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CLASSIFICATION AND N O M E N C L A T U R E O F
ENTEROBACTERIACEAE
F . Kauffmann a n d P. R. E d w a r d s
S t a t e n s S e r u m i n s t i t u t , C o p e n h a g e n , D e n m a r k (Chief: J . O r s k o v , M . D . )
and Communicable Disease C e n t e r , Public Health S e r v i c e , F e d e r a l
Security Agency, Atlanta, Georgia, U.S.A.
In the c l a s s i f i c a t i o n a n d n o m e n c l a t u r e of the E n t e r o b a c t e r i a c e a e
two d i f f e r e n t c o n c e p t s e x i s t , t o which we will r e f e r a r b i t r a r i l y a s the
b i o c h e m i c a l c l a s s i f i c a t i o n a n d a s the c l a s s i f i c a t i o n by a n t i g e n i c a n a l y s i s , a l t h o u g h s e r o l o g i c m e t h o d s e n t e r into the f o r m e r a n d b i o c h e m i c a l
methods into the l a t t e r .
1.
Biochemical Classification
In the b i o c h e m i c a l c l a s s i f i c a t i o n , a s typified i n the 4th e d i t i o n of B e r g e y ' s M a n u a l , only b i o c h e m i c a l m e t h o d s w e r e u s e d to d i s t i n g u i s h s p e c i e s . It s h o u l d be s a i d t h a t a g r a d u a l d e v e l o p m e n t of s e r o l o g i c m e t h o d s
h a d o c c u r r e d t h r o u g h the y e a r s a n d t h a t w o r k e r s r e l i e d o n t h e s e m e t h o d s to d i s t i n g u i s h t y p e s within c e r t a i n g e n e r a . F o r e x a m p l e , v a r i o u s
s e r o l o g i c a l l y d i s t i n c t t y p e s w e r e known in the g e n u s S a 1 m o n e 11 a ,
a n d t h e s e w e r e r e c o g n i z e d by a g g l u t i n a t i o n a n d a g g l u t i n i n a b s o r p t i o n
t e s t s . Yet in f o r m a l c l a s s i f i c a t i o n s only a b i o c h e m i c a l k e y w a s u s e d
to d i s t i n g u i s h s p e c i e s .
T h a t t h i s p r a c t i c e h a s p e r s i s t e d to the p r e s e n t d a y is shown by the
t r e a t m e n t of the g e n u s E s c h e r i c h i a i n the 6th e d i t i o n of B e r g e y ' s
Manual. T h r e e s p e c i e s a r e recognized, a s follows:
Species
Escherichia coli
Esche richia freundii
Escherichia intermedia
Citrate
Utilization
HZS
Production
t
t
t
T h e above i l l u s t r a t e d t r e a t m e n t of the g e n u s E s c h e r i c h i a r e p r e s e n t s a r e t r e a t f r o m f o r m e r e d i t i o n s of the m a n u a l in which e l a b o r a t e
b i o c h e m i c a l k e y s w e r e e m p l o y e d a n d a m u c h l a r g e r n u m b e r of s p e c i e s
w a s r e c o g n i z e d . F r o m t h i s i t i s obvious t h a t the e d i t o r s f e l t t h a t it
w a s not p r o f i t a b l e to d e l i n e a t e a l a r g e n u m b e r of i l l - d e f i n e d t y p e s
b a s e d upon b i o c h e m i c a l c h a r a c t e r i s t i c s which h a d no r e a l s i g n i f i c a n c e .
It is o b v i o u s also t h a t the t h r e e a b o v e - m e n t i o n e d s p e c i e s a r e , i n f a c t ,
l a r g e g r o u p s o r s u b g e n e r a s i n c e they a r e c o m p o s e d of m a n y b i o c h e m i c a l a n d s e r o l o g i c t y p e s . In t h e c l a s s i f i c a t i o n of the b a c t e r i a by m e t h o d s s u c h a s t h o s e o u t l i n e d , the e l e m e n t a r y u n i t s of which t h e s p e c i e s
a r e c o n s t i t u t e d c a n n o t be d i s t i n g u i s h e d a s t h e y a r e by the p r a c t i c a l u s e
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of antigenic a n a l y s i s , If, f o r e x a m p l e , one w i s h e s to d e t e r m i n e the
occurrenceof a p a r t i c u l a r c u l t u r e of E . c o l i i n infantile e n t e r i t i s , it
is i m p o s s i b l e to do s o by b i o c h e m i c a l m e t h o d s s i n c e s u c h m e t h o d s
s e r v e to distinguish only the s p e c i e s of E s c h e r i c h i a , e a c h of which
m a k e s up a l a r g e group of d i s t i n c t antigenic t y p e s . E v e n though a l l
biochemical methods which a r e p r e s e n t l y employed a r e applied to the
study of s u c h c u l t u r e s , only b i o c h e m i c a l g r o u p s , e a c h c o m p o s e d of
n u m e r o u s antigenic t y p e s , c a n be delineated. Such a s y s t e m will not
a n s w e r the p r e s e n t n e e d s of the scientific w o r k e r , of the c l i n i c a l b a c t e r i o l o g i s t , n o r of the epidemiologist.
2.
C l a s s i f i c a t i o n by Antigenic A n a l v s i s
While g r e a t r e l i a n c e had been placed in s e r o l o g i c t e s t s f o r m a n y y e a r s ,
the e r a of antigenic a n a l y s i s i n the E n t e r o b a c t e r i a c e a e m a y b e
s a i d to have begun with the pioneer work of B r u c e White which h a s been
c o n f i r m e d , r e f i n e d , and extended by m a n y w o r k e r s . A s in the o l d e r
method of c l a s s i f i c a t i o n , the b a c t e r i a a r e divided by b i o c h e m i c a l m e t h ods into g r o u p s c o m p a r a b l e to the recognized g e n e r a . T h e s e g r o u p s
a r e f u r t h e r divided into O - g r o u p s and s e r o t y p e s through the r e c o g n i tion of their constituent antigens by s e r o l o g i c m e t h o d s which have been
proven to be r e l i a b l e and to yield c o m p a r a b l e r e s u l t s i n the hands of
m a n y p e r s o n s . The c r i t i c i s m often h a s b e e n m a d e that p e r s o n s i n t e r e s t e d in antigenic a n a l y s i s have failed to employ b i o c h e m i c a l m e t h o d s
which would differentiate types o r s p e c i e s . Such c r i t i c i s m s a r e not
justified s i n c e a l l b i o c h e m i c a l methods which have b e e n found to yield
valuable information r e g u l a r l y a r e employed i n the w o r k . A f t e r the
division of the g r o u p s into O - g r o u p s on the b a s i s of t h e i r h e a t s t a b l e
s o m a t i c a n t i g e n s , the o r g a n i s m s a r e divided into s e r o t y p e s through
recognition of t h e i r K ( c a p s u l e o r envelope) a n d H ( f l a g e l l a r ) a n t i g e n s .
In s o m e i n s t a n c e s these s e r o t y p e s a r e divided f u r t h e r on the b a s i s of
t h e i r b i o c h e m i c a l p r o p e r t i e s o r by their r e a c t i o n s to s t a n d a r d b a c t e r i ophage s .
T h i s division into types and subtypes i s n e c e s s a r y to d e t e r m i n e the
epidemiological r e l a t i o n s h i p s of biochemically s i m i l a r c u l t u r e s , to investigate the pathogenic significance of individual t y p e s , a n d to obtain
a c l e a r p i c t u r e of the composition of the g e n e r a and of the f a m i l y .
A s r e g a r d s the s e p a r a t i o n of the f a m i l y into g r o u p s o r g e n e r a , it m u s t
be e m p h a s i z e d that no s h a r p l y defined g r o u p s e x i s t in n a t u r e . S t i l l ,
for p r a c t i c a l p u r p o s e s , i t i s d e s i r a b l e to divide the f a m i l y into s e v e r a l
g r o u p s . T h i s c a n be done by biochemical m e t h o d s , although s u c h
g r o u p s m u s t be e s t a b l i s h e d a r b i t r a r i l y and i t m u s t be kept i n m i n d that
m a n y c u l t u r e s which a r e studied will not c o n f o r m to any of t h e s e a r b i t r a r i l y defined g r o u p s . F u r t h e r division of the g r o u p s should be done
by s e r o l o g i c study of the b a c t e r i a .
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E a c h of the g r o u p s o r g e n e r a is c o m p o s e d of h u n d r e d s of s e r o l o g i c
t y p e s , which f r o m a p r a c t i c a l s t a n d p o i n t a r e c o n s t a n t a n d which c a n be
quickly a n d a c c u r a t e l y r e c o g n i z e d b y s e r o l o g i c m e t h o d s . It i s not n e c e s s a r y to d e s i g n a t e a l l t h e s e s e r o t y p e s by s p e c i a l n a m e s n o r to r e g a r d
t h e m a s s p e c i e s . It i s sufficient to n u m b e r t h e m o r , b e t t e r s t i l l , to
give t h e i r a n t i g e n i c f o r m u l a s . T h e s e t y p e s f o r m the e l e m e n t a r y u n i t s
e s t a b l i s h e d by the m e t h o d s e m p l o y e d a n d a r e the b a s i s of a n t i g e n i c
c l a s s i f i c a t i o n . It i s s u f f i c i e n t to d e s i g n a t e the O - g r o u p s into which the
t y p e s f a l l with c a p i t a l l e t t e r s (as in the c a s e of the g e n u s S a l m o n e l l a )
o r by a r a b i c n u m e r a l s . T h e a l p h a b e t d o e s n o t c o n t a i n s u f f i c i e n t l e t t e r s
to d e s i g n a t e a l l the O - g r o u p s of s o m e of the g r o u p s . F o r e x a m p l e , i n
the E s c h e r i c h i a g r o u p m o r e than 120 O - g r o u p s now a r e r e c o g n i z e d .
F r o m the f o r e g o i n g i t is o b v i o u s t h a t we h a v e two e n t i r e l y d i f f e r e n t s y s t e m s with which we m u s t d e a l . The f i r s t of t h e s e , t h e b i o c h e m i c a l
c l a s s i f i c a t i o n , is a p u r e l y taxonomic c o n c e p t a n d c a n be c o n s i d e r e d
only a s a c o n v e n i e n c e f o r the p u r p o s e s of the o r d e r l y c l a s s i f i c a t i o n of
b a c t e r i a i n which e a c h f a m i l y m u s t be divided into t r i b e s a n d e a c h
t r i b e into g e n e r a a n d in which e a c h genus m u s t h a v e a type s p e c i e s .
On the c o n t r a r y , the a n t i g e n i c c l a s s i f i c a t i o n f u l f i l l s t h e n e e d s of the
s c i e n t i s t who is studying the phylogeny a n d e v o l u t i o n of b a c t e r i a a s well
a s the n e e d s of the c l i n i c a l l a b o r a t o r y a n d of the e p i d e m i o l o g i s t . T h e
two s y s t e m s a r e i n no way c o m p a r a b l e a n d s h o u l d not be u s e d f o r the
same purposes.
The q u e s t i o n n a t u r a l l y a r i s e s a s to w h e t h e r t h e s e two s y s t e m s m a y be
c o r r e l a t e d and w h e t h e r i t i s p o s s i b l e to e r e c t a n a n t i g e n i c c l a s s i f i c a tion. To a c e r t a i n e x t e n t i t i s p o s s i b l e to do t h i s , although i t i s n e c e s s a r y to include s o m e g r o u p s not h i t h e r t o u s e d in conventional c l a s s i f i c a t i o n s . It is obvious a l s o t h a t the g r o u p s a t p r e s e n t r e c o g n i z e d do not
include a l l of t h e d i v e r s e s t r a i n s of the e n t e r i c b a c t e r i a . Any p r o p o s e d
grouping m u s t be b a s e d only o n e x i s t i n g knowledge with the r e a l i z a t i o n
that f u r t h e r g r o u p s p r o b a b l y will be a d d e d . F u r t h e r , it i s quite p o s s i ble that s o m e g r o u p s d e l i n e a t e d a t p r e s e n t will be m e r g e d . With the
p r e s e n t s t a t e of knowledge, the c h a r a c t e r i z a t i o n of e n t e r i c b a c t e r i a
m u s t of n e c e s s i t y r e m a i n i n a s o m e w h a t fluid s t a t e .
It is not b e l i e v e d that O - g r o u p s o r s e r o t y p e s c o n s t i t u t e s p e c i e s . P r o b a b l y s p e c i e s should be e s t a b l i s h e d a t the l e v e l of the b i o c h e m i c a l l y d e f i n e d s u b g r o u p s o r s u b g e n e r a . T h u s e a c h s p e c i e s would be m a d e up of
n u m e r o u s s e r o t y p e s defined b y s e r o l o g i c a n a l y s i s . T o d e s i g n a t e s p e c i e s a t t h i s l e v e l should c a u s e no difficulty i n a n y of the g r o u p s o t h e r
than S a l m o n e l l a . F o r t u n a t e l y , s u c h s e r o t y p e s a s have b e e n r e c o g n i z e d i n g r o u p s o t h e r than S a 1 m o n e 1 l a h a v e b e e n d e s i g n a t e d only by
l a b o r a t o r y n u m b e r s o r by a n t i g e n i c f o r m u l a s . E x a m p l e s of s u c h l a b e l ing a r e P r o t e u s X 2 and X 19 a n d C o l i 1:1:7. In s u c h c a s e s the d e s ignation of b i o c h e m i c a l s p e c i e s p r e s e n t s no p r o b l e m .
Unfortunately, i n the c a s e of the S a l m o n e l l a g r o u p , s e r o t y p e s t h a t
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w e r e distinguished many y e a r s ago have been labeled a s s p e c i e s , e . g .
S. b o v i s m o r b i f i c a n s , S. t y p h i m u r i u m , e t c . Here we e n counter the conflict between traditional usage a n d developing knowledge.
It is now known that it i s impossible to distinguish the v a r i o u s s e r o t y p e s
by biochemical methods, and such biochemical keys a s have been p r o posed for the genus have no b a s i s in fact. T h e r e f o r e , we a r e left with
two alternatives: (1) To s t a t e that s p e c i e s i n the genus S a l m o n e l l a
cannot be defined, o r (2) to propose a v e r y s i m p l e biochemical key
which will s e r v e to distinguish the accepted type s p e c i e s (S.c h o 1 e r a e s u i s , S. t y p h i , a n d S . k a u f f m a n n i i . Since these t h r e e s p e c i e s w e r e differentiated by serologic methods a n d not by biochemical
methods, it a p p e a r s that the classification i s not valid f r o m a taxonom i c standpoint. T h e r e f o r e , if s p e c i e s a r e to be established in the genUS the following method s e e m s to be the l e a s t illogical:
Salmonella
Species
Arabinose
Sarmonella choleraesuis
Salmonella typhi
Salmonella enterica
-= negative, t = promptly positive,
negative
Trehalose
Gas
Production
-
,*
t
t or
*=
-
t
t**
r a r e l y positive,
t**
** =
rarely
The t e r m S . k a u f f m a n n i i of B o r m a n , S t u a r t , and Wheeler is rejecbed since i t was not biochemically defined. In i t s place is substituted
S. e n t e r i c a which is biochemically defined. The r e s u l t is, f o r a l l
practical p u r p o s e s , the s a m e as that proposed by B o r m a n e t a l . The
genus, then, is divided into two biochemically defined s e r o t y p e s which
respectively r e p r e s e n t the type s p e c i e s and the b e s t known m e m b e r of
the genus, and into a large group of s e r o t y p e s which cannot be biochemically differentiated one f r o m another.
To give a n idea how such a biochemical classification would look, we
p r e s e n t a s h o r t d r a f t in Table 1. Keys f o r the differentiation of groups
and s p e c i e s a r e given in Tables 2 and 3. We do not r e c o m m e n d s u c h a
classification, but we would a g r e e that a classification s i m i l a r to that
given below could be used side by side with a n antigenic classification,
but only with the realization that the biochemical classification was to
be used f o r purely taxonomic purposes.
It should be emphasized that the opinions e x p r e s s e d h e r e i n a r e only the
views of the w r i t e r s and should not be taken a s e x p r e s s i n g the opinions
of o t h e r s . The t e r m s used in the biochemical classification m a y o r m a y
not be valid ones and should be subjected to review by p e r s o n s f a m i l i a r
with b a c t e r i a l taxonomy.
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T a b l e 1.
Bioc he m i c a 1 C l a s s if i c a t i o n
(For t a x o n o m i c p u r p o s e s )
Genus o r G r o u p
Species
1.Salmonella
1. S a l m o n e l l a c h o l e r a e s u i s
2. S a l m o n e l l a t y p h i
3. S a l m o n e l l a e n t e r i c a
2.Arizona
1.Arizona arizonae
3. Be t h e s d a
1. B e t h e s d a b a l l e r u p e n s i s
~
4. E s c h e r i c h i a
5. K l e b s i e l l a
1.Escherichia coli
1. K l e b s i e l l a p n e u m o n i a e
(or aerogenes)
2 , K 1 e b s i e 1l a r h i n o s c l e r o m a
tis
6. S h i g e l l a
1. S h i g e l l a d y s e n t e r i a e
2. S h i g e l l a f l e x n e r i - b o y d i i
3. S h i g e l l a s o n n e i
7. P r o v i d e n c i a
1. P r o v i d e n c i a p r o v i d e n c i a e
8. P r o t e u s
(1)
e ventua lly
combined
i n t o one
genus ?
-
eventually
combined
into o n e
- genus
1. P r o t e u s h a u s e r i
a. v a r . v u l g a r i s
b. v a r . m i r a b i 1 i . s
2. P r o t e u s m o r g a n i i
3. P r o t e u s r e t t g e r i
If i t i s d e c i d e d by the N o m e n c l a t u r e C o m m i t t e e t h a t the g e n e r i c
n a m e B a c t e r i u m i s to b e u s e d , then t h e E s c h e r i c h i a a n d
K l e b s i e 1 l a g r o u p s s h o u l d be c o m b i n e d i n o n e g e n u s a n d c a l l e d
respectively B a c t e r i u m c o l i and B a c t e r i u m a e r o g e n e s .
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<(
*,*
***** *****
s
96
Table 2 .
B i o c h e m i c a l D i f f e r e n t i a t i o n of G r o u p s
(Typical behaviour of m o s t i m p o r t a n t t y p e s )
-Salmonella
Arizona
Be the s d a
E s c h e r ic hia
Kle b s iella
Shigella
Providencia
Proteus
Mannitol tose Indole
t
t
t o r x
t
t o r x
t
t
t
t
v
-orx
v
-orx
t
V
v
atin
t
-
H7S
v
t
-
v
Gas
- o r x
-
x
v
t
Motility
V
V
+
t
+
-
t
-
+
Urea*
-
t
t
V
t
t
t
V
V
*
= U r e a r e a d a f t e r 6 h r s . incubation
t = P r o m p t l y positive
x = L a t e and i r r e g u l a r l y positive o r negative
- - Negative
v = Variable
Table 3.
B i o c h e m i c a l D i f f e r e n t i a t i o n of S p e c i e s
Klebsiella pneumoniae
Klebsiella rhinoscle romatis
t**
$higella dysenteriae
Shigella flexneriboydii
Shigella sonnei
Prot
var.
Prot
var.
Prot
Prot
eus hauseri
vulgaris
eus hauseri
mirabilis
eus morganii
eus rettgeri
t
X
**
t**
-t
t
t
t
+
t
t
t
t
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INTERNATIONAL BULLETIN
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Addendum - It i s realized that no provision i s made in the above c l a s sification for the motile f o r m s a t p r e s e n t classified a s A e r o b a c t e r ,
n o r of the f o r m s classified by B o r m a n , S t u a r t and Wheeler a s P a r a c o l o b a c t r u m a e r o g e n o i d e s . It is felt that p r e s e n t knowledge
of these f o r m s is insufficient to justify their placement in a definite
group. It i s not c l e a r whether the genus K l e b s i e 1 l a should be expanded to e m b r a c e these f o r m s o r whether the definition of the genus
E s c h e r i c h i a should be modified to allow t h e i r inclusion.
A c k n o w l e d g e m e n t - The w r i t e r s a r e indebted to D r . S. T. Cowan
f o r reviewing and criticizing the m a n u s c r i p t .
References
B e r g e y ' s Manual of Determinative Bacteriology, F o u r t h Edition, 1934,
B a l l i e r e , Tindall & Cox, London.
B e r g e y ' s Manual of Determinative Bacteriology, Sixth Edition, 1948,
The Williams & Wilkins Company, B a l t i m o r e .
B o r m a n , E . K . , C.A. S t u a r t , and K. M. Wheeler: J . of B a c t e r . 48,
351-367, 1944.
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