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引起。严重的病例可能导致剩下的唯一治疗选择是手术，这可能导致耳聋（R. White pers comm）。因此，迫切需要新的抗菌治疗方法。噬菌体是一种杀死细菌的病毒，相对来说，它是抗生素的替代品，但细菌对噬菌体的耐药性很容易产生。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。掠食性细菌Bdellovibrio bacteriovorus可能就是这样一种途径。弓形杆菌弧菌以敏感的革兰氏阴性菌为食，包括许多耐抗生素病原体，包括假单胞菌、大肠杆菌和弧菌等。我们之前的研究表明，蛭弧菌可以有效地降低鸡体内沙门氏菌的定植水平，并伴有临床症状的改善；随后又建立了包括志贺氏菌、克雷伯氏菌、弧菌和耶尔森氏菌在内的体内感染模型。蛭形弧菌具有自我复制和自我限制的特性，两者都优于传统抗生素。在动物试验中，蛭形弧菌不会造成伤害，最坏的情况是引起暂时的轻度炎症。蛭弧菌已被证明能有效地杀死生长在生物膜中的猎物，因此它有可能用于治疗局部感染，如外耳感染。这个项目的目的是确定Bdellovibrio治疗由铜绿假单胞菌引起的犬外耳炎的潜在疗效，并在我们最近发表的一些工作的基础上，研究Bdellovibrio和噬菌体联合捕食者治疗的附加潜力。主要目标：1：量化从犬中耳炎病例中分离的铜绿假单胞菌的Bdellovibrio体外捕食能力，包括纯培养和混合培养，含有从相同临床样本中分离的其他细菌物种。2：以先前的数据为基础，在体外实验中，通过将Bdellovibrio与噬菌体结合杀死大肠杆菌可以导致完全根除猎物，研究Bdellovibrio和a)假单胞菌噬菌体对抗纯假单胞菌培养物，或b)针对第二种细菌的噬菌体对抗假单胞菌和第二种细菌的混合培养物的组合效应。3：评估在上皮细胞存在的情况下，蛭弧菌捕食假单胞菌细胞形成生物膜结构的效果。利用已发表的基于组织培养的方法，铜绿假单胞菌在上皮细胞单层上形成生物膜12,13，将研究蛭弧菌在细菌猎物和上皮细胞存在下的捕食行为，例如在治疗过程中会遇到的情况。总之，这些目标将揭示蛭弧菌的潜在功效，以及噬菌体在复杂的多物种细菌群体中的联合治疗。它将告诉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一种动物和人类病原体，该项目可能为各种感染的治疗带来新的方法。

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.