of an eosin-methylene-blue a g a r plate. Instead of the ordinarily expected metallic colonies of Bact. coli or the mueoid growth of Bact. aerogenes, it was extremely interesting to find numerous smooth fiat grey or white colonies which even replaced the coliform organisms, as in Case 3 when it was the f o u r t h month before Bact. coli appeared. These organisms were repeatedly isolated in Cases I and 3 up to 6 months of age, a f t e r which they a p p e a r e d less frequently. F o r the
SNYDER:
BACT. ALXAL,ESCENS IN STOOLS OF NOR,NIAL INFANTS
343
other members of the fecal flora in these babies the author's thesis ~~ may be consulted. To show the regularity of the presence of Bact. alkalescens the results of the periodic examinations are given in Table I. TABLE I FtlEQUENCY
OF
ISOLATION
OF
Ba,6t.
A~]6c~le8(~e~8
IN
TKE
~FECES
OF
THREE
BABIES
UNDEI~ ONE YEArr OF A~E WEEKS CASE
~1 GI s lo 1~]14 16117118119
a (7404) *Serial
+ numbers
+
+
+
+
for laboratory
+
p,~,124128 a2136140 44:14:8
+
cultures
o
of hospital
+1
Ol +
material.
LABORATORY STUDIES
Preliminary work showed these suspicious forms on rosin-methyleneblue agar to be short nonmotile rods that did not ferment laetose or liquefy gelatin. Fermentation of mannite tentatively classified the groups as strains of Bact. dysenteriae (Flexner). However, extended cultural studies revealed the constant production of acid in dulcitol which identified the strains as B a c t alkalescens (Andrewes * and Bergey~*). Acid was not produeed in laetose in two weeks, which reaction differentiated these strains from the mutating Sonne and Dispar types. The cultural reactions were repeated at the Hygienic Laboratory and compared with two strains of BacL alkalescens sent by Professor E. D. G. Murray, of McGill University, one of which was tile u r i n a r y strain (49226) isolated by Starkey and the other a subculture of Andrewes, original BacL alkalescens. The urinary strain (7172) of Snyder and Harmer was also included. The cultural reactions of these strains and Bact. dysenteriae (Plexner) are given in Table II. T A B L E II CULTURAL ]~EACTIONS OF ~TI~AINS OF BfbOt. AlkaIesceus COIIPAt~:D W I T H Bact. Dysenteriae (~LEXNEF~)
Case 1 (7204) Case 2 (7166) Case 3 (7404:) 7172 49226
Bact. a~lca~esce~s Bact. dyseq~teriae (Ftexner)
3
~
A
A
~
22A A A A
a
A A A
I
o
Ao A A A
AIPA
A A A
0
o?
oA A ~ I
0 0 0 0
A A 0
5~
O
~ 0
A A A A A
2 A O
O O O O O O O
0 0
o
+ + +
o+ O
+ +
Alk. Alk. Alk. Alk. Alk. A]k. Alk.
Table I I shows the uniform fermentation with acid but no gas of glucose, levulose, galactose, maltose, xylose, mannitol, and dulcitol by the
344
THE JOUR.NAL OIO PEDIATRICS
strains Of Bact. alkalescens, whereas B,ct. dysentcriae (Flexner) did not produce acid in xylose or du]citok The reactions with mannitol and dulcitol are in accord with the excellent differential chart given by Andrewes and I n m a n } ~ Milk was also t u r n e d alkaline more r a p i d l y with the Bact. cd/calescens than with the F l e x n e r type. Another characteristic of Bact. aZka~escens as pointed out by Andrewes is its susceptibility to agglutination b y acid after the method of Michaelis. 2~ This feature is in contrast to the inagglutinability of dysentery bacilli by these acid concentrations (Michaelis2~). All the present strains of Baet. alkalescens could be shown to be completely flocculated in buffers of p H 3.2-2.4. The Flexner strain was not agglutinated by respective hydrogen ion concentrations. Serologieally, Andrewes found Bact. alkalescens to stimulate antibody formation with difficulty. ]in his monograph with Inlnan, however, he noted that good agglutination could be obtained by incubation at 55 ~ C. for t w e n t y to twenty-four hours. H e felt that this delayed reaction by Bact. alkalescens and lactose fermenters (B. ambiguous) was so striking as to be regarded of special significance. Also, a common antigen with the Flexner type, especially at 55 ~ C., was present. Well, Smith and F r a s e r , Popoff- and Spanswick, Welch and Miekle, a n d Neter were likewise able to obtain not only specific antibodies but also showed a common antigen with Flexner which could be absorbed. P r e l i m i n a r y tests with the strains at time of isolation showed 7204 and 7404 to agglutinate with a known Flexner antiserum but not with Shiga, Park, Hiss Y, and Sonne antisera. Strain 7166 was not agglutinated b y the Flexner antiserum. The reactions of ttnese strains with an antiserum p r e p a r e d against Strain 7404 are listed in Table I I I . TAtTlE IIi[ CZOSS-AGGLUTJ[NATION TESTS OI,' STRAINS OF Bac t . At]~cdesce'Y~s ISOLATED 1~I%0.~ IN?~ANTS WITII AN 2AkNTISEP~USI .D]%EPARED AGAINST ONE OF TIIE FECAL STI~AINS, 7404 SEI~U EI D I L U T I O N S
Cont. 7204 7166 7404
. ++§247247247
++++
q-+++
+4-++
(horn.) Flexner
/++++1++++1++++1++++1++++/++++/
-t-+l
-/
-
H e r e again the relation of Strain 7404 to the F l e x n e r type is shown, but there was no macroscopic agglutination with the other two strains. A f t e r these tests the cultures were placed on agar slants and sealed for more t h a n a year, when they were opened for additional study. Routine transfers were made f r o m one- to three-month intervals. All strains w h e n first plated on nutrient agar showed the colonies to be mostly smooth with some colonies showing slightly wrinkled edges i n forty-eight
SNYDEI%:
BACT.
ALKALESCENS
IN
STOOLS
OF
NOI'~i\{AL I N F A N T S
345
to seventy-two hours. Recently the Andrewes and 7166, 7404, 49226 strains were found to be completely dissociated into the R colony form. P r e p a r a t i o n of antisera against the Flexner type and Strains 7172, 7204, 7166, and 7404 was done with heat-killed saline suspensions as antigens which were given subcutaneously in rabbits in 2.0 c.e. doses ten times at four-day intervals, and the rabbits were bled for serum a few days a f t e r the last injection. Macroscopic tests showed no agglutination of the strains of B a c t . a l k a l e s c e n s by the Flexner antiserum. Strain 7172 was not flocculated by its own or the other antiscra, but the Andrewes, 7166, 7204, and 7404 strains and the Flexner type agglutinated up to a ] :40 serum dilution, and occasionally partial clumping as high as 1:320 by antisera 7204, 7166, and 7404 was observed. Antisera p r e p a r e d against the Andrewes and 49226 strains showed a similar lack of agglut, inins. The tests on the whole were v e r y inconclusive. Since plain broth cultures flocculated somewhat better than saline suspensions, new rabbits were inoculated with forty-eight-hour broth cultures of the Andrewes and 7204, 7172, 7166, 7404 strains, which were given either subcutaneously (2 e.c.) at four-day intervals or three weekly series of daily intravenous doses (0.5 to 2 e.c.) with a week's interval between the series. The agglutination reactions with these new antisera were not significantly different f r o m the preceding ones. Failing satisfactory agglutination tests, recourse was made to the complement fixation test as recommended by Starkey. Portions of 7204, 7172, Andrewes and Plexner antisera were heated to 56 ~ C. for t h i r t y minutes and mixed with saline suspensions diluted to a point determined not to be anticomplementary. I n the presence of complement and a hemolytic system the results are recorded in Table I V for a 1:20 sermn dilution. TABLE COMP],E~ENT BACTEI~IAL S Y S T E S I :
IV
FIXA!PlON T E S T S W I T I I
~O~(Y~6T$'g, Tg& fl,~/~Cd~68(J6'[b3
A N T I G E N 0.2 C,C. SALINE S U S P E N S I O N
SEr~U~t 0.2 c.c. (]-20) CO~PnE~ENT 0.2 C.C. (1-20) ItEI~,IOLYTIC SYSTE.~{: INCUBATION:
AIV[BOCEPTOR 0.2 C.C. ( 1 - 2 0 0 ) F~.B.C. ( S I I E E P ) 0.5 C.C. 2 PEN CENT S U S P E N S I O N BACTEI{IAL SYSTENI INCUBATED I N A WATEI% BATH 30 M I N U T E S AT 37~ ~IES~OLYTIC SYSTE~,I ADDED AND INCUBATED E01~ AN ADDITIONAL 3 0 1V[INUTES
9
7172 7204
o
o
o
o
Baet. alkalcscens Bact. dysenter4ae
o
o
o
o
(Flexner) C : Complete -hemolysis. O ---- N o h e m o l y s i s .
o o~
59
cc ce
c c
cc
346
T t I N , J O U R N A L OF P E D I A T R I C S
Serum dilutions of 1:5 and 1:10 gave the same results; namely, that there were apparently specific antibodies produced by the Bact. alkalescens strains which did not fix complement in the presence of Flexner antigen. DISCUSSION
The interesting feature in this study was tile isolation of Bact. alkalescens, a dysentery bacillus, from the stools of three normal infants in only one of whom occurred a marked gastroenteritis. In this instance it seemed probable that these organisms were the cause of the illness, since there were no other pathogenic forms demonstrated. Their presence was not chance finding since they were repeatedly obtained and in one ease were the only gram-negative aerobic nonsporulating rods in the stools up to the f o u r t h month, when a few coliform bacteria appeared. Except for the possibility in Case 3 in which a similar organism was found in the father's stool, the source of infection could not be determined. These findings support the slight pathogenicity of Bact. alkalescens as reported in the literature. However, the chance of increased ~irulenee through transmission remains, and thus a public health problem is raised. On a cultural basis the fermentation of dulcite seems to be the chief differential criterion and for routine diagnostic purposes should serve to separate Bact. alkalescens from the Flexner, Hiss Y, Park, and Strong varieties. Serologically, however, the problem, is complicated by the inability of some strains either to stimulate the production of antibodies or to agglutinate with known serum. Neter (personal communication) thinks there are three types of agglutination response with this species: first, a high titer agglutination; second, a delayed though high titer agglutination; and third, sera with little or no ability to agglutinate. A hig'h titer serum (2567) prepared by Neter failed to agglutinate any of the present Bact. alkalescens strains, but it did agglutinate the Flexner strain to a serum dilution of 1:1280, and its homologous antigen 1. :5,120. Complement fixation tests with this serum showed Neisser-Weehsbur~ types of antigen-antibody combination with the Andrewes and 7404 strains in sermn dilutions of 1:80 and 1:160 but not at 1:20 and 1:40. Strains' 7166, 7172, 49226, and 7404 did not fix complement, but the Flexner strain absorbed the complement in a sermn dilution of 1:160. Recent observations on these strains showed all but 720r to be completely or nearly completely dissociated into the rough phase on the basis of colony morphology. The Flexner type remained smooth. Undoubtedly this change explains some of the difficulties and divergent results encountered in the serological tests, but considering the inability to produce antibodies even with freshly isolated smooth strains, it would appear that bacterial variation was not the whole story. In an exchange of strains and antisera of Bact. a~l~alescens with Dr. Neter, both of us obtained little or no agglutination with the present strains as antigens and his high titer Bact. alkalescens (2567) antiserum as the antibody. "However,
SNYDEP~:
BACT. ALKALiESCENS IN STOOLS O.P NOIgl~AL INFANTS
347
Neter pointed out, as Andrewes had already indicated, that extensive agglutination took place at 55 ~ C. in forty-eight hours with nil strains except 7172. This delayed reaction could be confirmed. F u r t h e r studies are being made to determine other variables besides temperature acting u p o n the antigen-antibody reactions of Bact. alkalescens. At present it would seem that while strains of Bact. alkalescens exist that are capable of producing high titer antisera, the strains u n d e r study do not possess this power. As a result of low and irregular agglutination response, no absorption tests were made; only the gross relationship of the strains of Bact. alkalescens to each other and the Flexner type was indicated. SUMMARY
Strains of dysentery bacilli were frequently isolated from the stools of three infants under one year of age. 0 n l y one of these babies had any history of gastrointestinal trouble. The identity of these organisms with a subculture of the original strain of Bact. alkalescens was shown by culturaI tests. The serologic picture was complicated by the inability of the strains to stimulate the production of agg]utinins or other antibodies so that adequate titers for absorption tests could be made. Nevertheless, tile strains seemed to have an antigen in common which was also shared by the Flexner type in the agglutination test but not in the complement fixation reaction. REFERENCES J. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.
Andrewes, F. W.: Lancet 1: 560, 1918. Shiga, K.: Centralbl. f. Bakt. 23: 599j ]898. Wollstein, IV[.: J. 1VI. Research 10: 11, 1903. Davison, W. C.: Medicine 1: 389, 1922. Soule, M. H , and Heyman, A.: J. Lab. & Clin. Mud. 36: 281, 1933. Duval~ E.: Studies from Rockefeller Inst. 2: 121, 1904, Collins, /(. R.: J. Infect. Dis. 2: 620, 1905. Veeder, 9. S., Kilduff% R., and Denny, O. T.: Am. J. Dis. Child. 4: 75, 1912. Well, A . J . : Centralbl. f. Bakt. Orig. I, 112: 376~ 1928. Popoff, N. W., and Spanswick, M. P.: ,l. Lab. & Clin. Med. 16: 437~ 1931. Smith and Fraser, A. M.'. J. Path. & Baet. 31: 511, 1928. Weleh~ I-I., and Mickle, :F. L.: Am. J. Pub. ]-Iealth 2~: 219, ]93&. Murray, M. F., and Pike, :R. 3/I.: Clin. Misc. iVlary I. Bassett Hospital 1: 69, 1934. Starkey, D. I-I.: Canad. 1Vf. A. J. 31: 42, 1934. ~ackenzie, D. W., and ]~atner, ~ . : J. Urol. 31: 671, 1934. Snyder, M. L , and l:Ianne% J.: J. Infect. Dis. 60: 51, 1937. Neter, E.: J. Infect. Dis. 61: 338, 1937. Neter~ E., a~d Rappo]e, F.: Arch. Path. 25: 298, 1938. Neter, E.: J. Urol. 39: 727~ 1938. Snyder, 1V[.L.: A S~udy of the Normal Fecal Flora of I n f a n t s Under One Year of Age, :1935~ Univ. of Colorado Library, Boulder, Colo.
2]. ]3ergey, D. H.: ~anual of Determluative ]3aeteriology, ed. ~, Baltimore, 1934, Williams and Wilkins Company. 22. Andrewes, F. W., and Inman, A. C.: Special Rep. Med. Res. Committee, No. 42, 1919. 23. 3/[iehae]is, L.: 2~. Miehaelis, L.:
Deutsche meal. Wehnsehr. 37: 969, 1911. Deutsche reed. Wehnsehr. 43: lgO6, 19;I.7.
HYGIENE LABORAT01~Y~ "[J'NIVEr~SITY OF MICHIGAN"