164 华 北 农 学 报 27 卷 8 3 ETEC K88 SDS 56 PK88-4NaCl 25 24 1 2 70% 1 1. 1 K88 1. 6 CVCC1527 C83705 O60 K88ac 1. 6. 1 Multiplicity of infection CVCC192 C8

华 北 农 学 报 2 012,2 7 ( 4 ) : 1 6 3-167 K88 1 1 2 1 1 2 1. 210014 2. 210095 ETEC K88 K88 PK88-4 DNA 60 kb 60 ph 5 ~ 10 0. 01 10 min 40 min 40 5 h 100% PK88-4 K88 K88 K88 S435 A 1000-7091 2012 04-0163 - 06 Isolation and Characterization of a Lytic Bacteriophage of Enterotoxigenic Escherichia coli K88 WANG Ran 1 HAN Han 1 2 ZHANG Hui 1 BAO Hong-duo 1 WANG Tian 2 1. Institute of Food Safety Jiangsu Academy of Agriculture Sciences Key Lab of Food Safety Monitoring and Management Ministry of Agriculture Nanjing Nanjing Agricultural University Nanjing 210014 China 2. College of Animal Science and Technology 210095 China Abstract To isolate and characterize the lytic bacteriophage that might be used in prevention and treatment of porcine postweaning diarrhea due to enterotoxigenic E. coli K88 ETEC-K88. E. coli expressing the K88 adhesin on their surface is a common cause of diarrhea in newborn and weaned piglets. Mixtures of 3 strains of ETEC K88 were used as hosts for isolation of phages in sewage from 22 pig farms. The isolated phage was characterized at the microbiological and molecular levels. The optimal multiplicity of infection MOI one-step growth curve Thermoand ph stability lytic effective in vitro of the isolated bacteriophage were investigated. One phages that lysed ETEC K88 was isolated by double-layer agar plate method and named PK88-4. The phage produced large clear plaques. The electron microscope showed the phage had necks and contractile tails and therefore belonged to the Myoviridae. The estimated genome size was about 60 kb and the restriction enzyme fragments suggested that it was dsdna. The optimal MOI was 0. 01. The phage was stable at ph 5-10 and under 60 and could kill 100% host strain within 2 hours with MOI higher than 0. 001. The new isolated phage named PK88-4 are potential candidates for prophylaxis and therapy of porcine post-weaning diarrhea due to ETEC K88. Key words Enterotoxigenic E. coli K88 Lytic bacteriophage Biological properties Lytic activities ETEC 1 35% ~ 40% 2011-05 - 08 CX 11 2069 BK2012788 1973-1958 -

164 华 北 农 学 报 27 卷 8 3 ETEC K88 SDS 56 PK88-4NaCl 25 24 1 2 70% 1 1. 1 K88 1. 6 CVCC1527 C83705 O60 K88ac 1. 6. 1 Multiplicity of infection CVCC192 C83549 O149 K81. K88ac CVCC1524 C83698 O8 K91 K88 22 ETEC K88 0. 000 1 0. 001 0. 01 0. 1 10 100 37 1. 2 LB 9 PEG8000 Amresco 5 h 3 2 DNase Ⅰ RNaseA Sigma K 1. 6. 2 12 Amresco HindⅢ TOYOBO ETEC K88 MOI = 10 1. 3 LB 2 LB 10 37 0 0. 22 μm 10 ml 10 min 10 ml 2 LB 1 ml K88 CaCl 2 1 mmol /ml 37 151 r /min 12 h 4 10 000 g 10 min 0. 22 μm 2 - ETEC 3 LB 37 50% ETEC 4 ETEC K88 6 ~ 8 h 5 0. 9 ml SM EP 1 h 4 19 6 0. 1 ml K88 2 7 7 ~ 8 1. 4 9 10 min Bacteriophage 1 2% PTA ph 7. 0 10 min 1. 5 9 DNaseI RNaseA 37 30 min K Hind Ⅲ MOI MOI 11 37 10 min 13 000 g 30 s 3 2 1. 6. 3 3 10 10 PFU /ml 40 50 60 70 0. 1 ml 80 30 ~ 60 min 0. 1 ml 3. 5 ml 50 0. 6% LB 3 2

4 期 王 冉 等 : 大 肠 杆 菌 K88 噬 菌 体 的 分 离 鉴 定 及 其 生 物 学 特 性 165 1. 6. 4 3 10 10 PFU /ml ph 2 3 3 DNase Ⅰ RnaseA Mung 4 5 6 7 8 9 10 11 12 37 Bean Nuclease PK88-4 2 h RnaseA Mung Bean Nuclease 3 2 1. 7 1. 7. 1 ETEC K88 60 kb CVCC1527 OD 600 0. 5 2. 5 10 8 CFU /ml MOI 0. 000 1 0. 00 1 0. 01 0. 1 1 37 30 min OD 600 1. 7. 2 ETEC K88CVCC1527 MOI 2 PK88-4 0. 000 1 0. 01 0 37 Fig. 2 Transmission electron micrograph 0 5 h 50 μg /ml morphology of PK88-4 Bar = 100 nm AMP SPSS 13 version 2 2. 1 3 K88 5 ~ 6 1 1 PK88-4 Hind Ⅲ Marker 0. 7% DNaseⅠ PK88-4 DNA PK88-4 1 2 PK-88-4 HindⅢ M1 M2 2 kb + 15 kb Marker 10 kb Marker Lane 1 and 2 are the banding patters of phage. PK88-4 DNA digested with HindⅢ Lane M1 and M2 are 2 kb + 15 kb ladder marker and 1 kb ladder marker respectively. with Hind Ⅲ 1 PK88-4 2. 3 Fig. 1 Plaques of PK88-4 2 PK88-4 MOI = 0. 01 PK88-4 L 64 nm W 66 4. 2 10 10 PFU /ml PK88-4 nm L /W 1 100 nm 18 nm 0. 01 1 PK88-4 A1 IC- TV 8 PK88-4 2. 2 PK88-4 CsCl 10 min 40 min 40 4 3 Fig. 3 PK88-4 Electrophoresis of phage genome digested

166 华 北 农 学 报 27 卷 Tab. 1 1 Determination of optimal multiplicity of infection MOI MOIPFU / CFU TiterPFU / ml 100 2. 5 10 7 10 8. 8 10 8 1 1. 2 10 9 0. 1 3. 8 10 10 0. 01 4. 2 10 10 0. 001 1. 3 10 10 0. 000 1 7. 7 10 9 Fig. 4 4 One step growth curve 2. 4 PK88-4 40 50 60 min60 30 min 60 60 min 10 8 PFU /ml 70 PK88-4 80 30 min PK88-4 PK88-4 60 5 PK88-4 ph 5 ~ 10 ph = 2 12 PK88-4 6 2. 6 MOI = 0 OD 600 MOI = 0. 01 0. 001 0. 000 1 OD 600 OD 600 MOI = 1 0. 1 MOI 5 h OD OD 600 < 0. 1 7 Fig. 7 7 PK88-4 Lytic kinetics of PK88-4 Fig. 5 5 PK88-4 Resistance of the PK88-4 to temperature Fig. 6 6 PK88-4 Resistance of the PK88-4 to acidity and alkalinity CFU /ml PK88-4 0 h 5 h ETEC K88 P = 0. 05 The bacterial concentrations are expressed as the number of CFU /ml of culture. The speckled and barrier columns represent concentrations of the ETEC K88 at 0 h and 5 h after the administrations of PK88-4 respectively. The standard error are indicated with brackets. Different letters indicate significant differences at P = 0. 05. Fig. 8 8 PK88-4 Bactericidal effects of phage PK88-4 in vitro

4 期 王 冉 等 : 大 肠 杆 菌 K88 噬 菌 体 的 分 离 鉴 定 及 其 生 物 学 特 性 167 2. 7 PK88-4 2. 5 10 8 CFU /ml 5 h MOI = 0. 000 1 5 logmoi = 1 0. 01 K88 BK2012788 100% MOI CX 11 2069 MOI P < 0. 05 10 6 PFU /ml 50 μg /ml AMP P > 0. 05 K88 3 1 Nagy B Fekete P Z. Enterotoxigenic Escherichia coli in veterinary medicine J. International Journal of Medical 3. 1 K88 Microbiology 2005 295 6-7 443-454. 2. J. 1987 6 25-29. 3. C. 1992 115-122. 4 Payne D Moore N Lambert P. Use of an enzyme-linked immunosorbent assay ELISA to measure the expression of the K88 fimbrial antigen by enterotoxigenic Escherichia coli ETEC J. Journal of Immunological Methods 1993 159 1-2 283-289. 5 Fairbrother J M Nadeau E Gyles C L. Escherichia coli in K88 postweaning diarrhea in pigs an update on bacterial types pathogenesis and prevention strategies J. Animal PK88-4 Health Research Reviews / Conference of Research Worker K88 in Animal Disease 2005 6 1 17-39. 15 6. 95 K88 BpD J. Jamalludeen 10 0149 GJ1 2008 39 4 472-477. 7 Amezcua R Friendship R M Dewey C E et al. Presentation of postweaning Escherichia coli diarrhea in southern GJ9 PK88-4 A1 BpD 15 GJ1 GJ9 10 Ontario prevalence of hemolytic E. coli serogroups involved and their antimicrobial resistance patterns J. Canadian Journal of Veterinary Research 2008 66 73-78. A3 PK88-4 60 kb K88 BpD 100 kb GJ1 8 Sulakvelid A Alavidze Z Morris J G. Bacteriophage Therapy J. Antimicrobial Agents and Chemotherapy 2001 GJ9 40 ~ 50 kb 45 3 649-659. PK88-4 K88 9 Sambrook J Russell D W. M.. 3. 2002 27-30. 10 Jamalludeen N Johnson R P et al. Isolation and characterization of nine bacteriophages that lyse O149 entero- 3. 2 toxigenic Escherichia coli J. Veterinary Microbiology 2007 124 1-2 47-57. 11. J. 2009 4 2 88-90. 12 Lu Z Breidt F J R Fleming H P et al. Isolation and characterization of a Lactobacillus plantarum bacteriophage ΦJL-1 from a cucumber fermentation J. International Journal of Food Microbiology 2003 84 2 225-235. 13. 1 f2 30 ~ 35 min 13 J. Ecp65 23 min 11 Bp6 25 ~ 27 2003 25 23 2106-2108. 14. min 14 BpD 30 min 15 MVSS MVBS J. 2008 44 4 10-11. 21 min 27 min 16 15. K88 D. 2009. PK88-4 10 min 16 Viscardi M Perugini A G Auriemma C et al. Isolation and characterisation of two novel coliphages with high potential to control antibiotic-resistant pathogenic Escherichia coli EHEC and EPEC J. International Journal of Antimicrobial Agents 2008 31 152-157. PK88-4 1 10 000 K88 5 h 100% 8