College of Animal Science and Veterinary Medicine, Jilin University, Changchun, People's Republic of China.
PLoS One. 2012;7(3):e31698. doi: 10.1371/journal.pone.0031698. Epub 2012 Mar 1.
Bacteriophage could be an alternative to conventional antibiotic therapy against multidrug-resistant bacteria. However, the emergence of resistant variants after phage treatment limited its therapeutic application.
METHODOLOGY/PRINCIPAL FINDINGS: In this study, an approach, named "Step-by-Step" (SBS), has been established. This method takes advantage of the occurrence of phage-resistant bacteria variants and ensures that phages lytic for wild-type strain and its phage-resistant variants are selected. A phage cocktail lytic for Klebsiella pneumoniae was established by the SBS method. This phage cocktail consisted of three phages (GH-K1, GH-K2 and GH-K3) which have different but overlapping host strains. Several phage-resistant variants of Klebsiella pneumoniae were isolated after different phages treatments. The virulence of these variants was much weaker [minimal lethal doses (MLD)>1.3×10(9) cfu/mouse] than that of wild-type K7 countpart (MLD = 2.5×10(3) cfu/mouse). Compared with any single phage, the phage cocktail significantly reduced the mutation frequency of Klebsiella pneumoniae and effectively rescued Klebsiella pneumoniae bacteremia in a murine K7 strain challenge model. The minimal protective dose (MPD) of the phage cocktail which was sufficient to protect bacteremic mice from lethal K7 infection was only 3.0×10(4) pfu, significantly smaller (p<0.01) than that of single monophage. Moreover, a delayed administration of this phage cocktail was still effective in protection against K7 challenge.
CONCLUSIONS/SIGNIFICANCE: Our data showed that the phage cocktail was more effective in reducing bacterial mutation frequency and in the rescue of murine bacteremia than monophage suggesting that phage cocktail established by SBS method has great therapeutic potential for multidrug-resistant bacteria infection.
噬菌体可以替代传统的抗生素疗法来治疗多药耐药菌。然而，噬菌体治疗后耐药变体的出现限制了其治疗应用。
方法/主要发现：在本研究中，建立了一种名为“逐步（SBS）”的方法。该方法利用噬菌体耐药细菌变体的出现，并确保选择裂解野生型菌株及其噬菌体耐药变体的噬菌体。通过 SBS 方法建立了裂解肺炎克雷伯菌的噬菌体鸡尾酒。这种噬菌体鸡尾酒由三种噬菌体（GH-K1、GH-K2 和 GH-K3）组成，它们具有不同但重叠的宿主菌株。在不同噬菌体处理后，分离出几种肺炎克雷伯菌的噬菌体耐药变体。这些变体的毒力明显较弱[最小致死剂量（MLD）>1.3×10（9）cfu/小鼠]，低于野生型 K7 对照（MLD=2.5×10（3）cfu/小鼠）。与任何单一噬菌体相比，噬菌体鸡尾酒显著降低了肺炎克雷伯菌的突变频率，并在 K7 株小鼠挑战模型中有效挽救了肺炎克雷伯菌菌血症。足以保护菌血症小鼠免受致命 K7 感染的噬菌体鸡尾酒最小保护剂量（MPD）仅为 3.0×10（4）pfu，明显小于（p<0.01）单一单噬菌体。此外，延迟给予这种噬菌体鸡尾酒仍然有效保护免受 K7 挑战。
结论/意义：我们的数据表明，噬菌体鸡尾酒在降低细菌突变频率和挽救小鼠菌血症方面比单噬菌体更有效，这表明通过 SBS 方法建立的噬菌体鸡尾酒对多药耐药菌感染具有很大的治疗潜力。
