3 平成23年 研究論文 /研究論文2(061‐067)
Contents
1. EHEC O111 NmEC 42 O 111 0111 1 15 http www mhlw go jp stf houdou 2r98520000 01dpi2 att 2r9852000001dpm2 pdf Infectious Diseases Surveill2. EHEC 0111 0111 Table 3 NmEC STEC XM EHEC NmEC STEC mEC XM EHEC XM EHEC STEC Vi EHEC CT SBMAC CIX CT SMAC 4 LAMP
3. 4 NmEC 42 LAMP 0 917 1 000 NmEC EHEC 0157 EHEC 0O26 EHEC O111 EHEC O111 LAMP VT 1 EHEC O111 2 NmEC mEC
4. mEC EHEC O111 EHEC O111 129 2011 Auvray EHEC O111 EHEC EHEC O111 EHEC 0111 EHEC O111 EHEC O111 42C NmEC mEC
5. 42C EHEC O111 Drys dale NmEC 42 mEC 42C EHEC O111 Kanki O157 EHEC NmEC 42 modified TSB universal pre enrichment broth EHEC O111
6. 1 000 mEC 36 mEC NmEC 42 LAMP 1 000 100 EHEC O111 Blais NmEC 37C 42C 0157 H7 66 42 EHEC O111 EHEC O111 CT SBMAC CT SMAC mEC 36C STE
7. 2 Stat View Student Newman Keuls 3 3 1 25g ESC 4 14 3cfu 110512 6 2cfu LAMP 3 1 000 4 4 Table1 42C NmEC mEC 7 1 000 4 4 36C mEC EHEC O111 4 EHEC O111 CIX CT SMAC Table 1 STEC XM EHEC CT SBMAC EHEC O111 1
8. 6 0cfu 30 0cfu 0 5 24 2 41 5C 41 5 42 5C 10 cfu LAMP 0111 65 Table 3 The numbers of non E coli O111 colonies with similar morphological characteristics to EHEC O111 on medium from samples uninoculated with EHEC O111 Medium Enrichmenf Plating method CIX CHROMagar STEC XM EHEC Vi EHEC CT SMAC CT SBMAC NmEC Direct plating 25 6 6 13 25 24 IMS platng 30 12 14 22 33 24 mEC Direct plating 25 18 7 12 31 24 IMS platng 28 12 2 12 24 18 Enriched at 42C The numbers of non E coli O111 colonies but suspected as EHEC O111 LAMP NmEC mEC
9. 0 917 11 12 NmEC mEC 1 000 12 12 Table 2 NmEC mEC 1 000 12 12 NmEC 1 000 12 12 NmEC mEC 0 833 10 12 0 917 11 12 Table 2 LAMP mEC CIX CT SBMAC 0 750 9 12 0 833 10 12 NmEC mEC 1 000 12 12 NmEC mEC 0 833 1
10. 10 Ecoli O111F 0111 2 2 2 2 4 EHEC O111 ESC 4 VT 25g 3 24 O111 0111 63 4 TSB10ml 37C 18 3ml PBS27ml 50ml 1
11. 169 4 VT O157 026 O111 2010 36 2011 5 0157 0O26 EHEC O111 62
12. DNA Loopamp Loopamp LA 320C O111 1ml EHEC O111 10ul 20ul SMAO CT SMAC CT SMAC CT CT SBMAC STEC XM EHEC ViEHEC CTX 36 1 C 18 24 EHEC O111
13. 10 10 2 0 1 ml 0 5ml TSA24 0 1ml 36 1 C 24 36 1 18 20 EHEC O111 ESC 4 1 ml 10 100 0 1ml 10 10 cfu 25g mEC 225ml
14. 100 0 1ml 10 TSA Sample 25 g Inoculation level of EHEC O111 cfu 25 g Low 6 0 High 30 0 NmEC 225 ml mEC 225 ml 42 1C 22h Enrichment culture LAMP assay Direct plating IMS plating Negative Positive Agar CT SMAC CT SBMAC CIX CHROMagar STEC XM EHEC Vi EHEC 36 1 C 18 24h Presumed EHEC O111 colonies were confirmed by O111 latex aggutination test Fig 2 Schematic overview of the detection of EHEC O111 in beef sample by plating methods and LAMP method on a collaborative study NmEC mEC 3 225 ml 1 42 1 22 LAMP LAMP CT SMAC CT SBMAC CIX STEC XM EHEC ViEHEC
15. EHEC O111 EHEC O111 2 2 1 EHECO111 2 ESC4 VT1 VT2 110512 VT2 25g TSB 10ml 36C18 PBS 10 10 0 2ml Sample 25g Strain and inoculation level cfu 25 g EHEC O111 Toyama 110512 6 2 EHEC O111 ESC4 143 N
16. 11 12 1 000 12 12 1 000 12 12 1 000 12 12 Direct plating CIX 0 917 11 12 000 12 12 0 917 11 12 0 750 9 12 1 000 12 12 1 000 12 12 CHROMagar STEC 0 917 11 12 000 12 12 0 917 11 12 0 833 10 12 1 000 12 12 1 000 12 12 XM EHEC 0 833 10 12 000 12 12 0 917 1 12 0 833 10 12 1 000 12 12 1 000 12 12 ViEHEC 0 917 11 12 000 12 12 0 917 11 12 0 833 10 12 1 000 12 12 1 000 12 12 CT SMAC 0 917 11 12 000 12 12 0 917 11 12 0 833 10 12 1 000 12 12 1 000 12 12 CT SBMAC 0 917 11 12 000 12 12 0 833 10 12 0 750 9 12 1 000 12 12 1 000 12 12 IMS platmg CIX 0 917 11 12 000 12 12 0 917 11 12 0 833 10 12 1 000 12 12 1 000 12 12 CHROMagar STEC 0 917 11 12 000 12 12 0 917 11 12 0 833 10 12 1 000 12 12 1 000 12 12 XM EHEC 0 917 11 12 000 12 12 0 917 11 12 0 917 11 12 1 000 12 12 1 000 12 12 ViEHEC 0 917 11 12 000 12 12 0 917 11 12 0 917 11 12 1 000 12 12 1 000 12 12 CT SMAC 0 917 11 12 000 12 12 0 833 10 12 0 833 10 12 1 000 12 12 1 000 12 12 CT SBMAC 0 917 11 12 000 12 12 0 833 10 12 0 750 9 12 1 000 12 12 1 000 12 12 Enrichment broth temperature Low level 6 0 CFU 25g high level 30 0 CFU 25g Number of positive samples number of samples inoculated with EHEC O111 There were significant difference betwl agars at low and high inoculation levels 3 2 25g
17. 000 4 4 42C NmEC mEC 1 000 4 4 Table 1 36C mEC 4 EHEC 0111 CIX SMAC CT SMAC 1 000 4 4 CT SBMAC O 111 64 129 2011 Table 1 Sensitivity and specificity of detection methods for EHEC 0O111 on a single laboratory evaluation Method Sensitivity Specificity NmEC 42 mEC 42 C mEC 36 C NmEC 42C mEC 42C mEC 36C LAMP assay 1 00 4 4 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 Direct plating CIX 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4
18. CHROMagar STEC 00 4 4 00 4 4 0 00 0 4 1 00 4 4 1 00 4 4 1 00 4 4 XM EHEC 00 4 4 00 4 4 0 00 0 4 1 00 4 4 1 00 4 4 1 00 4 4 ViEHEC 00 4 4 00 4 4 0 25 1 4 1 00 4 4 1 00 4 4 1 00 4 4 SMAC 00 4 4 00 4 4 0 75 3 4 1 00 4 4 1 00 4 4 1 00 4 4 CT SMAC 00 4 4 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 CT SBMAC 00 4 4 00 4 4 0 00 0 4 1 00 4 4 1 00 4 4 1 00 4 4 IMS platimg CIX 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 CHROMagar STEC 00 4 4 00 4 4 0 50 2 4 1 00 4 4 1 00 4 4 1 00 4 4 XM EHEC 00 4 4 00 4 4 0 75 3 4 1 00 4 4 1 00 4 4 1 00 4 4 Vi EHEC 00 4 4 00 4 4 0 50 2 4 1 00 4 4 1 00 4 4 1 00 4 4 SMAC 00 4 4 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 CT SMAC 00 4 4 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 1 00 4 4 CT SBMAC 00 4 4 00 4 4 0 75 3 4 1 00 4 4 1 00 4 4 1 00 4 4 Inoculation level Toyama110512 strain 6 2 CFU 25g ESC4 strain 14 3 CFU 25g 5Enrichment broth temperature Number of positive samples number of samples inoculated with EHEC O111 each strain was inoculated in two samples Number of negative samples number of samples uninoculated with EHEC O111 Table 2 Sensitivity and specificity of detection methods for EHEC O111 on a collaborative study Method Sensitivity Specificity NmEC 42C mEC 42 C NmEC 42C mEC 42C Low level High level Low leve High level LAMP assay 0 917 11 12 000 12 12 0 917
19. 0 12 0 917 11 12 NmEC 1 000 12 12 Table 2 mEC CT SBMAC 0 750 9 12 0 750 9 12 0 917 11 12 NmEC mEC 1 000 12 12 Student Newman Keuls NmEC mEC LAMP mEC NmEC p 0 05 Table 2 LAMP
20. Bull Natl Inst Health Sci 129 61 67 2011 61 Original 0111 Detection methods of enterohemorrhagic Escherichia coli Olll in beef A collaborative study Midori Hiroi Kayoko Ohtsuka Shinji lizuka Kenichiro Taga Kanji Sugiyama Yoshiko Sugita Konishi Yukiko Hara Kudo To establish a detection method for enterohemorrhagic Escherichia coli EHEC O111 in meat a single laboratory evaluation and a collaborative study were conducted focusing on comparisons of the efficien cies in combination with enrichment a direct plating method and a plating method with immunomagnetic separation IMS plating method using various agar media for EHEC O111 loop mediated isothermal amplification LAMP assay targeting the Verocytotoxin VT gene as a molecular detection method On a single laboratory evaluation enrichment in modified EC at 36C was inferior to that in modified EC supplemented with novobiocin NmEC and mEC at 42C to isolate EHEC O111 by plating methods On a collaborative study there were no significant differences between combinations of enrichment in NmEC at 42C LAMP assay and enrichment in mEC at 42C LAMP assay The combinations of enrichment in NmEC at 42C direct plating and enrichment in NmEC at 42C IMS p
21. C XM EHEC ViEHEC SMAC CT SBMAC Ta ble 1 Fratamico EHEC O111 Fratamico EHEC O111 mEC 36C NmEC mEC 42 C
22. ance Center National 2 Institute Infectious Diseases Symptoms associated with EHEC infection by serotype 2010 Infect Agents Surv Rep 32 127 2011 0157 026 1102004 18 11 2 Blais B W Booth R A Phillippe L M and Yamazaki H 7 Food Microbiol 36 221 225 1997 Orth D Grit K Dierich M P and Wirzner R 7 Res Microbiol 158 105 111 2007 Fratamico P M Bagi L K Cray W C Jr Narang N Yan X Medina M and Liu Y Foodborne Pathog Dis 8 601 607 2011 Hara Kudo Y Konishi N Ohtsuka K Hiramatsu R Tanaka H Konuma H and Takatori K Int J 7oog Microbiol 122 156 161 2008 Auvray F Lecureuil C Tache J Leclerc V 3 4 5 6 7 8 Deperrois V and Lombard B Lett 4pp Micro biol 45 646 651 2007 Drysdale M MacRae M Strachan N J C Reid T M S and Ogden I D 7 Appl Microbiol 97 220 224 1997 Kanki M Seto K Harada T Yonogi S and Kumeda Y Lett Appl Microbiol 53 167 173 2011 9 10 67
23. lating were superior to combinations of enrichment in mEC at 42C direct plating and enrichment in mEC at 42C IMS plating p lt 0 05 There were no significant differences among the six different agar media by the direct plating and IMS platng methods As a result it was suggested that the following methods are adequate for detection of EHEC O111 in beef combinations of enrichment in NmEC at 42C and direct plating and IMS plating methods or LAMP assay as a screening assay to detect VT gene followed by direct platng and IMS platng methods Keywords enterohemorrhagic Escherichia co7 O111 beet LAMP assay direct plating method IMS plating method 1 23 4 O111 EHEC O111 5 27 16 To whom correspondence should be addressed Yukiko Hara Kudo Division of Microbiology National Institute of Health Sciences 1 18 1 Kamiyoga Setagaya ku Tokyo 158 8501 Japan Tel amp Fax 81 3 3 3700 9497 E mail ykudo nihs gojJp 1 Shizuoka Institute of Environment and Hygiene 2 Saitama Institute of Public Health 3 Yokohama Quarantine Station Center for Inspection of Im ported Foods amp Infectious Diseases Yokohama 4 Kobe Quarantine Station Center for Inspection of Imported Foods amp Infectious Diseases Kobe
24. mEC 225 ml mEC 225 ml 22h 42 1C 42 1C 36 1C Enrichment culture LAMP assay Direct plating IMS plating Negative Positive Agar CT SMAC SMAC CT SBMAC CHROMagar STEC XM EHEC Vi EHEC CIX 36 1 C 18 24 h Presumed EHEC O111 colonies were confirmed by O111 latex aggutination test Fig 1 Schematic overview of the detection of EHEC O111 in beef sample by plating methods and LAMP method on a single laboratory evaluation 129 2011 10 0 2ml TSA 10 36 1 C 24 2 225ml modified EC mEC mEC NmEC 1 mEC 42 1 36 1 NmEC 42 1 22 Loop mediated amplification LAMP DNA
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