ノカルジア属細菌の分子生物学的同定法と抗菌薬感受性に関する検討

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Nocardia AB'+CDEFG0HIJ? K12AELELM3
HIN? *2OPQRFB4STFR+5U6V7WUXYZT[ \]
'+8(A? 9^? V>_]`FabSTcdZ? K
=:AG0HI[ :;HA? *2; Nocardia 26 ef8gh<
i
=jkf>lU? m?B]fE][
*2;ST] 26 eAh<
i <
i3? @n? AB=Cop@
qN N. asteroides 12 e? N. nova 8 e? N. farcinica 6 eST][ =
EFA? r.DsSTFR 16S rDNA fER] restriction fragment length polymorphism (RFLP) fERFE]ct? iH N. asteroides ST] 12 e
uv 4 eJ N. asteroides, 4 eJ N. farcinica, 4 eJ N. transvalensis Rujko`][
=? h<
iqN N. nova ST] 8 euv 2 eJ N. nova, 1 eJ N.
farcinica, wN 5 eAFGo`][ iH N. farcinica ST] 6 eA
=QRFUxF N. farcinica HST][ cTZ Nocardia fE]ct? mqNIyz{JBZTU? |}~
-? ~|? |-? -? ST ? HQQRJ
ST][ :;dZ? Nocardia 16S rDNA fER] RFLP JEJ
ST][
Key words: Nocardia, RFLP, conventional identiﬁcation, antimicrobial susceptibility testing
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Nocardia AKLM
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TEL: 03῍3762῍4151
FAX: 03῍5493῍5415
E-mail: kazu@med.toho-u.ac.jp
YZT[ \]? Nocardia J12STF©FU
Y3ªHmAM3HIN? cTJ
'+88«3ESo`FR[ ¬
ro`F? 16S rDNA fER] restriction fragment length polymorphism (RFLP) opZ®
H Nocardia JRdDsST
FR3)¯7)[ Conville ZA? °±²[ HinP1I, DpnII,
*2&
»¼
Vol. 16
No. 2
2006. 21
¼NL
82
SphI, BstEII Nocardia RFLP mox-14 Solution (TaKaRa) 100 ml !_ `
N. asteroides, N. nova, N. farci-
10 .ab 12,000 rpm 5 .`!
nica, N. transvalensis c
V OPlm 12,000 rpm 2 .
8)
cde
! Nocardia N. farcinica "
de fg!hijk 300 ml U
fg!hk 10 ml n
#$%&'
o rgTTdp DNA q<
(!

%"
PCR r
) * Nocardia !!" "+
2)
#$%&')
, '-(
p DNA q< 0.5 ml PCR r< 50 ml AdNTP
!./0()+#*+,1&'
Mixture
23-4 56"./
Bu#er 2I primer setsB m Primer "
2.5 U 10
Conville ! 1 primer Upstream
Nocardia 26 7!0!123894
+.:35946+
12.5 nmol Taq porymerase
7;%,
[5ῌ-CGA-ACG-CTG-GCG-GCG-TGC-TTA-AC-3ῌ] 50
! 89<=:++#;
pmol
<
CCA-CAA-GGG-GG-3ῌ] 50 pmol Downstream 2
* >?@='-
./ Nocardia A3894
+
Downstream 1 [5ῌ-CCT-GTA-CAC-CGA-
[5ῌ-ACC-TGT-ACA-CCA-ACC-ACA-AGG-GGG-3ῌ]
" N. asteroidesB .:35946+!2 N.
50 pmol 8)
farcinica >
94 1 . 65 45 s 72 1 . 40 ~uZQ
?CD ?
E@CFGH%
94 5 .rc
72 10 .t PCR ,1 1e
tPr Q 1 kb DNA marker (BioLabs) 1.
u_¡v` wx sOdM\un
Nocardia K! PCR ]5Sy(P
5 6 " Nocardia A B C D E 7 6 7
3)
RFLP
N. nova, IFM 0284 N. farcinica, IFM
¢ PCR ]5" z£¤U (HinP1I, DpnII, SphI, Bst-
0319 N. asteroides, IFM 0239 N. otitidiscaviarum,
EII) (BioLabs) !{8 NuSieve 3 : 1 aga-
(IFM 0290
IFM 0236 N. brasiliensis, IFM 0333 N. transvalen-
rose 2 Q (FMC Cprporation) u_¡v
sis) 2I./7 26 7 AABC92IFGC
`|}]5¥Tn¦[PT7;
9HI7 : 1998J2002 J!./B !0
2.
4.
+#" eQ§{vT (ABK), Wf~uZiT
KLMKNOPQRQSTK!UV WXPQ
(MINO), uWRM (IPM), r¦l~vT (SPFX),
Y2I b-LZ[\PS]3!2L^_M
¨©l~vT (LVFX), ª[ddM (CAZ), eW
N` abcdeWfghiTYj
{vT (AMK), iR«in (LZD), rQ¬hpl~
hklfT.O!2m Nocardia )
«PQ/pihpiM (ST) ® 9 ~
(P
#¯;°D"±@²DE³´µ (NCCLS)
! Q
Y]3R AenopP
Q eLqfPr siripPQ tLZpPr
%DE+!E¶ WPLPN|TpT<
KQXPr ufvpPQ \QpPr \TfPr
[\ (Difco) 89<=:+! MIC <
LMfPr wQqpPQB j Sx5.yR Ae
11)
zoT {SuT |}l~TOT OvT l~
<¨Pp¢·Q! 5 ml 48 ]
TOT TUB 45!V ZsTYWR
2I 72 ]! MIC >
@= !UV uWRM (IPM), pL\
" S. aureus (ATCC 29213), E. faecalis (ATCC
uvT (TOB), {k\uvT (KM), 5-QLv
29212), E. coli (ATCC 25922), P. aeruginosa (ATCC
@ 35 3 [^ 107 cfu/ml ¸DE7
Q (5-FU) !!%;!!1
27853) ¹
.E!)(P
`m9), 10)
3.
PCR 1)
YXY
<Z[\ 48J72 ][^ Catri22
8359º» Vol. 16
No. 2
2006.
#¯!"
z|
1qr*pHI78
1)
83
'()*+ 1 kb ,-*./$%01
23 (Fig. 1). 4* 26 56*
26 Nocardia )789: ";<+ 1 kb ,
12 N. asteroides, 8
-*$%0123 (data not shown) =
N. nova, 6 N. farcinica >?@ @ 26 Nocardia #A
2)
26 16S rDNA Nocardia (N. asteroides, N. brasili-
ensis, N. farcinica, N. nova, N. otitidiscaviarum, N.
transvalensis) !"# 16S rDNA $%&
16S rDNA BCDE DpnII, HinP1I, SphI, BstEII FGHI<* '(*J" RFLP KL9:
3)
16S rDNA RFLP 6 MN
16S rDNA DpnII, HinP1I, Bst-
EII, SphI 4 MNBCDEFG Fig.
2 *O@IPQ RFLP RSTUVW
Table 1 * 16S rDNA $%01BCDEXYYZ
[\]^_#` N. asteroides 12 Qa 4 N. asteroides, 4 N. farcinica, 4 N. transvalensis RFLP RSTU/b /c *
Pd N. nova 8 Qa 2 N. nova,
1 N. farcinica RFLP RSTU/b ? ed 5 *f"#g RFLP RSTUgHh
#/i< jkl!
m/
bHI<gO@? ^ N. farcinica #" 6 g RFLP KL
Fig. 1.
PCR uv=> w1 kb Ladder, 1x|}
~Tyz{
M: 1 kb TT Lane 1: N. asteroides,
Lane 2: N. brasiliensis, Lane 3: N.
farcinica, Lane 4: N. nova, Lane 5: N.
otitidiscaviarum, Lane 6: N. transvalensis
Fig. 2.
*f"#<Hh#
N. farcinica /RSTU
` Table 2 **fnI=
> RFLP KL*PI=>op`
4)
!"#$
Fig. 3 * RFLP KL N. asteroides 4
N. farcinica 11 N. transvalensis 4 qr
BCDEs!tuv=> w100 bp Ladder, 2x Nusieve yz{
1: DpnII, 2: HinP1, 3: BstEII, 4; SphI
1
Vol. 16
No. 2
2006. 23
tuvwxy
84
PQRSTUVWXYYZ RFLP [\<@
Table 1.
Restriction enzymes used
Species
DpnII
N.
N.
N.
N.
N.
N.
N.
N.
asteroides type strain
brasiliensis
farcinica
nova
nova variant
otitidiscaviarum
transvalensis
pseudobrasiliensis
Unclassified
700,
700,
705,
640,
700,
455,
705,
525,
200,
200,
200,
200,
200,
250,
200,
200,
HinP1I
95
95
95
95, 60
95
200, 95
95
175, 95
420,
420,
420,
420,
420,
420,
645,
420,
455, 250, 200, 95
350,
350,
225,
350,
350,
350,
350
350,
225
225
175, 125, 55
225
225
150, 75
BstEII
SphI
Uncut
730, 270
Uncut
835, 165
730, 270
Uncut
Uncut
Uncut
225
420, 225, 175, 125, 55
]^_àbcLde RFLP dcLfghi
Table 2.
Identification from RFLP pattern
Conventional
identification
N. nova (8 strains)
N. asteroides (12 strains)
N. farcinica (6 strains)
N. farcinica
(11 strains)
N. asteroides
(4 strains)
1
4
6ῌ
4ῌ
N. nova
(2 strains)
N. transvalensis
(4 strains)
Unclassified
(5 strains)
2ῌ
5
4
ῌ_àbcLde RFLP d(fgj
Fig. 3.
Nocardia kl (N. farcinica, N. asteroides, N. transvalensis) 5
MINO N. transva-
()*
+, - 3 ./01234
lensis N. asteroides 4
5 CAZ 64 mg/ml 678
3 N. farcinica 11 7 % MIC 9
IPM !"#:;<=>?@4
!"#
5( SPFX A,$B CD
26 $% N. farcinica 1 &'
EFGHIG@JKLM,%N%O N.
24 mCnop_qars Vol. 16
No. 2
2006.
¸¹º=»ZN
!"
Y¼"I
85
farcinica12 8 1 mg/ml MIC (r ()]alB8 J9 CT AMK MIC 2 mg/ml zNi/aaOvfAk
ABK Pk
Nocardia 6F
U,V 37 WZQ?QR+ LS;O
ST $
UT,V UD#
6
% & '()*+,(
UVWV , 6
, 1X/7
- ."/0,1(2345-67
UV YZ 6
89:-;<
UPk~RSV
LZD Nocardia =" MIC 1>4 mg/
Pk~ J9 CT Iva
ml .? @ABCD"
<J[f\w
[89:
"#
Q?U]6"89:^
!"#
5)
E
6F
% _ÌvQ? Nocardia =_`
N. farcinica ῌ῍῎ῒΐ῕ῑ῔
GHIJK
Nocardia = LMN
0Z 6
!" N.
asteroidesO RFLP P#Q? N. farcinica
!
D#
E XQ"
8H
}~ ST $Q IPM 67Y"Q?
a
$RST% (Fig. 4).
῏
U
$V 42 W
UX
'V () *YZ[\D
&
ῐ
G7 b=Iv" Nocardia =
U]+,V 2 ^_Q? 38`-./a0b1
c|6
2 34c56Fa 4 ^ 15 7&d8
e$ ¡wf¢gh£¤
8Z e9 X :fCT ; g<=9>hi
"
j?@7AklB8
ä©ª«=e$
6F
#m nC
!d.? =
!¥i
¦a§."
bIv
DE/a6EF
e9 CT ; gop&F"
Nocardia =¦#
1Gq>hi/aarstuIvwY"
+Jj:." Streptomyces, Actinomyces, Rho-
x yz{a|!8HI6EF
}
dococcus, Gordonia 67
~ JK
"
lm ®)«n¯ b-9°±*²³
*YZ[\D] 6 ^ 4 7LM
Fig. 4.
6" }l
¬kd.
N. farcinica Q"$J9 CT Q[89:´µ
7X@oZN¶·
Vol. 16
No. 2
2006. 25
»q¼r
86
Nocardia _ N. transvalensis 0
H7
II'5J0@70v-39-:;_7 sV
45
!"#
$%
&
J:I'7 N. asteroides 39-:;_m
&'()*+ ,-."#
K)*LME+[9-:hR>)*#7
&/*&012)*,3 IPM,
U*2), 12), 13)+
!4'&)*56
$%
TOB, KM, 5-FU
3)*7%89-:;*9), 10)+
4'56
'(&<)*=>
3: ? 4'56
" )
Nocardia 5J&)*4'N0 IO
I75JP8_&}-*,3
E QR 3\6 3w&0 1 S&@_J:
@
9-*,3EU*+ I'5J&:0 ST =
*+ Nocardia D'0E
7TbUV3:9-*,37O;7 ']
3E34'&)*
"FG7HI B1
'(34'56
; I&/*W
;
'73
B
'AB3C73
7
M
&!
J:;*3;KL&0
;
N%O*34'56
PQ
; /RS
!E -'XI '&
)*4'56
)*,37Yv;+
'(T")*#
efa00 N. asteroides, N. farcinica, N. trans-
V PCR ;: No-
valensis .3: TbUV3: ST =
cardia D' DNA $%& 'WX()$
0 b-y (IPM, CAZ), y
Y'()* RFLP *
7;
(MINO), (AMK, ABK), IZ+[9-:;*3)\7)+ Conville 0 28 ],
¡¡ (LVFX, SPFX), ¢£
^-].&"#&/*3 16S
4' (LZD) &Z;:4'56
_+
7U*+ ,/K
rDNA ;_ RFLP /à0 25 ]12
efa0_]&h;:0 NCCLS Aq¤ ST
PQ7b3 3 ]c
= IPM, AMK, MINO &h;:
]0}-
*PQ3
J_,3+
[:;*8)+ , 3 ]0."# N.
J_7 CAZ MIC &h;:0)¥:]7 64
asteroides, N. otitidiscaviarum, Nocardia D'3
mg/ml S3;K;Z~_+ b2 ¡
9-:;_EU*7 RFLP 0-d-
¡&h;:0 LVFX &:[\S]7
N. farcinica, N. asteroides, 43;KPQ&
J:;_+ ef5ga00 "#
&/*3 RFLP PQ7b3_]0 N.
~_& ¦ciprofloxacin q¤
MIC 4 mg/ml S§ SPFX &h;:0 19 ]
9]
7 MIC 1 mg/ml 56
~:;_+ ,F
asteroides 12 ]
4 ] N. nova 8 ]
2 ] N.
G3/Ib3)*/K& Nocardia 5J&:
farcinica 0 6 ]
6 ]3;KPQUJ_+ 6&
SPFX 7*ÜJ_]75g+[©;I
"# N. nova 39-_]&hi*b
Zª}9-*14), 15)+ ef 4 MRSA 4«
3778I j3k!7 RFLP &h;:
¬
^' (VRE) 3:-d-_m9
4&99-_+ ,&l:0 ,-4
-:;* ABK, LZD &Z;:E56

]7 N. vova variant U*m
E%O-*,
_7 ABK 0 1 mg/ml, LZD 0 4 mg/ml )¥
3 Bn 16S rRNA m:o (146\156) :]7`®9-:hR ,-,^
pq;<rst
*
7~¯9-_PQUJ_+ LZD Nocardia
'(uJ:;*+
efa00 "# N. asteroides
D' ¦140 ]§ h/x N. brasiliensis ¦25 ]§ &
39-_ 12 ]K 4 ]7 N. asteroides, 4 ]
)*4'°
a0_FG&h;:E MIC90 7
7 N. farcinica, 4 ]7 N. transvalensis RFLP Y
-d- 1\4 mg/ml, 2 mg/ml 3+[9-:hR 5
3b3_+ ,-v&+[:w_/K&
g56
PQ0,-&V;FGUJ_16), 17)+
N. asteroides 3 N. farcinica, N. transvalensis .
"#&0 =g0 TOB 56
h/xy
Nocardia D'&/*5J3:0±²³ a
z{> |g0C?h
bcdef
/x@$%&A_9), 10)+ ,-'(&h
-:;*+ 6&3´
;:"#3 RFLP B-7}-_
µ¶3:0 N. asteroides complex (N. asteroides
C04DU*7 '(&4'56
PQ
sensu stricto type VI, N. farcinica, N. nova, N. ab-
;*,3E9~_FG3E%O-*+ v
scessus) · N. farcinica 7¸U*,37+[9-
26 nI,ôp#¹º Vol. 16
No. 2
2006.
gh?5JiH7;,37j
5Jµ¶3
*klm
ZKLCWEG0HIºC»¼Q>?
1), 12), 18)20) Kageyama 19922001 Nocardia 303 2) Nocardia !"#$%& N.
asteroides %'()* 10 7 N. farci-
5)
nica 14 9 N. brasiliensis 4 4 )* +,-* ./
0-$'1)23 N. transvalensis % 45'(67)89
6)
:%;21) <=>?64
5%@
A B'(CDEFG
0HI N. asteroides HI;%
RFLP N. farcinica )%
J-*
Nocardia KLC
N. asteroides M N.
farcinica 8
!"#NOPQC
7)
R6SJ6 RFLP -T
UVWXYZ[\]^-HI%QQ
7-*_6à
bc>?J Nocardia KLCHI
d
8)
16S rDNA RFLP ef%P;
gh Nocardia KLC N. asteroides, N. farcinica, N. nova, N. brasiliensis, N. otitidiscaviarum,
N. transvalensis -T 10 CRij%klm$
'PQ%S
A nopqrs
t*CQQ7-*_6
à1), 2), 12) bu; Nocar-
9)
10)
11)
dia KLCHI[0 vBwx[
>yz{|}-T~L>?
_%)23
῍
1)
12)
ῌ
Beaman, B.L., L. Beaman, 1994. Nocardia species: host-parasite relationships. Clin. Microbiol. Rev. 7: 213ῌ264.
2) Kageyama, A., K. Yazawa, J. Ishikawa, et al.
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y 23: 877ῌ884.
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>y 29: 111ῌ119.
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% Z
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Usefulness of RFLP Method for Identification of Recent Isolates of
Nocardia spp. and Antimicrobial Susceptibility Testing
Rie Shibuya,1) Kazuhiro Tateda,1), 2), Soichiro Kimura,1) Yoshikazu Ishii,1) Hinako Murakami,2)
Reiko Shimatsu,1) Fusako Kashitani,1) Morihiro Iwata,2) Tetsuya Matsumoto,3)
Kazuhiro Kimura,4) Koh Uchida,4) Koichiro Nakata,4) Setsuko Kubo,5)
Yuzuru Mikami,5) Keizo Yamaguchi1), 2)
1)
2)
3)
4)
5)
Department of Microbiology and Infectious Diseases, Toho University School of
Medicine; Corresponding author
Clinical Laboratory, Toho University Omori Medical Center
Department of Microbiology, Tokyo Medical University
Respiratory Medicine, Internal Medicine, Toho Unviversity School of Medicine
Research Center for Pathogenic Fungi and Microbial Toxicoses, Chiba University, Chuo-ku, Chiba 260ῌ8673, Japan.
In this study, we examined usefulness of RFLP method for identification of clinical isolates of Nocardia
spp. (standard strains and clinical isolates), comparing to conventional biochemical assays. Twenty-six
strains of clinical isolates were conventionally identified as N. asteroides (n12), N. nova (n8), N. farcinica
(n6). In contrast, RFLP method using bacterial 16S rDNA demonstrated N. asteroides (n4), N. farcinica
(n4), N. transvalensis (n4) in 12 strains of N. asteroides conventionally identified. Also RFLP results
showed N. nova (n2), N. farcinica (n1), unclassified (n5) in 8 strains of N. nova conventionally
identified, whereas all 6 strains N. farcinica indicated identical results. Antimicrobial susceptibility testing
showed that the first-line antibiotics, such as minocycline, amikacin, imipenem and ST, in addition to newer
antimicrobials (linezolid, arbekacin), were generally active against clinical isolates of Nocardia spp.,
although a small variation was observed among strains and species. These data suggest that RFLP method
for identification of Nocardia organisms may be a useful technique in the clinical microbiological laboratory setting.
28 Vol. 16
No. 2
2006.

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