Raya Raul R, Oot Rebecca A, Moore-Maley Ben, Wieland Serena, Callaway Todd R, Kutter Elizabeth M, Brabban Andrew D
The Evergreen State College; Olympia, WA USA.
Bacteriophage. 2011 Jan;1(1):15-24. doi: 10.4161/bact.1.1.14175.
In preparing sheep for an in vivo Escherichia coli O157:H7 eradication trial, we found that 20/39 members of a single flock were naturally colonized by O157:H7-infecting phages. Characterization showed these were all one phage type (subsequently named CEV2) infecting 15/16 O157:H7, 7/72 ECOR and common lab strains. Further characterization by PFGE (genome∼120 kb), restriction enzyme digest (DNA appears unmodified), receptor studies (FhuA but not TonB is required for infection) and sequencing (>95% nucleotide identity) showed it is a close relative of the classically studied coliphage T5. Unlike T5, CEV2 infects O157:H7 in vitro, both aerobically and anaerobically, rapidly adsorbing and killing, but resistant mutants regrew within 24 h. When used together with T4-like CEV1 (MOI ∼2 per phage), bacterial killing was longer lasting. CEV2 did not reproduce when co-infecting the same cell as CEV1, presumably succumbing to CEV1's ability to shut off transcription of cytosine-containing DNA. In vivo sheep trials to remove resident O157:H7 showed that a cocktail of CEV2 and CEV1 (∼10(11) total PFU) applied once orally was more effective (>99.9% reduction) than CEV1 alone (∼99%) compared to the untreated phage-free control. Those sheep naturally carrying CEV2, receiving no additional phage treatment, had the lowest O157:H7 levels (∼99.99% reduction). These data suggest that phage cocktails are more effective than individual phage in removing O157:H7 that have taken residence if the phage work in concert with one another and that naturally resident O157:H7-infecting phages may prevent O157:H7 gut colonization and be one explanation for the transient O157:H7 colonization in ruminants.
在为一项体内清除大肠杆菌O157:H7的试验准备绵羊时，我们发现一个单一群落的39只绵羊中有20只被感染O157:H7的噬菌体自然定植。特征分析表明，这些噬菌体均为同一噬菌体类型（随后命名为CEV2），可感染15/16的O157:H7、7/72的大肠杆菌参考菌株（ECOR）以及常见实验室菌株。通过脉冲场凝胶电泳（PFGE，基因组约120 kb）、限制性内切酶消化（DNA似乎未被修饰）、受体研究（感染需要FhuA而非TonB）和测序（核苷酸同一性>95%）进一步分析表明，它是经典研究的大肠杆菌噬菌体T5的近亲。与T5不同，CEV2在体外需氧和厌氧条件下均能感染O157:H7，能迅速吸附并杀死细菌，但抗性突变体在24小时内会重新生长。当与T4样的CEV1一起使用时（每个噬菌体的感染复数约为2），细菌杀灭持续时间更长。当CEV2与CEV1共同感染同一细胞时，CEV2无法繁殖，推测是因为CEV1具有关闭含胞嘧啶DNA转录的能力。体内绵羊试验以清除定植的O157:H7，结果显示，与未处理的无噬菌体对照组相比，口服一次CEV2和CEV1的混合制剂（总计约10¹¹ 噬菌斑形成单位）比单独使用CEV1更有效（减少>99.9%），单独使用CEV1时减少约99%。那些自然携带CEV2且未接受额外噬菌体处理的绵羊，其O157:H7水平最低（减少约99.99%）。这些数据表明，如果噬菌体协同作用，噬菌体混合制剂在清除已定植的O157:H7方面比单个噬菌体更有效，并且自然定植的感染O157:H7的噬菌体可能会阻止O157:H7在肠道定植，这可能是反刍动物中O157:H7短暂定植的一个原因。
