(PFGE) Clostridium di$cile

2009 205 (PFGE) Clostridium di$cile 1) 3) 2) 2) 2) 2, 4) 5) 1) 2) 3) 4) 5) 21 5 22 21 8 31 2004 1 2008 12 5 Clostridium di$cile (C. di$cile) 340 248 A /B 141 (56.9) A /B 26 (10.5) A /B 81 (32.7) 136 (PFGE) PFGE 4 132 51 A L 12 85 (62.5) 39 47 (34.6) 35 (25.7) A bu#er 200 mm C A /B A B J PFGE 5 C. di$cile Key words: Clostridium di$cile, PFGE Clostridium di$cile 7.6 1) 20 40 2, 3) C. di$cile b- C. di$cile (4668650) 65 TEL: 0527442614 FAX: 0527442613 E-mail: t-ookura@med.nagoya-u.ac.jp (CDI) 1) C. di$cile A B A B binary toxin 4, 5) (PFGE) 6) C. di$cile DNA PFGE 79) PFGE Vol. 19 No. 4 2009. 7

206 PCR PFGE 10) PFGE 5 C. di$cile PFGE 1. 2004 1 2008 12 5 CCMA 4 11) PCR C. di$cile 1080 10 CCMA 97 136 PFGE 2. (1) A 1 13,500 rpm 10 125 ml 30 (2) PCR C. di$cile PCR C. di$cile A B C. di$cile Gumerlock PG 48 (5 - CTCTTGAAACTGGGAGACTTGA- 3 ) PG 49(5-ACTGAGAGTAGCTTTA-3) 12) A B Kato NK2(5-CCCAATAGAAGATT- CAATATTAAGCTT- 3 ) NK3(5-GGAA- GAAAACAACTTCTGGCTCACTCAGCT- 3 ) 13) A B NK9(5-CCACCAGCTGCAGCCA TA-3 ), NK 11 (5-TGATGCTAATAATGAATCT AAAATGGTAAC-3) NKV011(5-TTTTGA 14, 15) TCCTATAGAATCTAACTTAGTAAC-[S 2] 3) A B HK 2 TE (ph 8.0) McFarland 1.0 3.0 100 10 14,000 rpm EXTaq 1 20 ml PCR 0.2 mm, EXTaq 0.4 unit 2 ml PCR PG48 PG49 NK2 NK3 95 20 55 120 35 NK9, NK11 NKV011 95 20 60 120 35 PCR TAE 2 100 V 30 PCR C. di$cile A B (3) PFGE PFGE Alonso 10) CCMA 1 (BHI) 37 18 BHI 3,000 rpm 20 Washing Solution (0.15 M NaCl, 0.01 M EDTA) 3 Pett IV (10 mm Tris HCl, 1 M NaCl) McFarland 4.0 100 ml 1.5 100 ml 2 mg/ml Lysis 10 mm TrisHCl, 1 M NaCl, 100 mm EDTA, 0.5 sodium N-dodecanolsalcosinate (SDS) 0.2 deoxycholate, 0.5 Brij-58 500 ml 37 2 K 75 U/ml (ES solution: 0.25 M EDTA, 1 SDS) 500 ml 50 18 1mm 1 mm phenylmethylsulfonyl fluoride (PMSF) K TE 3 SmaI 30 1 DNA 200 mm 0.9 SmaI lladder 8 Vol. 19 No. 4 2009.

Clostridium di$cile PFGE 207 1. 5 2004 233 53 22.7 237 19 8.0 2005 261 65 24.9 309 41 13.2 2006 341 57 16.7 343 52 15.2 2007 342 83 24.3 453 92 20.3 2008 384 82 21.4 612 123 20.1 1,561 340 22.3 1,954 327 16.7 2. A 110 (8.8) 164 (13.2) 274 (22.0) 99 (7.9) 873 (70.1) 972 (78.0) 209 (16.8) 1,037 (83.2) 1,246 (100) 3. A /B 67 (34.2) 53 (27.0) 120 A /B 4 (2.0) 16 (8.2) 20 A /B 17 (8.7) 39 (19.9) 56 88 108 196 62.20 55.80 72.30 (Bio-Rad) (CHEF-DR II: BIO-RAD) 200 mm 0.5TBE 430 20 PFGE Alonso 10) 0.9 0.5TBE 200 mm K (5) PFGE Finger Printing II (BIO-RAD) 80 PFGE A S 1 PFGE (1) 5 A 1 233 384 16.7 24.9 237 612 8.0 20.3 2 A 78.9 Vol. 19 No. 4 2009. 9

208 1. PFGE M: lambda ladder marker, 1 NL 5090, 2 NL5092, 3 NL5094, 4 NL5122 A 8.8 29.9 16.8 22.0 (2) 1 5 C. di$cile 340 248 A /B 141 (56.9), A /B 25 (10.1), A /B 81 (32.7), binary toxin 1 3 248 196 62.2 PCR 55.8 72.3 A /B 4 A /B 17 (3) PFGE DNA 4 PFGE 1 9) DNA 45 2 Alonso 10) 2. Clostridium di$cile132 PFGE AL: PFGE 80 PFGE PFGE 340 PFGE 97 136 4 4 10 Vol. 19 No. 4 2009.

Clostridium di$cile PFGE 209 4. PFGE PFGE 2004 2005 2006 2007 2008 85 (62.5) 47 (34.6) 4 (2.9) A A /B 35 (23) 1 (1) 5 (5) 19 (10) a 10 (8) a B A /B 14 (14) 3 (3) 6 (6) 1 (1) 4 (4) C A /B 12 (8) 1 (1) 3 (2) 4 (3) b 4 (3) b D A /B 6 (4) 4 (2) 1 (1) 1 (1) E A /B 3 (3) 1 (1) 2 (2) F A /B 3 (3) 1 (1) 1 (1) 1 (1) G A /B 3 (3) 1 (1) 1 (1) H A /B 2 (2) 2 (2) I A /B 2 (2) 1 (1) 1 (1) J A /B 2 (2) 2 (2) K A /B 2 (2) 2 (2) L A /B 2 (2) 1 (1) 1 (1) MS P A /B 12 (6) 3 (2) 3 (2) c 6 (3) c P A /B 2 (1) 2 (1) A /B 33 (30) 2 (2) 10 (10) 7 (7) 14 (11) NT A /B 4 (4) 1 (1) 2 (2) 1 (1) 136 (108) 6 (4) 8 (8) 32 (30) 44 (32) 46 (37) : 97 11 PFGE ac: 2 132 PFGE 51 2 4 PFGE A L A 35 (25.7) B 14 (10.3) C 12 (8.9) 4 PFGE M S 2 33 30 2 PFGE A 6 23 7 12 6 9 4 97 21 C. di$cile 10 PFGE 11 PFGE 4 PFGE 108 PFGE C 12 P 2 A /B A /B (4) PFGE 4 PFGE PFGE A L 136 85 (62.5) M S 47 (34.6) H J K 2 5 A 23 35 2007 2008 2 164 B 141 (56.9) PCR A /B 3 A /B 120 67 55.8 A /B A /B 21 Vol. 19 No. 4 2009. 11

210 PCR 2 209 99 (47.4) C. di$cile B CDI C. di$cile 32.7 A CDI PFGE 6) C. di$cile PFGE MRSA DNA 45 A 35 B 1 R 2 7 A 35 Kato 2001 3 C. di$cile 120 87 (73) 7) Sawabe 1999 2004 5 C. di$cile 148 55 DNA 55 PFGE 33 8) Sawabe 8) PFGE Bidet 9), Corkill 16) PCR 1 G PCR PFGE PCR 8, 10, 17, 18) DNA 3 PFGE 2004 2005 2006 2008 PFGE A 35 (25.7) 2004 1 1 2006 5 5 2007 19 10 2008 10 8 B 14 (10.3) 2008 PFGE C 12 (8.8) A /B A /B 19) A /B A B PFGE 3 20) 21 2 10 PFGE 10 11 PFGE 11 12 Vol. 19 No. 4 2009.

Clostridium di$cile PFGE 211 PFGE K 2 1 PFGE 2 PFGE 4 7 80 PCR 1) Kato, H., H. Kita, T. Karasawa, et al. 2001. Colonisation and transmission of Clostridium di$cile in healthy individuals examined by PCR ribotyping and pulsed-field gel electrophoresis. J. Med. Microbiol. 50: 720727. 2) Bartlett, J. G. 2006. The new epidemic of Clostridiumdi$cile-associated enteric diseases. Ann. Intern. Med. 145(10): 758764. 3) Cookson, B. 2007. Hypervirulent strains of Clostridium di$cile. Postgrad. Med. J. 83(979): 291295. 4) Martin, H., B. Willey, D. E. Low, et al. 2008. Characterization of Clostridium di$cile strains isolated from patients in Ontario, Canada, from 2004 to 2006. J. Clin. Microbio. 46: 2999 3004. 5) Fenner, L., R. Frei, M. Gregory, et al. 2008. Epidemiology of Clostredium di$cile-associated disease at University Hospital Basel including molecular characterisation of the isolates 20062007. Eur. J. Clin. Microbiol. Infect. Dis. 27: 12011207. 6) Tenovor, F. C., R. D. Arbeit, R. V. Goering, et al. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J. Clin. Microbiol. 33: 22332239. 7) Kato, H., N. Kato, K. Watanabe, et al. 2001. Analysis of Clostridium di$cile isolates from nosocomial outbreaks at three hospitals in diverse areas of Japan. J. Clin. Microbiol. 39: 13911395. 8) Sawabe, E., H. Kato, K. Osawa, et al. 2007. Molecular analysis of Clostridium di$cile at a university teaching hospital in Japan: A shift in predominant type over a five-year period. Eur. J. Clin. Microbiol. Infect. Dis. 26(10): 695703. 9) Bidet, P., V. Lalande, B. Salauze, et al. 2000. Comparison of PCR-ribotyping, arbiratory primed PCR, and pulsed-field gel electrophoresis for typing Clostridium di$cile. J. Clin. Microbial. 38: 24842487. 10) Alonso, R., A. Martin, T. P. Martin, et al. 2005. An improved protocol for pulsed-field gel electrophoresis typing of Clostridium di$cile. J. Med. Microbiol. 54: 155157. 11) 2002. Clostridium di$cile 12: 115121. 12) Gumerlock, P. H., Y. J. Tang, F. J. Meyers, et al. 1991. Use of the polymerase chain reaction for the specific and direct detection of Clostridium di$cile in human feces. Rev. Infect. Dis. 13: 10531060. 13) Kato, N., C.Y. Ou, H. Kato, et al. 1991. Identification of toxigenic Clostridium di$cile by the polymerase chain reaction: J. Clin. Microbiol. 29: 3337. 14) Kato, H., N. Kato, K. Watanabe, et al. 1998. Identification of toxin A-negative, toxin B- positive Clostridium di$cile by PCR. J. Clin. Microbiol. 36: 21782182. 15) Kato, H., N. Kato, S. Katow, et al. 1999. Detections in the repeating sequences of toxin A gene of toxin A-negative, toxin B-positive Clostridium di$cile strains. FEMS Microbiol. Lett. 175: 197203. 16) Corkill, J. E., R. Graham, C. A. Hart. 2000. Pulsed-field gel electrophoresis of degradationsensiteve DNAs from Clostridium di$cile PCR ribotype 1 strains. J. Clin. Microbiol. 38: 2791 2792. 17) Corne, H., W. Klaassen, A. Hanneke, et al. 2002. Molecular fingerprinting of Clostridium di$cile isolates: Pulsed-field gel electrophoresis versus amplified fragment length polymorphism. J. Clin. Microbiol. 40: 101104. 18) Killgore, G., T. Angela, S. Jhonson, et al. 2008. Comparison of seven techniques for typing international epidemic strains of Clostridium di$cile: Restriction endonuclease analysis, pulsed-field gel electrophoresis, PCR-ribotyping, multilocus sequence typing, multilocus variable-number tandem-repeat analysis, amplified fragment length polymorphism, and surface layer protein A gene sequence typing. J. Clin. Microbiol. 46: 431437. 19) 2004. toxin A toxin B Clostridium di$cile 78: 312319. 20) Fawley, W. N., P. Paunell, P. Verity, et al. 2005. Vol. 19 No. 4 2009. 13

212 Molecular epidemiology of Clostridium di$cile infection and the significance of subtypes of the United Kingdom epidemic strain (PCR Ribotype 1). J. Clin. Microbiol. 43: 26852696. Analysis of Clostridium di$cile Strains Isolated in a Hospital Using an Improved Procedure of Pulsed-Field Gel Electrophoresis Toshi Nada 1), Masahiro Suzuki 2), Teruko Ohkura 1), Yukiko Nakanishi 1), Mariko Mochizuki 1), Hisashi Baba 1, 3), Tetsuya Yagi 4) 1) Department of Clinical Laboratory, Nagoya University Hospital 2) Department of Microbiology and Medical Zoology, Aichi Prefecture Institute of Public Health 3) Department of Infectious Diseases, Nagoya University Hospital 4) Center of National University Hospital for Infection Control, Nagoya University Hospital From January 2004 to December 2008, we isolated 340 Clostridium di$cile strains from fecal sample, and we tested for toxin type of 248 strains using PCR method. The isolates comprise of 141 toxin type A / B (56.9), and 25 A /B (10.5), A /B (32.7), and one binary toxin type. One hundred thirty six Clostridium di$cile isolates recovered from feces of 97 patients, were analyzed by pulsed-field gel electrophoresis (PFGE). They were classified into 51 PFGE types using the identity criteria of 80 similarity in the dendrogram analysis. Eighty-five strains (62.5), which were isolated from di#erent patients, were deviced into 12 PFGE types (from type A to type L), and the other 47 strains showed unique 40 PFGE types. Although PFGE type A (35 strains) were the most predominant genotype, these strains showed non-typable DNA degradation until the addition of 200 mm thiourea into 0.9 agarose gel and TBE running bu#er. 12 strains of PFGE type C and 2 strains of PFGE type P were toxin A /B, which showed false negative results for rapid toxin detection test. As strains of PFGE type A, B, and J were isolated from the same ward in a year, these strains might spread via nosocomial transmission. Some strains with same PFGE types were isolated consistently for 25 years, suggesting that they are surviving in the hospital environment or in the human gastrointestinal tract for such a long time. 14 Vol. 19 No. 4 2009.