ῌE*Y/+.½ 2010
9
ῌ῎
ῐ῍
Clostridium !"1)#$%
2)#2)
1)
&'()*
2)
-./,.01234567#12
+,
89 21 6 : 19 ;< 9 21 11 : 2 ;=
>?
@ABCDEFG
tridium K< L/M.NK
H!IJK Clos-
IOPQ"#BR$NI%S
TU H!&'(< V)*'W+X%YZ 16S ribosomal RNA [,\-.]^
T Clostridium hathewayi, Clostridium clostridioforme, Clostridium bolteae, Clostridium citroniae, Clostridium aldenense, Clostridium symbiosum 6 _`< a/b (API 20
A, Rapid ID32A) I0#T/M.N%YZcd1eYfgYCR$TU Indole
W/_` C. citroniae C. aldenense hif< j 2 _R$N
`< rhamnose,
b-glucuronidase, b-galactocidase @2TU Indole 3W/ 4 _Ck C. symbiosum
l@ a-galactocidase himTU C. hathewayi `< cellobiose 4< a-arabinosidase %
YZ b-N-acetylglucosaminidase 4YfR$n5himTU C. clostridioforme C. bolteae R$K`< N@6o%fR$`p7himTU qT< C. symbiosum `
cd1eI#BrJs8H!tAR$n5himTU
Key words: Clostridium clostridioforme, Clostridium bolteae, Clostridium hathewayi, Clostridium citroniae, Clostridium aldenense
῏
ῑ
B Clostridium symbiosum58) @iU
tB@
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rRNA) [,\-.XY\/+.&N
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tridioforme, Clostridium bolteae9), Clostridium hathe-
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§1, 16)U T@m< ¨
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< C. clos-
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forme
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R$@p7
symbiosum I© 6 _@Qªj
BU
FGH (629῍2261) &''IJ
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CzRt
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+µhJn5B_$i`i
WX_¶·qhN@¸h¹º
E*Y/+.»¼ Vol. 20
No. 1
2010.
9
10
10 Vol. 20
No. 1
2010.
Clostridium clostridioforme group »2
11
saminidase (b-NAG) 8 }1W=3>$~
0 m 20A W#'h5/ 72 h5~0
!"#$%%&
m *+,W?@ C. clos-
'()*+, API 20A -
tridioforme 1 ?t
. Rapid ID32A /
01(%20
1 *+, C. clostridioforme ~
xA20
1.
&
2004 11 3% 2006 9 3 23 4356
2789 : ;< =: 1.
'
(Fig. 1)
g#'
!"#-i@W
>?% @ AB
I+,KWB
CDEFGH,
$C@D%!¡W.
IJKLMN
O - . 16 S ribosomal RNA P Q ! (1,486 R
% j(/.UW 26 U (43¢) 1,489 bp) ! $ 2 S Q T C.
EW C. hathewayi (63¢) £ C. citroniae (67¢)
hathewayi 16 U C. clostridioforme 10 U C. bolteae
j(@¤A1¥%1 C. aldenense (20¢) £ C.
11 U C. citroniae 6 U C. aldenense 5 U C. sym-
symbiosum (17¢) WF% (Table 2) 60 U
biosum 12 UV 60 U/
- / AC #$nopqr
2.
24 h5 48 h5 72 h5#'0u,/G¦0
W XYGZG[\X]^_;<`
u,1-w§U~¨U1H©
"#$ aAC #$ %&bF,[cdJ+*[e[f 0 72 h5#'W 48 UWIª 3R5 mm u,
g#'h a3 %5#'f !"#-i"
/ « 12 UW 2 mm rD%¨u,
#$ @-.j()*/kl0 m +2
/@¤0¬E1(~ ¨u,/
U/ AC #$nopqrs#'0 24 h5
@¤0UW C. symbiosum @
48 h5 72 h5#'tu,$v a-w @
W ® 24 h5#'WJ¯°
f /x.0
%±@ K²1££L³0u, -wu
!"#$%
,®W-w ´k~ Bacteroides ?µ0
y/0z/{|}1~0*+, API
1 72 h5¶#'W 0·J¯1M¸N~
3.
20A a20A %&\Kf ~z23/y4
(Fig. 1) #OW 'P#W££¹
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$v/6789
Wu,@!¡/.%
0 :W'1 GIJ
;~~/<0 32A W agalactosidase (a-GAL), b-galactosidase (b-GAL), bgalactosidase-6-phosphate (b-GP), a-glucosidase (a-
2.
()*+,$ Clostridium $
-.$/0(12+$34
20RU*+, 32A -. 20A
65/ Table 1 º0
GLU), b-glucosidase (b-GLU), a-arabinosidase (a-
C. hathewayi 16 U 20 A - i x A W
ARA), b-glucuronidase (b-GUR), b-N-acetylgluco-
36.4R98.5¢ 32A W 99.9¢~¥xA
Fig. 1.
Cellular morphologies and colonies of C. clostridioforme group and C. symbiosum
a) Spore stain of C. hathewayi
b) Gram stain of C. clostridioforme
c) Gram stain of C. bolteae
d) Gram stain of C. citroniae
e) Gram stain of C. aldenense
f) Gram stain of C. symbiosum
g) C. clostridioforme group and C. symbiosum on Anaero-Columbia agar with rabbit blood after 72
h of incubation. Colonies of C. clostridioforme group (right) are circular with slight irregular
edges and attained a diameter of 3ῌ5 mm. In comparison, C. symbiosum (left) forms small colony.
%&>=¼½ Vol. 20
No. 1
2010. 11
46746301
46756330
46756331
46756730
46756731
00100020
00604000
40044231
42144231
42644030
44044230
44704020
46754010
46754031
46754230
00000000
00000001
04514331
20010020
44104331
44754031
10000010
10100031
54104030
54144031
54554031
54556031
10000000
10454200
14510201
50554220
00000000
00100000
40010001
41000000
41010001
41014000
C. clostridioforme (10)
C. bolteae (11)
C. citroniae (6)
C. aldenense (5)
C. symbiosum (12)
Profile No.
C. hathewayi (16)
Species
(No. of isolates)
12 Vol. 20
No. 1
2010.
necrophorum/nucleatum 68.0
stercoris/eggerthii 99.9
clostridioforme
stercoris/eggerthii
ῌ
P. intermedia/disiens
C. cadaveris
E. limosum 97.6
C. cadaveris
P. propionicus/avidum 50.0
F.
B.
C.
B.
F. necrophorum/nucleatum 95.9
C. botulinum/sporogenes
P. intermedia/disiens 97.0
C. clostridioforme
C. clostridioforme
C. clostridioforme
6
1
1
2
1
1
1
1
1
2
1
1
1
1
1
1
4
3
1
1
1
1
1
2
2
4
7
ῌ
C.histolyticum 51.3
C. clostridioforme 99.9
B. ureolyticus
C. clostridioforme
C. clostridioforme 97.5
56.0
49.2
98.5
36.4
62.8
1
1
1
1
1
1
1
1
1
1
clostridioforme
clostridioforme
clostridioforme
clostridioforme
clostridioforme
P. propionicus/avidum 58.0
Fusobacterium mortiferum 86.1
C. clostridioforme
C. clostridioforme, B. distasonis
B.distasonis
Fusobacterium mortiferum 65.6
Fusobacterium mortiferum 98.9
B. distasonis 27.9
C. clostridioforme 89.0
C. clostridioforme 11.7
C.
C.
C.
C.
C.
C.
C.
C.
C.
C.
clostridioforme
clostridioforme
clostridioforme
clostridioforme
clostridioforme
99.9
99.9
99.9
99.9
99.9
Species and ῍ ID obtained by Rapid ID32A
0000000000 ῌ
0100000000 C. septicum 44.3, C. fallax 26.1, C. botulinum 13.9
4100200000 C. clostridioforme 87.7
4120200000 C. clostridioforme 96.3
4120600000 C. clostridioforme
0040200000 F. necrophorum 91.1
0040600000 F. necrophorum 99.8
4040200000 F. necrophorum 84.6
4500000000 C. beijerinckii/butyricum 89.4
4540000000 C. beijerinckii/butyricum 52.0, C. fallax 18.2, C. clostridioforme 16.2
4750000000 C. tertium 36.6, C. beijerinckii/butyricum 35.4, C. baratii 23.2
4150000000 C. baratii 49.8, C. clostridioforme 32.6
4550000000 C. clostridioforme 56.3, C. beijerinckii/butyricum 27.9
4550040000 C. clostridioforme 99.9
4571040000
4575044000
4577040000
4577044000
4577044020
No. of
Profile No.
isolates
Rapid ID32A
Identification by use of API 20A and Rapid ID 32A
Species and ῍ ID obtained
by API 20A
API 20A
Table 1.
1
11
1
2
2
1
4
1
3
7
1
3
5
2
1
1
5
6
3
No. of
isolates
12
C. bolteae (11)
C. clostridioforme (10)
C. hathewayi (16)
Species
(No. of isolates)
46754010
42644030
00604000
42144231
46754031
00100020
44704020
46754230
40044231
44044230
9
14
17
22
31
36
37
39
56
62
00000000
00000000
04514331
44754031
00000000
00000000
00000001
00000001
44104331
20010020
00000001
46756330
46756731
46756730
46756730
46756731
46746301
46756731
46756731
46756731
46756731
46756330
46756730
46756331
46756730
46756331
46756731
7
8
12
18
19
20
21
32
35
43
44
46
47
57
58
61
2
6
16
24
27
29
40
41
49
52
54
API 20A
profile
No.
Strain
No.
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IND
GLU
MAN
L AC
SAC
MAL
SAL
XYL
ARA
ESC
CEL
MNE
ML Z
RAF
SRO
RHA
TRE
Spore
4540000000
4540000000
4500000000
4540000000
4500000000
4540000000
4500000000
4750000000
4540000000
4540000000
4540000000
4550040000
4550040000
4150000000
4150000000
4550000000
4550000000
4550000000
4550000000
4550000000
4150000000
4577044000
4575044000
4577044000
4577044020
4577044020
4577040000
4577040000
4577040000
4577044000
4577044000
4577040000
4577040000
4577044020
4577044000
4571040000
4577044000
Rapid
ID 32A
profile
No.
Results of biochemical reaction in commercial identification kits for strains identified by 16S rRNA sequencing
ῌ
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ῌ
a-GAL
b-GAL
a-GLU
b-GLU
a-ARA
b-GUR
b-NAG
MNE
RAF
IND
PAL
LGA
PyrA
Table 2.
Clostridium clostridioforme group
13
Vol. 20
No. 1
2010. 13
14 54554031
54556031
54144031
10000010
54104030
10100031
10454200
50554220
14510201
10000000
50554220
00000000
41000000
41000000
00000000
00000000
40010001
00000000
00000000
00000000
41010001
00100000
41014000
10
11
34
42
45
51
3
4
15
25
30
1
5
13
23
26
28
33
50
53
55
59
60
C. aldenense (5)
C. symbiosum (12)
Vol. 20
No. 1
2010.
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0100000000
0100000000
0100000000
0100000000
0100000000
0000000000
0100000000
0100000000
0100000000
0100000000
0100000000
0100000000
4100200000
4120200000
4130600000
4120200000
4120600000
0040600000
0040600000
0040200000
0040600000
0040600000
4040200000
Rapid
ID 32A
profile
No.
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a-GAL
b-GAL
a-GLU
b-GLU
a-ARA
b-GUR
b-NAG
MNE
RAF
IND
PAL
LGA
PyrA
Abbreviations: IND, indole production; GLU, glucose; MAN, mannitol; LAC, lactose; SAC, sucrose; MAL, maltose; SAL, salicin; XYL, xylose; ARA,
arabinose; ESC, esculine hydrolysis; CEL, cellobiose; MNE, mannose; MLZ, melezitose; RAF, ra$nose; SOR, sorbitol; RHA, rhamnose; TRE, trehalose;
a-GALῌ a-galactosidase; b-GALῌ b-galactosidase; a-GLUῌ a-glucosidase; b-GLUῌ b-glucosidase; a-ARAῌ a-arabinosidase; b-GURῌ b-glucuronidase;
b-NAGῌ b-N-acetylglucosaminidase; PAL, alkaline phosphatase; LGA, leucyl-glycine arylamidase; PyrA, pyroglutamic acid arylamidase; ῍, positive
reaction to biochemical; ῎ῌ negative reaction.
C. citroniae (6)
API 20A
profile
No.
Strain
No.
Species
(number of isolates)
(Continued)
IND
GLU
MAN
L AC
SAC
MAL
SAL
XYL
ARA
ESC
CEL
MNE
ML Z
RAF
SRO
RHA
TRE
Spore
Table 2.
14
Clostridium clostridioforme group w$m%
15
20A >?@ABC
!Q."g
C. clostridioforme C. clostridio-
forme 10 20A 4 C. clostridioforme #+*DE$Ih,
-
$.D
1 Fu-
iHj#
32A b-GLU
mortiferum C. clostridioforme $%&
C. bolteae 1
sobacterium
!"# 32A $%& C. clostridio-
Dk&#
forme 32.6'99.9(
))# C. bolteae 11 20A 2 ῌ
97.5'99.9(#
*
+,
l R R m %4) C. clostridioforme
-. 32A 7 C.
group C. symbiosum 74no&'
῍
clostridioforme 16.2(
pqr(s)t)
$RSufr()*+v
,
Clostridium species / C.
w,+r(s
-.r(
xC:yU/01
@
citroniae 012 C. aldenense 3&4 20A Cz{|2
}~30
45&'
56&,
.
&'s6 60.4(D\
uf7;r(
!#
32A C. citroniae $%& Fusobacterium
86& - C. hathewayi 9:
!D
necrophorum C.
E+c)
C. clostridiofome ) C. symbiosum aldenense C. clostridioforme ;<&'[.c
c
# C. symbiosum 789:0&
@9:WC Clostridium
=r
4 C. clostridioforme #
(s0&>no
;
<89:=>?@ABCD Table 2
c
R0&
C. clostridioforme
E+ -
Table 3 FG+H
group ?$@A%&56&,
B
I3&JKLMN+ C. symbiosum
CD+&
12 O+&NPQ>?@ABCDE+
89:1 Bacteroides ) Fusobac-
89: 32A b-GAL $%&
terium $c *
$RS profile No. 0100000000
cB Clostridium QEF-
0000000000 C. clostridioforme
+G
JKm%
group HT# -
BUVWC
HnoI;J AC ;J0
X. C. symbiosum a-GAL &
cO 43(?zW(KF
# 20A 4*
+
QY
j
C
C. hathewayi 62(
6 C. bolteae H))Z#
FD@6 +#&
LpM,$c
C. clostridioforme group [S
C. hathewayi, C.
N5DOcj
citroniae, C. aldenense 3 .3&89
-
FP0&4
C
:\]F 5), 9), 10), 15) ^_
WC1FQR ¡18)
Ryu ¡18)
xUU
\] (Table 3) `Q+
¢B£U (VCM) r¤
S¥TU Fusobacte-
$RS
C. hathewayi 20A 0a lactose
rium ) Bacteroides Dk$6VW FM ;J
(LAC), salicin (SAL), melezitose (MLZ), ra$nose
)x¦§?B¨©Xª{©¦yU (BBE) ;JY
(RAF), cellobiose (CEL), 32A b-GAL, a-GAL,
« ¡Di¬$Zj
b-NAG, a-ARA 100(# +
+
.Hb4I
CEL, b-
C. clostridioforme ®1[¯&S
NAG, a-ARA BUVWCX
cO°G.±H89
.
cI$%&# C. citro-
:¨W²³W©´_& -
¨W
niae, C. aldenense $%&BUVWCDX+
²³W©´_& C. clostridioforme 3
.X# c 2 .b4
&4µ[
H I rhamnose (RHA) b-GUR, b-
,
QE]^m¶·Z# ¸c
GAL d<e
fd
JK
&}^]¹W1c
<e
#
<.3&
H89:D_+
Q
C. clostridioforme C. bolteae 3&
!O6\5
\]1ºTW012F^_
`ab»]^]¼½ Vol. 20
No. 1
2010. 15
16 Vol. 20
No. 1
2010.
῎
100
100
100
100
100
100
100
0
100
63
100
100
100
69
100
94
100
0
100
100
100
94
100
100
94
GNR
63
100
0
100
100
100
0
0
70
100
0
20
0
῍
100
100
100
100
῎
0
῍
῍
῍
῍
῍
῍
50
60
0
0
40
70
80
0
60
60
60
30
0
90
80
GNR
30
70
0
Isolates
(n῏10)
100
97
98
῍
96
69
῍
῍
0
GNR
Reference
῎
0
῍
50
50
0
0
100
100
0
῎
100
100
῍
0
GNR
Reference
C. clostridioforme
73
100
100
0
0
100
9
0
0
0
27
9
18
0
18
27
18
0
27
27
18
27
0
27
36
GNR
55
9
0
Isolates
(n῏11)
῎
0
῎
100
100
0
0
17
16
83
῍
70
71
῎
0
GNR
Reference
C. bolteae
100
0
17
0
0
0
0
67
0
0
0
0
0
0
0
100
67
0
67
83
33
33
17
67
83
GNR
67
50
100
Isolates
(n῏6)
῎
100
0
0
0
0
0
100
1
0
0
100
GNR
Reference
C. citroniae
a
b
῎
0
100
100
0
80
0
20
40
0
0
0
0
0
0
80
0
40
0
20
60
80
80
0
60
40
GNR
20
60
100
0
92
0
0
0
0
0
0
0
0
0
῍
0
0
0
0
0
0
42
33
0
8
0
25
0
8
0
GNR
17
0
0
Isolates
(n῏12)
c
Tested by
῍
῎
῎
῎
῎
῎
῎
῎
῎
GNR
Reference
C. symbiosum
Tested by API 20A.
῎
92
100
100
0
92
100
0
77
8
0
100
GNR
Reference
C. aldenense
Isolates
(n῏5)
Numbers represent percentages of positive result. ῍, positive reaction to biochemical; ῎ῌ negative reaction.
Rapid ID 32A.
Gram stain appearance
Spores
Esculine hydrolysisb
Indole productionb
Fermentation ofb:
Lactose
Salicin
Melezitose
Sorbitol
Ra$nose
Rhamnose
Glucose
Mannitol
Sucrose
Maltose
Xylose
Arabinose
Cellobiose
Mannose
Trehalose
Enzyme reactionc
b-Glucuronidase
b-Galactosidase
a-Galactosidase
b-N-Acetylglucosaminidase
a-Arabinosidase
a-Glucosidase
b-Glucosidase
Alkaline phosphatase
Leucyl-glycine arylamidase
Pyroglutamic acid arylamidase
Gas liquid chromatography
Butyric acid
Isolates
(n῏16)
C. hathewayi
Result for species (No. of strains tested)a
Comperison of phenotypic characteristics of five species in the C. clostridioforme group and C. symbiosum between the our isolates and
references5, 9, 10, 15)
Phenotypic
characteristics
Table 3.
16
Clostridium clostridioforme group z7
17
8pt:uvRUkl<S
C. clostridio-
{89G|}:z~?2$ C.
forme group C. hathewayi !"#
$
% C. clostridioforme &'(
(8
z$ C. clostridioforme C. bolteae 2 +
hathewayi
;
}:?<M$
Finegold A C. hathewayi, C. clostridio15)
$($ )*( C. clostridioforme group forme, C. bolteae 3 +7
PCR 0
+ & 32 A C. clostridioforme C.
aldenense
, C. clostridioforme -.- /
( *(
N$
16)
C. clostridioforme, C. bolteae 2 +=%
$&'4) >
U PCR 8 2 +=7
+0
C. clostridioforme group 1
[\A $
2
C. clostridioforme group M
3 C. symbiosum 45 72
RST<U?V
45678 9:;<-=
BB=-@ C.
>?' @$(
hathewayi @ C. clostridioforme/bolteae
A% B?
=O 56[\A
RST<U
C DE
=
FGH
. 2 + Finegold A-
N1)
I
JK
8L C. hathewayi, C. citro-
&A C. clostridioforme group 3 +
niae, C. aldenense M Warren A N$
K
$($ Warren A'RS
FG15) ! LAC, SAL, MLZ, RAF, RHA T<UVfRST<U?V@ 3 +A
O"PQRST<UVW#$ 1
20A Q 32A @$
$
%
8 16S rRNA ]H)*/
>
B 3 + 9698^?8(A C+
?
$($ CX bolteae C. clostridioforme M
$D
!" hh%8A 2 +
FG=3?'/Y
;
Z&G '([\A
Song A
A 2 +5!"E&
C. clostridioforme C. bolteae 16S rRNA ]H)
% Warren
*+ 3^,_?
@OB=- 8A= 16S
2 += FG
RST<U.8
E$
p RAF RHA =>?' -` sorbitol (SOR), MLZ
rRNA ] H ) * P F * / $ a LAC, SAL, esculin (ESC) b-GLU . b`
(Table 2)
SOR, MLZ . LAC, SAL, ESC a?
!"JK
p 8AM;
cd9) $($ !" 8e/$
@$
78m
f
+%>?' $;
g,(
7hi C. clostridio-
forme M hh Z&Gj0
$
f LAC, SAL, ESC
af%g,
(
C. bolteae SOR, MLZ
af
1(
(Table 3) Warren A% C. clostridioforme @kl<S LAC 27@
13
¡BG¢>? 7hi 3
H5£¤ Bacteroides
-QQ
¥u¦I
9§$
¨e ©¥ªJ«Y¬$K
L©¥ªaM?
¨eN>
[\ )$ Ryu JqQ VCM '©¥ª
LAC W#$
C. clostridioforme C. bolteae .?8/O$$
7
=3m
cd
!"%
¡ +M%>? !
13 8A 2 +5=
N®(A;
¨e¯
W#7456[\A
RS
°(? ®±U(A©¥ªJ«²x
LAC Mno
15)
T<Ua 3 +pi C. hathewayi
Bacteroides O£¤©¥ªaMJ«
1,Jqr7.E72$ C. clos-
tridioforme C. bolteae 2 +s%.E
?
1R
7r7
¯°(?' E. coli O0PµS¶
g, C. hathewayi b` 2 +5
³2$ PQ´
$
1J«
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Clostridium clostridioforme group 19
Morphological and Biochemical Identification of Cigar-Shaped Clostridia
Yasushi Daimon,1) Kaori Tanaka,2) Kunitomo Watanabe2)
1)
2)
Clinical Laboratory, Kyoto Prefectural Yosanoumi Hospital
Division of Anaerobe Research, Life Science Research Center, Gifu University
Clostridium clostridioforme has been known to be phenotypically heterogeneous species with cigarshaped morphology. Recent taxonomic studies have shown that C. clostridioforme represents five distinct
species in the C. clostridioforme group: Clostridium hathewayi, C. clostridioforme, Clostridium bolteae,
Clostridium citroniae, and Clostridium aldenense. A total of 60 strains isolated in our laboratory as the
cigar-shaped clostridia were identified through end-product analysis and 16S rRNA sequencing as follows:
C. hathewayi (16 strains), C. clostridioforme (10), C. bolteae (11), C. citroniae (6), C. aldenense (5), and C.
symbiosum (12). Biochemical characteristics for those isolates were investigated using commercially
available identification kits. C. citroniae and C. aldenense were distinguished from each other by the results
for rhamnose, b-glucuronidase, b-galactocidase (C. citroniae : , , ῌ, C. aldenense ῌ, ῌ, , respectively),
although both of them were indole positive. C. hathewayi was di#erentiated from other species in C.
clostridioforme group by the positive reaction of cellobiose, a-arabinosidase, N-acetylglucosaminidase. It
was di$cult to distinguish between C. clostridioforme and C. bolteae by phenotypic characteristics. C.
symbiosum was distinguishable from C. clostridioforme group in colonial size.
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