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Helicobacter�������������������
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��� 20� 4 � 21���� �� 20� 8 � 1��
Helicobacter���!!"#$������%&'()*+ Helicobacter pylori +,-./'01�2� 3�� Helicobacter cinaedi ���"42567'8	:� H. cinaedi !;<#$� Helicobacter���!=>� ��������!.?�@ Helicobacter� 15
��ABC# H. cinaedi%D&'C 14 C!(>0� �)*��+EFG,��� HIJKLMNG,���-.OPQN���-.OPQN EX��� RST�� HP&'��� Vi HIJG,��� U/*��VQIG,��� W,XYLZ[\]^P[_[�JQ[`a HP G,��� 8 �!0b$�@ 8 �����cd� HIJKLMNG,��� Vi HIJG,��� VQIG,��%� E1;2��)e-� 7:+fgh3425i7'0>8���67�j��+k3�JQlN!m�$� EFG,��,-n8o+,-��+��9p2q:)1�@ &� 3
�����;)%HIJKLMNG,��)�9p2�<=�)e?�@ 9p��r�!s88>).?� ATP r�?@)%� +EFG,��W,X-.OPQN��t+9p$��A�r�?@B2�C 5�8+uv+Dws8�+!$� HIJKLMNG,��t��A� ATP r�%xy7'0>8�:9+j?�@ 7:+zE�mF{|!.c#� +EFG,��W,X-.OPQN��t��A�#}2�C 5�8+%~�m+-T$0>8�+!$� HIJKLMNG,��t��A%GHmF2Iy7'0>�@ �t���9:� HIJKLMNG,��%����� 9p�yJ�KL+W>0M'0>8#��:'�@
Key words: Helicobacter cinaedi� NO��� ����
� �Helicobacter �% 2008� 4 ��� 30 ��+&P
7'0W-� �':%�1}Q���� (entero-
hepatic) #���� (gastric) +&�:'8@ �Z9:&'7'8 Helicobacter species #$0%� gastric
Helicobacter species +;('8 Helicobacter pylori
22R)e82� enterohepatic Helicobacter species
+&P7'8 Helicobacter cinaedi, Helicobacter bilis,
Helicobacter canadensis, Helicobacter canis, Helico-
bacter fennelliae, Helicobacter pullorum n�:'0>81)@ j9)n H. cinaedi % 1980�S��� �:9
�AT��+,-U���2Dw$����Q���V� EF9:�&'W2"47'0>82, 3)@ 3�)%� U��X�j>��9:��W"4n6:'84)@(�� Y@LQN[� H. cinaedi 2Z��[�\)]�$� �\�^!�1�&$�"4ne85, 6)@ �Z��+%��� � OMNj¡�¢_9:n&'"42e8`_£a�^�)e8#Z¤+� b¥�^�#$0�c¦�:'0>8@ &�,cjd§9:� H.
cinaedi %%D��e+W>0f¨s©1��)e82� g��!;< Helicobacter���%�C2ªhjn�2#>@ ��9p%��«¬)$�®!i$�(�� jk�¯l!m�s8n�2#}� �m+%°]± n (thin spreading layer) +�ps8��9p�q:2ªh)e8@ �����%D�e)% Heli-
cobacter������9²q³j�Co.�qp2´�:'0>8@
Helicobacter���!!"#$�NO&'��%���$�2�7'0>82� �':�����
q�rstµ (¶464�8650) R·¸u�¹vgw 1�100�������������������TEL: 052�757�6780FAX: 052�757�6780E-mail: [email protected]
�g%D����º» Vol. 18 No. 4 2008.
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227
��� H. pylori �������7)� � � H.
pylori����� H. cinaedi�������������� �!� H. pylori"#�$%���&��'���&(�) � Helicobacter* 15$%+,-.�/ H. cinaedi�0�- 14-���1�2 8%�&�(�3�' ���� 4�56(��7��
�����1. ��� 89:;�2 <�=>�? .�/ Campylobacter
spp.���2 1��@���ABCD�2 <�=>�?� ��ABCD�2 EX <�=>�?�H. pylori����2�E�FGHIJKD:;�2 <�=>�?� LMNOPM HP��2 <QRST?� LMNOPM ViFGH:;�2 <QRST?� HCUVM HP:;�2 <�� BD?� WCG:;�2<XY>�? � 8%��2(�3 �� 89:;�2� ��ABCD�2� ��ABCD
�2 EX� LMNOPM HP��2� .�/HCUVM HP:;�2� 5%��2Z��89([� .�� Helicobacter*$%�\]�2^��_�]�`�(aZ7�� b�� FGHIJKD:;�2�LMNOPM ViFGH:;�2� WCG:;�2�3%�cd8e([� ��_��2�E�� �]f� tetrazolium violetghi��@��j$k��l��@m��n=opo���qr��� s](t7��
2. ���Helicobacter * 15 $ % + , -u H. bilis PAGU
599T (vLMG 18386T), H. canadensis PAGU 600T
(vCCUG 47163T), H. canis PAGU 598T (vNCTC
12379T), H. cinaedi PAGU 597T (vCCUG 18818T),
H. fennelliae PAGU 601T (vCCUG 7546T), H. pullo-
rum PAGU 602T (vNCTC 12824T), Helicobacter an-
seris PAGU 940T (vMIT 04�9362T), Helicobacter
brantae PAGU 941T (vMIT 04�9366T), Helicobacter
marmotae PAGU 943T (vMIT 98�6070T), Helicobac-
ter mastomyrinus PAGU 944T (vMIT 97�5574T),
Helicobacter ganmani PAGU 603T (vCCUG
43526T), Helicobacter hepaticus PAGU 604T (vLMG
16316T), Helicobacter muridarum PAGU 606T
(vLMG 13646T), Helicobacter pametensis PAGU
607T (vLMG 12678T), Helicobacter acinonychis
PAGU 609T (vLMG 12684T)(�@���!�� H. cinaedi�w�.�/w#��0�-
14-�x� ��
3. � �������a) 8� ��������������������yAz{|}UID��� �M~C��J��� �B}UW�c|��� 3%(�@�� �yAz{|}UID���� ���(n��� N2 80��CO2 10�� H2 10����yA�z{ � (Fig. 1)� �!���P����D�D(|}UID��z@.� ��(�� �� �M~C��J���� M~C��JFGH���&$������ <� yAST? ���}D <� yAST? (x� � � !"���(�� �� �� �B}UW�c|���� BBLB}UW�c|����A�� (BD)�#*�9k(�$ � �c|(%& �� �� �'�g!����B}UW�c|��(g����@��yAz{|}UID���(�@� 8%�1�2� Helicobacter* 15$%+,-.�/ H. cinaedi
�0�- 14-( 37�� 3� �� � $��l¡¢()£¤¥��� 3¦§��' �� ¨�� M~C��J���� B}UW�c|����.@�� ©ª�!�����E� enterohepatic Helicobacter
6$%+,-(�@� !"��' ��
Fig. 1. Gas displacement chamber system.�chamber (desiccator with valve), �pressure gauge, �vacuum pump, �compressed gas cylinder (N2 80�«CO2
10�«H2 10�)
*¬®�¯®¬°��±²b³�´µ+b�¶¬,-�·¸�¹*¬®�¯®¬°��±²b³�´µ+b�¶¬,-�·¸�¹
���0�º.T»¼ Vol. 18 No. 4 2008.8
228
b) ���������� ������� ���� ������� � ���� ��������������� �������� !�"# $%� &���'()*+ �,-# ./012�3��456789�� �������:;<=>?@AB9���� ������� � 3
C����DE"�� HelicobacterFGCHIJKLMNOP�QRS�T# �UVMWX�YXZ[�\S;] 200 ml�^GW�_`a��Mbc# ��������d�U"� H. cinaedi PAGU 597T�_`a��Mbc�^GW�ef"# g�hhijklmn�� o6pqrst@9� 37u# 3vw�U"# 1
vx�MIJyz�{|"��
c) ATP�����������������$%�IJ`a�IJ012�3��# �������# 456789��# =>?@AB9�����3C����DE"�� d�U"� H. cinaedi PAGU
597T:;<}~���X���3S PAGU 934�D�# ��9�9�t�t�86 (MHB)M� McF#1
�G����"# ��Mg�G�� 10���"��_��M��"�G�� 200 ml���C"�� �9������Mklmn# o6pqrst@9�E�� 37u# �2�# O�y���U"�� �U 1v�#3v�# 5v�M_��M�J"�G�� ATP '�¡¢"�� ATP '£H# _��MMHB 3 ml�bc��9��G��¤¥�c# G��R¦�§¨"
Table 1. Growth level of each media using gas displacement chamber.
SpeciesPAGU
No.
Blood Non blood
�Bloodagar
�Skirrow �SkirrowEx
�HPselective
�ColumbiaHP
�Helico-bacter
�ViHelico
�Pyloriagar
H. bilis 599T ©© ©© ©© ©© ©© ©© w ©©H. canadensis 600T ©© ©© ©© © ©© ©© w ©H. canis 598T ©© ©© ©© ©© ©© ©© w ©H. cinaedi 597T ©© ©© ©© ©© ©© ©© © ©©H. fennelliae 601T ©© ©© ©© ©© ©© ©© © ©©H. pullorum 602T ©© ©© ©© ©© ©© © w ©
H. anseris 940T ©© ©© ©© w © ©© © ©©H. brantae 941T ©© ©© ©© ©© ©© ©© w ªH. marmotae 943T ©© ©© ©© ©© ©© ©© w ©©H. mastomyrinus 944T ©© ©© ©© ª © ©© © ªH. ganmani 603T ©© w w © w w w wH. hepaticus 604T © ©© ©© © ©© w ©© ©H. muridarum 606T ©© © ©© w w ª ª ªH. pametensis 607T ©© ©© ©© ©© ©© ©© w ªH. acinonychis 609T ©© ©© ©© ©© ©© w © ©©
H. cinaedi 611 © ©© © ©© © ©© w ©©H. cinaedi 612 ©© ©© ©© ©© © © ª wH. cinaedi 615 ©© ©© ©© ©© ©© ©© © ©H. cinaedi 616 ©© ©© ©© ©© ©© ©© w wH. cinaedi 623 ©© ©© ©© ©© ©© ©© © wH. cinaedi 624 ©© w w w w ª ª ªH. cinaedi 630 ©© ©© ©© ©© ©© ©© © ©H. cinaedi 636 ©© ©© w ©© © © © ©H. cinaedi 640 ©© ©© © ©© © © w wH. cinaedi 641 © w w w w ©© w ©H. cinaedi 642 ©© ©© © ©© ©© ©© © ©©H. cinaedi 824 ©© ©© ©© ©© ©© ©© © wH. cinaedi 825 ©© ©© ©© ©© ©© ©© © ªH. cinaedi 934 ©© © © © ©© © w w
Growth level was evaluated in four stages. ©©: good growth, ©: middle growth, w: weak growth, and ª:no growth.
HelicobacterFGC«¬���)*HelicobacterFGC«¬���)*
v����®¯°± Vol. 18 No. 4 2008. 9
229
��� �� 50 ml���� Bac Titer-Glo Microbial
Cell Viability Assay (Promega) 50 ml�� �������������� (DTX 880. Backman
Coulter)������ �!"��#$%&'( 1)*� 3)*� 5)*&+,-&./
0'1���234�5678�9:;<=&.�>?�#
� �1. 8�������� �������3@�'(AB� 3)C'(���� D2��3E
�FG��# 8@�HI'1&./JKLM�NOP�'(��QR� Table 1, Fig. 2&ST# U�� VWXYZ�[.\]NO^Y_�'(�QR� Table
2&ST# `ab�cd�� 3@�'(e�f�gJKLM�NOP���'(hij�2kl�m?�#nopqr'1�st 7@�uv'1�f�g� we
Fig. 2. A summary of the growth level in eight media using gas displacement chamber.Number of strains evaluated by each growth level (xx, x, w, y) was showed.
Table 2. Growth level using Aneropack and Campypouch.
SpeciesPAGU
No.Gas
system
Blood Non blood
�Bloodagar
�Skirrow �SkirrowEX
�HPselective
�ColumbiaHP
�Helico-bacter
�ViHelico
�Pyloriagar
H. bilis 599T ANE x xx xx w xx w w wCAM xx xx xx w xx w w w
H. canadensis 600T ANE xx x w w x y y yCAM x w w w x x w w
H. canis 598T ANE xx xx x x xx y y yCAM x x x x x y y y
H. cinaedi 597T ANE xx xx xx xx xx xx x xCAM xx xx xx xx xx x x x
H. fennelliae 601T ANE xx xx xx xx xx xx xx xxCAM xx xx xx xx xx x x x
H. pullorum 602T ANE xx xx x x x y y yCAM x x x x x w y y
Each type strain of six species designated as an enterohepatic Helicobacter species was used. Growth levelwas evaluated in four stages, same as the legend in Table 1 (xx, x, w, y). ANE: Aneropack, CAM:Campypouch.
z{|}~�}{��~����~����~�{��~��l�z{|}~�}{��~����~����~�{��~��l�
)`��,D���� Vol. 18 No. 4 2008.10
230
���������� ����� ��������� ������ � !"# �$%��&'()*+,-�./��� 012341 Vi()*./��� 5�)./��� 36�78���9�:� ()*+,-�./���� ���;�<!"#
2. ���������� �()*+,-�./��:� =��./��>?@�������ABC 1DE>�FGHCI�JK 8L�LM�N" (Fig. 3)# ()*+,-�./���:OPQRRS TUVWCI'"X� ��Y�Z� [&'>78��Y�\R�]^%_`H"# aW�bc� ()*+,-�./��:defg24efh ��i��LM jk�<!"# l"����?m�noHp&I>I�qr stW"#
3. ATP�����������������=��./��ubv?@�������H"��� ATPwxyz%{!">a|� �i 3D}~����� ��H"# ��� ()*+,-�./�������yz�:� ��� (PAGU597): 3
D}~��p ���H� ����� (PAGU934)�: 5D}l� ATPwx�:Y�H" (Fig. 4)#
ATPwxyz>�e�� ������%noH">a|� =��./��ubv?@������Y���:�i 5D}�:����_ (coccoid form)��!CI"���H� ()*+,-�./��Y���: 5Df�i����_%��HCI'�� � !" (Fig. 5)#
Fig. 3. Comparison of the color change by the colony growth.Bacterial growth was observed at daily intervals for three days. H. cinaedi PAGU 597T wasincubated in a gas displacement chamber. Purple color depending on bacterial growth was easyto find on the Helicobacter agar from the first day.
Helicobacter��6����� ¡Helicobacter��6����� ¡
D¢���[£¤¥¦ Vol. 18 No. 4 2008. 11
231
� ���� ����������� ������
�������������������� !�!� Helicobacter�"#�$%&'���()!*++� ,!�"� CFU (Colony Forming Unit)
*-.�����/0��1�2� 3��� �45��� �67�8*9:;<���/0�=*ATP >?���!� @"�A"�>?B ���!<� ATP >? *-.� 3�����,CD�����/�� �EFG���H-I��JK���L�"���� 5��*6���M>?��N!� �+�O"(*�P!<;� Q�� RS�TU8�G���L�"�� 5�V��WX�M>?��N���+� �Y*� "�XYZ[(��D!<;�X���+\Y�� H. pylori ����]!;^_* ����YZ[(�YO"(*�P�����!Y�<;� ("��P*�`#� a#� bJcd� $e�����6f��ghg6cd�i%!<;��� O"(j�("�P�kl�!< “nonculturable
state” j�m/�2.� “bacterial cell death” �&n�2��;0'o�2�8) H. cinaedi X()�,*?�2����p+*qrY�� !���<s��/0t*� u*O"(*�P!<;�"��,"!
<X-v!<�6;wx?�2.� .y�/0�2� ��-06z1�Yqr��� RS�TU8�G����2{V��WXYZ[("�|D��<H.� 3���-.X,CD��4�<;��qrY�� ���}#~���=*s���5�/06���*H;<�7�*89�2������ g��s�{�,^_*H;<�����+ *X.y����2� Helicobacter�"#���*�a#�/0�!� ����+ ��;�"��,!6; �:����9��t� ;<=��>!<�Y��!�{V*-�<����?@#��P���=� (�����9!<X��*)�����X�A����X2� ��BC�DE*F��=� �45������t*��L*G"�� 200 ml �Z<�Y"��H#���;0���9;� ��*-.� p+6�+ ������ s�{�,�"X�I�������� �45��9!� 3 #�����J��� bJ �
��%T�*-�����X,K��2��;0kl�LY�� bJ ���%T�����M�W�N2 80 � CO2 10 � H210 �bJ� �!<;� Q��¡¢£¤UH-IK�%¥£¦�� O2 �§¨! CO2 ����$J©ª��=� ¡¢£¤U
Fig. 4. Evaluation of bacterial activity using ATP assay system.H. cinaedi PAGU 597T or PAGU 934 (clinical isolate) was cultured on each medium for 5 days.Bacterial cells were collected from each medium, on day-1, -3, and -5, and measure the activity.Data were means of triplicate. The bacterial activity of the cells collected from Blood agar andModified Skirrow agar after 5 days culture were dramatically decreased, whereas the cells fromthe Helicobacter agar were still increasing or slightly decreased. RLU: relative luminescenceunits.«¬« : Blood agar, �
�
: Skirrow agar, ��®� : Helicobacter agar.
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��6�W�Xº»¼ Vol. 18 No. 4 2008.12
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�� O2� 6�12�� CO2� 5�8������ �������� O2� 5�15�� CO2� 5�12������ ��������������� !"� 2#�� $!�%&'�()���*+�,�� Bergey’s Manual of Systematic Bacteriology� -H. pylori, H. mustelae, H. nemestrinae, H. acino-
mychis�./012�,34()5678�9: !;<=3>9��� ?@ -H. cinaedi��� ()�ABCD,�9�AB5EFG�: !;H=3>9�9)� IJ� ()���KL� HelicobacterMN#�ABOPG�!9QRS10)T,�� UV�WX�Y3Z!?[8J� �����\]^�_�� `ab�
� c�de�f� ghi��j�klZ�����������]�md5no8J�� Y�" BDpOxoid�q�r0���st]�md5u_v�w�8�xo8� ()5yz��{|5}~G�@$�RS=3>9�11)� HelicobacterMN#�� ���� ��hQ�()��5A�G�� ]�md�xo���=3��
HelicobacterMN#��<8JhQ]�������5�~8��� �������d����5�~G��!��9� H. cinaedi���� H. bilisp H.
canadensis�qT���d��AB��Z3�!;H=3>9�12, 13)� ��� ����xo8J�wlZ
Fig. 5. The morphological change of the bacterial cells.H. cinaedi PAGU 597T was cultured on each medium for 5 days. Bacterial cells were collectedfrom each medium, on day-1, -3, and -5, and observed the morphology with Gram-stained undermicroscope (�1,000).The cells collected from the Helicobacter agar on the day-5 sustained the spiral form, howeverthe majority cells from Blood agar and Modified Skirrow agar changed the form into coccoidshape.
HelicobacterMN#�������HelicobacterMN#�������
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233
������� 6������� H. fennelliae�� ����������� �� 5������������������ �! ���������"#��$� %&�'��()������*+�, -����.�/01� �! ���%2�'������3�� -�����4)�����5��*+6�7 -����8��9� 24:;������<��=�! > ?1@��ABC@D�� Helicobacter E���FG�HI@����$� ))J�'����KL�MNOP�� ViKL�Q��� RSLQ������,TU �! V � 3���� -�W�XY,� KL�MNOPQ���� -�Z��9��[@\�� �! ViKL�Q��D]^RSLQ��@�� _`abcde!fG&+g�"#� $chij% k&lm '(���'n ���! �� KL�MNOPQ��@�o�Nc)*'(_`ab'n ���"#&lj%'(���'n ��+�! "#&lj%'(���� p�qors�+,@'t -uv-. ���,01� �� /�@'n �wxy b+g�'0qors[��z{P�| kS�H|m +g}��c�6� >�12� -3~#�,������ -uv�@\�� ����+�[,������! �[�+�� HelicobacterE��� -uv���j@����4���9� ����5���!�3�F6���[�� KL�MNOPQ��k7��8m � HelicobacterE���HIFGD]^ -uv-�9:@D��. �D9� �;���,���/��,�<��!
� �1) Solnick, J. V., D. B. Schauer. 2001. Emergence of
diverse Helicobacter species in the pathogenesisof gastric and enterohepatic diseases. Clin. Mi-crobiol. Rev. 14(1): 59�97.
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pylobacter fennelliae (sp. nov.): two new Campy-
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7) �� �� �=��� �� >� �� 2000. Heli-
cobacter pylori HIFG��5� �KL�MNOP��� k7��8m� �?�� �A@}#HI������ 11(1): 27�32.
8) Kusters, J. G., M. M. Gerrits, C. M. Vanden-broucke-Grauls, et al. 1997. Coccoid form ofHelicobacter pylori are the morphologic mani-festation of cell death. Infect. Immun. 65(9):3672�3679.
9) Garrity, G. M., J. A. Bell, T. Liburn. 2005. Fam-ily II. Helicobacteraceae fam. nov. In Brenner, D.J., Krieg, N. R., Staley, J. T., and Garrity, G. M.(eds.), Bergey’s Manual of Systematic Bacteriol-
ogy. 2nd ed. Vol. 2. New York, Springer-Verlag.10) A���� �BC d� �¡ �� �� 2007� %
&�[�� Helicobacter cinaediD]^>�D¢�����,£EFGH@¤��F6� ¥¦§¨©� 81: 700�706.
11) Iª«� ¬=J� ®¯°K� �� 2005. H.
cinaedi, H. fenneliae, H. pullorum�/�,L±/� �A,@}# 32: 175�180.
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species isolated from bile, livers, and intestinesof aged, inbred mice. J. Clin. Microbiol. 33(2):445�454.
13) Fox, J. G., C. C. Chien, F. G. Rodgers, et al. 2000.Helicobacter canadensis sp. nov. isolated fromhumans with diarrhea as an example of anemerging pathogen. J. Clin. Micobiol. 38(7):2546�2549.
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Evaluation of Various Media for Rapid Detection of Helicobacter spp.
Junko Tomida, Mitsuaki Kashida, Kazuki Oinishi, Ryuichi Endo, Yuji Morita, Yoshiaki Kawamura
Department of Microbiology, Aichi-Gakuin University, School of Pharmacy
The evaluation of commercially available media for Helicobacter species has been carried out mainly byHelicobacter pylori strains. However the reports of Helicobacter cinaedi infection have been increasing, wedecided to investigate the performance of various media for growing several Helicobacter species. Weevaluated following 8 media with using the type strain of 15 species of the genus Helicobacter, and 14clinical isolates of H. cinaedi; Sheep blood agar (Nissui), Modified Skirrow agar (Nissui), Modified Skirrowagar EX (Nissui), HP selective agar (Eiken), Columbia Helicobacter pylori agar (BD), Helicobacter agar(Nissui), Vi Helico agar (Eiken), and Pylori agar (Kyokuto). Because the latter three media were containinghorse-serum and indicator-dye (tetrazolium salt), the bacterial growth was recognizable at a glance by theviolet-blue color on the translucent media. Within these three media, Helicobacter spp. was the best-grownon the Helicobacter agar. To evaluate the total activity of the growing bacteria, ATP assay system wasapplied. The bacterial activities of the cells collected from Blood agar and Modified Skirrow agar after 5days culture were dramatically decreased, whereas the cells from the Helicobacter agar were still increasedor slightly decreased. In addition, the cells collected from the Helicobacter agar after 5 days culturesustained spiral form, while the majority cells from Blood agar and Modified Skirrow agar changed theform into coccoid shape. From these data, we finally concluded that Helicobacter agar (Nissui) was usefulfor rapid detection and also sustaining the bacterial activity.
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