Investigating the use of Bdellovibrio bacteriovorus as a 'living antibiotic' to control Salmonella in pigs.

在全球范围内，非伤寒沙门氏菌属。每年造成9300万人感染，约27%是由于食用受污染的猪肉。沙门氏菌也是猪的病原体，每年给欧盟养猪业造成约6亿欧元的损失。沙门氏菌的多药耐药（MDR）菌株越来越多地导致猪的感染，并与人类的较高发病率和死亡率相关1。迫切需要抗生素的替代方法。其中一种方法是使用捕食性细菌噬菌蛭弧菌。蛭弧菌捕食一系列革兰氏阴性细菌病原体，包括沙门氏菌。我们以前证明，蛭弧菌可以显着减少鸡盲肠中的沙门氏菌肠炎;伴随着临床症状的改善2。随后，蛭弧菌已被应用于治疗由耶尔森氏菌3、志贺氏菌4、克雷伯氏菌5和弧菌6引起的感染。蛭弧菌比抗生素具有优势：它们是自我复制和自我限制的;仅在易感细菌内复制;抗性是与表型可塑性相关的短暂事件7。蛭弧菌在治疗试验期间不会伤害动物8，最坏的情况是引起轻微和暂时的炎症9。蛭弧菌存在于动物和人类的肠道中，至少是短暂的10，11;我们有证据表明，蛭弧菌可以从猪的肠道中分离出来。该项目的目的是将蛭弧菌应用于猪的治疗：通过从猪的肠道中分离蛭弧菌，表征这些细菌对猎物的偏好，并确定它们在不同猪肠道隔室的离体模型中对沙门氏菌的存活和捕食效率。此外，将通过测试一系列微胶囊和纳米胶囊技术来优化蛭弧菌进入肠道的递送。主要目的：1.从猪肠样品的不同肠道隔室分离蛭弧菌（从商业和现场屠宰场获得）。2.蛭弧菌分离株的遗传和表型分析，包括对一系列猪源沙门氏菌的捕食和捕食效率3.在离体肠道模型中测试蛭弧菌对沙门氏菌的捕食作用。同时分析了蛭弧菌的制备方法，包括微胶囊化和纳米胶囊化，以优化蛭弧菌的递送.通过宏基因组学分析蛭弧菌处理前后对猪肠道菌群的影响，将拓宽我们对蛭弧菌在哺乳动物肠道定植能力的认识，以及在复杂环境中对目标致病菌的影响。这将使我们能够进一步探索蛭弧菌对猪肠道微生物群的影响，并有助于优化蛭弧菌制剂用于体内治疗试验（超出本项目范围）。鉴于猪和人中多重耐药沙门氏菌的存在日益增加，该项目可能导致对这种感染的新的替代治疗，其可能是常规抗生素化疗不可替代的。Dis.（2018）.doi：10.1089/fpd.2017.2403; 2.Atterbury，Appl Env Microbiol（2011）77，5794 -5803; 3.Russo，Microorganisms 2018）.doi：10.3390/microorganisms7010002; 4.Willis，Curr.（2016）.doi：10.1016/j.cub.2016.09.067; 5.Shatzkes，MBio（2016）.doi：10.1128/mBio.01847-16; 6.Li，Int. J. Food Microbiol.（2011）.doi：10.1016/j.ijfoodmicro.2011.07.036; 7. Shemesh，Environ. Microbiol.（2004年）。doi：10.1046/j.1462-2920.2003.00530.x; 8.Westergaard，Appl Env Microbiol（1977）34，506 - 511; 9.Shatzkes，Sci. Rep.（2015）. doi：10.1038/srep12899; 10.Schwudke，Syst.Appl.Microbiol.（2001）.doi：10.1078/0723-2020-00042; 11.Iebba，PLoS One（2013）.doi：10.1371/journal.pone.0061608.

Globally, non-typhoidal Salmonella spp. is responsible for 93 million human infections perannum, ~27% due to consuming contaminated pork. Salmonella is also a pathogen of pigs, and costs the EU pig industry ~Euro 600 million per annum. Multidrug resistant (MDR) strains of Salmonella are increasingly responsible for infections in pigs, and are associated with higher morbidity and mortality in humans1. Alternative approaches to antibiotics are desperately needed. One such approach is the use of the predatory bacterium Bdellovibrio bacteriovorus.Bdellovibrio preys upon a range of Gramnegative bacterial pathogensincluding Salmonella spp.. We previously demonstrated that Bdellovibrio can significantly reduce Salmonella Enteritidis in the caeca of chickens; accompanied by improvements in clinical symptoms2. Subsequently, Bdellovibrio has been applied to treat infections caused by Yersinia3, Shigella4, Klebsiella5 and Vibrio6. Bdellovibrio has advantages over antibiotics:they are self replicating and self-limiting; replicating only within susceptible bacteria; and resistance is a transient event linked to phenotypic plasticity7. Bdellovibrio does not harm animals during therapeutic trials8, at worst eliciting a mild and temporary inflammation9. Bdellovibrio is present, at least transiently, in the intestinal tracts of animals and humans10,11; we have evidence that Bdellovibrio can be isolated from the intestinal tract of pigs. The aim of this project is to move towards applying Bdellovibrio therapeutically in pigs: by isolating Bdellovibrio from the intestine of pigs, characterising these bacteria with respect to prey preference and determining their survival and predatory efficiency against Salmonella in ex-vivo models of different pig intestinal compartments. In addition, the delivery of Bdellovibrio into the intestinal tract will be optimised by testing a range of micro- and nano-encapsulation techniques.Main objectives:1. Isolation of Bdellovibrio from different gut compartments of pig intestine samples (obtained from commercial and on-site abattoirs). 2. Genetic and phenotypic analysis of Bdellovibrio isolates, including preyrange and predation efficiency on a range of porcine-derived Salmonella spp.. 3. Testing Bdellovibrio predation on Salmonella in ex-vivo gut models. Also analysis of Bdellovibrio preparation methods including micro and nano-encapsulation to optimise Bdellovibrio delivery.4. Determining the effect of Bdellovibrio on the microbiota of the pig intestine by metagenomics analyses before and after Bdellovibrio treatment.This project will broaden our knowledge about Bdellovibrio's ability to colonise themammalian intestine, and affect target pathogenic species in a complex environment. It will allow us to further explore the effect of Bdellovibrio on the intestinal microbiota of pigs and helpus to optimise Bdellovibrio preparations for use in an in vivo therapeutic trial (outside the scope of this project). Given the increasing presence of multi-drug resistant Salmonella in both pigs and humans, this project may lead to a new, alternative treatment for such infections which may be recalcitrant to conventional antibiotic chemotherapy.References1.Parisi, Foodborne Pathog. Dis.(2018).doi:10.1089/fpd.2017.2403; 2.Atterbury, Appl Env Microbiol (2011)77,5794-5803; 3.Russo, Microorganisms 2018).doi:10.3390/microorganisms7010002; 4.Willis, Curr. Biol. (2016).doi:10.1016/j.cub.2016.09.067; 5.Shatzkes, MBio (2016).doi:10.1128/mBio.01847-16; 6.Li,Int. J. Food Microbiol. (2011).doi:10.1016/j.ijfoodmicro.2011.07.036; 7.Shemesh, Environ. Microbiol. (2004). doi:10.1046/j.1462-2920.2003.00530.x; 8.Westergaard, Appl Env Microbiol (1977) 34,506- 511; 9.Shatzkes, Sci. Rep. (2015). doi:10.1038/srep12899; 10.Schwudke, Syst. Appl. Microbiol. (2001).doi:10.1078/0723-2020-00042; 11.Iebba, PLoS One (2013).doi:10.1371/journal.pone.0061608.