Developing the predatory bacterium Bdellovibrio bacteriovorus as a novel therapy for treating canine otitis externa

犬中耳炎是一种细菌感染，通常由多重耐药铜绿假单胞菌1引起。严重的病例可能导致唯一剩下的治疗选择是手术2，3，这可能导致耳聋（R。白色persons）。因此，迫切需要新的抗菌治疗方法。噬菌体，即杀死细菌的病毒，是抗生素的相对充分研究的替代品，然而细菌对噬菌体的耐药性很容易出现。其他不会导致病原体产生这种遗传编码耐药性的“活”抗菌药物可能是更好的选择。捕食性细菌噬菌蛭弧菌可能就是这样一种方法。噬菌蛭弧菌通过捕食敏感的革兰氏阴性菌而生长，包括许多耐药病原体，包括假单胞菌属、E.大肠杆菌和弧菌等。我们先前已经表明，蛭弧菌可以有效地降低沙门氏菌在鸡4中的定殖水平，并伴随着临床症状的改善;随后是涉及志贺氏菌5、克雷伯氏菌6、弧菌7和耶尔森氏菌8的体内感染模型。蛭弧菌既能自我复制又能自我限制，这两种优势都超过了传统的抗生素。在动物试验中，蛭弧菌不会造成伤害，最坏的情况是引起暂时的轻微炎症。蛭弧菌已被证明能有效地杀死生长在生物膜中的猎物10，因此它有可能用于对抗局部感染，如外耳感染。该项目的目的是确定蛭弧菌疗法治疗由铜绿假单胞菌引起的犬外耳炎的潜在功效，并研究涉及蛭弧菌和噬菌体的组合捕食者疗法的附加潜力，建立在我们最近发表的一些工作11上。主要目标：一曰：定量蛭弧菌在纯培养物和混合培养物中杀死来自犬中耳炎病例的体外铜绿假单胞菌分离株的捕食能力，所述混合培养物含有从相同临床样品共分离的其他细菌物种。2：建立在以前的数据表明，杀死E。通过蛭弧菌属与噬菌体的组合对大肠杆菌的感染可以导致在体外测定中完全根除猎物，以观察蛭弧菌属与a）假单胞菌属噬菌体对假单胞菌属纯培养物的组合效果，或B）靶向第二细菌物种的噬菌体对假单胞菌属和第二物种的混合培养物的组合效果。3：评估蛭弧菌捕食在上皮细胞存在下形成生物膜结构的假单胞菌细胞的功效。使用已发表的基于组织培养的方法，其中铜绿假单胞菌在上皮细胞单层上形成生物膜12，13，将研究在细菌猎物和上皮细胞存在下的蛭弧菌捕食，例如在治疗期间会遇到的。这些目标将揭示蛭弧菌的潜在功效，以及在复杂的多物种细菌群体中使用噬菌体的组合疗法。它将告知我们在未来的体内治疗试验中采取的最佳方法（超出本项目的范围）。由于铜绿假单胞菌是动物和人类的病原体，该项目可能会导致各种感染的新的治疗方法。

Canine otitis media is a bacterial infection commonly caused by multi-drug resistant Pseudomonas aeruginosa1. Severe cases can result in the only remaining therapeutic option being surgery2,3, which may result in deafness (R. White pers comm). As such, novel approaches to antibacterial therapies are desperately needed. Bacteriophage, viruses that kill bacteria, are relatively well-studied alternatives to antibiotics, however resistance of the bacteria to bacteriophage can arise readily. Other 'living' antibacterials that do not result in such genetically-encoded resistance by the pathogen may represent a better alternative. The predatory bacterium Bdellovibrio bacteriovorus may be one such approach. Bdellovibrio bacteriovorus grows by preying upon susceptible Gram-negative bacteria, including many antibiotic-resistant pathogens, including Pseudomonas spp, E. coli, and Vibrio spp amongst others. We have previously shown that Bdellovibrio can effectively reduce the colonisation levels of Salmonella in chicks4, accompanied by an improvement in clinical symptoms; this has been followed by in vivo infection models involving Shigella5, Klebsiella6, Vibrio7 and Yersinia8 spp. Bdellovibrio are both self-replicating and self-limiting, both advantages over conventional antibiotics. In animal trials, Bdellovibrio does not cause harm8, at worst eliciting a temporary mild inflammation9. Bdellovibrio has been shown to efficiently kill prey growing in biofilms10, and as such it has potential for use against topical infections, such as external ear infections. The aim of this project is to determine the potential efficacy of Bdellovibrio therapy for the treatment of canine otitis externa caused by Pseudomonas aeruginosa, and to study the added potential of combinatorial predator therapy involving both Bdellovibrio and bacteriophage, building upon some of our recently published work11. Main objectives: 1: Quantify the predatory ability of Bdellovibrio to kill in vitro Pseudomonas aeruginosa isolates from cases of canine otitis media, both in pure and mixed cultures, containing other bacterial species co-isolated from the same clinical samples. 2: To build on previous data showing that killing of E. coli by the combination of a Bdellovibrio with a bacteriophage could result in complete eradication of prey in an in vitro assay, to look at the combinatorial effects of Bdellovibrio and either a) a Pseudomonas phage against a pure culture of Pseudomonas, or b) a phage targeting a second bacterial species, against a mixed culture of Pseudomonas and a second species. 3: Assess the efficacy of Bdellovibrio predation on Pseudomonas cells forming biofilm structures in the presence of epithelial cells. Using a published tissue culture-based approach whereby P. aeruginosa forms biofilms on monolayers of epithelial cells12,13, Bdellovibrio predation in the presence of both bacterial prey and epithelial cells will be studied, such as would be encountered during therapy. Together these objectives will reveal the potential efficacy of Bdellovibrio, and combinatorial therapies with bacteriophage in complex, multi-species populations of bacteria. It will inform us as to the best approaches to take in future in vivo therapeutic trials (outside the scope of this project). As Pseudomonas aeruginosa is both an animal and human pathogen, this project may lead to new therapeutic approaches for a variety of infections.