(PFGE) Clostridium di$cile

Transcription

1 (PFGE) Clostridium di$cile 1) 3) 2) 2) 2) 2, 4) 5) 1) 2) 3) 4) 5) Clostridium di$cile (C. di$cile) A /B 141 (56.9) A /B 26 (10.5) A /B 81 (32.7) 136 (PFGE) PFGE A L (62.5) (34.6) 35 (25.7) A bu#er 200 mm C A /B A B J PFGE 5 C. di$cile Key words: Clostridium di$cile, PFGE Clostridium di$cile 7.6 1) , 3) C. di$cile b- C. di$cile ( ) 65 TEL: FAX: 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

2 206 PCR PFGE 10) PFGE 5 C. di$cile PFGE CCMA 4 11) PCR C. di$cile CCMA PFGE 2. (1) A 1 13,500 rpm ml 30 (2) PCR C. di$cile PCR C. di$cile A B C. di$cile Gumerlock PG 48 (5 - CTCTTGAAACTGGGAGACTTGA- 3 ) PG 49(5-ACTGAGAGTAGCTTTA-3) 12) A B Kato NK2(5-CCCAATAGAAGATT- CAATATTAAGCTT- 3 ) NK3(5-GGAA- GAAAACAACTTCTGGCTCACTCAGCT- 3 ) 13) A B NK9(5-CCACCAGCTGCAGCCA TA-3 ), NK 11 (5-TGATGCTAATAATGAATCT AAAATGGTAAC-3) NKV011(5-TTTTGA 14, 15) TCCTATAGAATCTAACTTAGTAAC-[S 2] 3) A B HK 2 TE (ph 8.0) McFarland ,000 rpm EXTaq 1 20 ml PCR 0.2 mm, EXTaq 0.4 unit 2 ml PCR PG48 PG49 NK2 NK NK9, NK11 NKV PCR TAE V 30 PCR C. di$cile A B (3) PFGE PFGE Alonso 10) CCMA 1 (BHI) 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 ml 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 ml 37 2 K 75 U/ml (ES solution: 0.25 M EDTA, 1 SDS) 500 ml mm 1 mm phenylmethylsulfonyl fluoride (PMSF) K TE 3 SmaI 30 1 DNA 200 mm 0.9 SmaI lladder 8 Vol. 19 No

3 Clostridium di$cile PFGE , , 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) (Bio-Rad) (CHEF-DR II: BIO-RAD) 200 mm 0.5TBE PFGE Alonso 10) TBE 200 mm K (5) PFGE Finger Printing II (BIO-RAD) 80 PFGE A S 1 PFGE (1) 5 A A 78.9 Vol. 19 No

4 PFGE M: lambda ladder marker, 1 NL 5090, 2 NL5092, 3 NL5094, 4 NL5122 A (2) 1 5 C. di$cile A /B 141 (56.9), A /B 25 (10.1), A /B 81 (32.7), binary toxin PCR A /B 4 A /B 17 (3) PFGE DNA 4 PFGE 1 9) DNA 45 2 Alonso 10) 2. Clostridium di$cile132 PFGE AL: PFGE 80 PFGE PFGE 340 PFGE Vol. 19 No

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

6 210 PCR (47.4) C. di$cile B CDI C. di$cile 32.7 A CDI PFGE 6) C. di$cile PFGE MRSA DNA 45 A 35 B 1 R 2 7 A 35 Kato C. di$cile (73) 7) Sawabe C. di$cile 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 PFGE A 35 (25.7) B 14 (10.3) 2008 PFGE C 12 (8.8) A /B A /B 19) A /B A B PFGE 3 20) PFGE PFGE Vol. 19 No

7 Clostridium di$cile PFGE 211 PFGE K 2 1 PFGE 2 PFGE PCR 1) Kato, H., H. Kita, T. Karasawa, et al Colonisation and transmission of Clostridium di$cile in healthy individuals examined by PCR ribotyping and pulsed-field gel electrophoresis. J. Med. Microbiol. 50: ) Bartlett, J. G The new epidemic of Clostridiumdi$cile-associated enteric diseases. Ann. Intern. Med. 145(10): ) Cookson, B Hypervirulent strains of Clostridium di$cile. Postgrad. Med. J. 83(979): ) Martin, H., B. Willey, D. E. Low, et al Characterization of Clostridium di$cile strains isolated from patients in Ontario, Canada, from 2004 to J. Clin. Microbio. 46: ) Fenner, L., R. Frei, M. Gregory, et al Epidemiology of Clostredium di$cile-associated disease at University Hospital Basel including molecular characterisation of the isolates Eur. J. Clin. Microbiol. Infect. Dis. 27: ) Tenovor, F. C., R. D. Arbeit, R. V. Goering, et al Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: Criteria for bacterial strain typing. J. Clin. Microbiol. 33: ) Kato, H., N. Kato, K. Watanabe, et al Analysis of Clostridium di$cile isolates from nosocomial outbreaks at three hospitals in diverse areas of Japan. J. Clin. Microbiol. 39: ) Sawabe, E., H. Kato, K. Osawa, et al Molecular analysis of Clostridium di$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): ) Bidet, P., V. Lalande, B. Salauze, et al Comparison of PCR-ribotyping, arbiratory primed PCR, and pulsed-field gel electrophoresis for typing Clostridium di$cile. J. Clin. Microbial. 38: ) Alonso, R., A. Martin, T. P. Martin, et al An improved protocol for pulsed-field gel electrophoresis typing of Clostridium di$cile. J. Med. Microbiol. 54: ) Clostridium di$cile 12: ) Gumerlock, P. H., Y. J. Tang, F. J. Meyers, et al Use of the polymerase chain reaction for the specific and direct detection of Clostridium di$cile in human feces. Rev. Infect. Dis. 13: ) Kato, N., C.Y. Ou, H. Kato, et al Identification of toxigenic Clostridium di$cile by the polymerase chain reaction: J. Clin. Microbiol. 29: ) Kato, H., N. Kato, K. Watanabe, et al Identification of toxin A-negative, toxin B- positive Clostridium di$cile by PCR. J. Clin. Microbiol. 36: ) Kato, H., N. Kato, S. Katow, et al Detections in the repeating sequences of toxin A gene of toxin A-negative, toxin B-positive Clostridium di$cile strains. FEMS Microbiol. Lett. 175: ) Corkill, J. E., R. Graham, C. A. Hart Pulsed-field gel electrophoresis of degradationsensiteve DNAs from Clostridium di$cile PCR ribotype 1 strains. J. Clin. Microbiol. 38: ) Corne, H., W. Klaassen, A. Hanneke, et al Molecular fingerprinting of Clostridium di$cile isolates: Pulsed-field gel electrophoresis versus amplified fragment length polymorphism. J. Clin. Microbiol. 40: ) Killgore, G., T. Angela, S. Jhonson, et al Comparison of seven techniques for typing international epidemic strains of Clostridium di$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: ) toxin A toxin B Clostridium di$cile 78: ) Fawley, W. N., P. Paunell, P. Verity, et al Vol. 19 No

8 212 Molecular epidemiology of Clostridium di$cile infection and the significance of subtypes of the United Kingdom epidemic strain (PCR Ribotype 1). J. Clin. Microbiol. 43: Analysis of Clostridium di$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