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。每年造成9300万人感染，其中约27%是由于食用受污染的猪肉造成的。沙门氏菌也是猪的一种病原体，每年给欧盟养猪业造成约6亿欧元的损失。多药耐药(MDR)沙门氏菌菌株越来越多地导致猪的感染，并与人类更高的发病率和死亡率1相关。人们迫切需要抗生素的替代方法。一种这样的方法是使用捕食性细菌--细小芽孢杆菌，该细菌捕食包括沙门氏菌在内的一系列革兰氏阴性细菌。我们先前证明，蜈蚣弧菌可以显著减少鸡盲肠内的肠炎沙门氏菌；伴随着临床症状的改善2。随后，蜈蚣弧菌被应用于治疗由耶尔西尼亚3、志贺氏菌4、克雷伯氏菌5和弧菌6引起的感染。蜈蚣弧菌比抗生素有优势：它们是自我复制和自我限制的；只在敏感细菌中复制；抗药性是与表型可塑性有关的短暂事件7。在治疗试验中，蜈蚣弧菌不会对动物造成伤害，最坏的情况是会引起轻微的暂时炎症。蜈蚣弧菌至少暂时存在于动物和人类的肠道10，11；我们有证据表明，可以从猪的肠道中分离出蜈蚣弧菌。该项目的目的是通过从猪的肠道中分离出蜈蚣弧菌，根据捕食者的喜好确定这些细菌的特征，并在不同猪肠室的体外模型中确定它们对沙门氏菌的存活和捕食效率，从而在猪的治疗上应用蜈蚣弧菌。此外，通过测试一系列微胶囊和纳米胶囊技术，将优化蛭弧菌进入肠道的传输。主要目标：1.从不同肠道隔间的猪肠道样本(从商业和现场屠宰场获得)中分离蛭弧菌。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.在体外肠道模型上测试蜈蚣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，包括微囊化和纳米胶囊化，以优化蛭弧菌的输送。通过元基因组学分析来确定蜈蚣弧菌在治疗前后对猪肠道微生物区系的影响。这个项目将拓宽我们对蜈蚣弧菌在哺乳动物肠道的定植能力的了解，并在复杂的环境中影响目标致病物种。这将使我们能够进一步探索蜈蚣弧菌对猪和Helpus肠道微生物群的影响，以优化用于活体治疗试验(不在本项目范围内)的蛭弧菌制剂。鉴于多重耐药沙门氏菌在猪和人中的存在日益增加，该项目可能导致一种新的替代疗法来治疗这些感染，这些感染可能是传统抗生素化疗的顽固性。参考1.Parisi，食源性致病菌。(2018年).doi：10.1089/fpd.2017.2403；2.Atterbury，应用环境微生物(2011年)77,5794-5803；3.Russo，微生物(2018年).doi：10.3390/Micro有机体7010002；4.威利斯，Curr.比奥尔。(2016年).doi：10.1016/j.cu.2016.09.067；5.Shatzkes，mBio(2016年).doi：10.1128/mBio.01847-16；6.Li，Int.J.食品微生物。(2011年).doi：10.1016/j.ijfoodmicro.2011.07.036；7.谢梅什，环境。微生物。(2004)。DOI：10.1046/j.1462-2920.2003.00530.x；8.韦斯特加德，应用环境微生物学(1977年)34,506-511；9.沙兹克斯，科学。众议员(2015)。DOI：10.1038/sref12899；10.Schwudke，系统。APPL微生物。(2001年).doi：10.1078/0723-2020年-00042；11.Iebba，PLoS One(2013年).doi：10.1371/Joural.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.