A tripartite strategy for controlling Clostridioides difficile

肠道细菌可以通过多种方式交换DNA，以抵抗抗生素并导致无法治愈的感染。我们的研究旨在了解抗生素耐药基因(ARGs)如何以及何时与其他细菌交换，并找到新的治疗剂，最终应该会减少这些情况，并使感染更容易治疗。这项研究的重点是艰难梭状芽胞杆菌，这是一种重要的人类病原体，可导致高发病率和高死亡率的感染。在动物和环境中发现的艰难梭菌能够引起人类疾病。破坏肠道微生物平衡的抗生素治疗是艰难梭菌感染(CDI)的主要危险因素，因此CDI的抗生素治疗往往失败。此外，艰难梭菌由于涉及与其他细胞、细菌病毒(称为噬菌体)和外部DNA(如转座子)的接触的基因交换事件，不断进化为对抗生素具有抗药性。其中一些事件比其他事件发生得更频繁，可能是因为环境因素，了解这些因素对于控制ARG交换事件很重要。为此，我们在不同的环境条件下研究了艰难梭菌细胞与其他细菌、噬菌体和外部DNA的接触，这些环境条件模拟了肠道条件，如抗生素的存在和pH的变化，并测量了细胞之间ARG交换的差异。我们正在研究杀死或增强艰难梭菌杀灭抗生素的新药物，作为治疗感染的可能方法。我们研究的两种这样的试剂是噬菌体和阳离子多肽。噬菌体是细菌的天敌，到目前为止发现的许多噬菌体都可以通过基因改造有效地杀死艰难梭菌。我们通过移除阻止细菌有效杀死的噬菌体基因来做到这一点，迫使噬菌体在感染后复制并分解其细菌宿主细胞。阳离子多肽是一小段带正电的蛋白质，可以破坏细菌的细胞壁或DNA。在这项研究中，我们将测试一种合成的阳离子多肽，通过将它们混合在一起并将它们添加到活跃生长的细胞中，来提高抗生素对艰难梭菌的活性。我们还在寻找保护患者免受CDI复发的药物，CDI是大约20%的CDI患者的严重问题。益生菌是一种无害的细菌，可以帮助我们的肠道抵御病原体的侵袭。我们正在测试不同类型的益生菌阻止艰难梭菌在模拟CDI患者自然微生物环境的人类肠道模型中定植的能力。这是通过从健康人的粪便样本中培养细菌，在类似于人类大肠的pH、营养和无氧条件下的三个烧瓶中进行的。然后在烧瓶中加入抗生素和艰难梭菌以建立“感染”，加入更多的抗生素来去除艰难梭菌并模拟复发的感染。然后将益生菌添加到系统中，并检查益生菌是否可以预防艰难梭菌。我们还研究了无害的艰难梭菌(缺乏产生毒素的能力)与产生毒素的艰难梭菌竞争和预防感染的能力。到目前为止，我们已经发现了一种无害的菌株，可以在广泛的实验条件下阻止艰难梭菌超级菌株的生长和毒素产生，这在以前还没有得到证明。这种控制艰难梭菌获得ARG、生长和重新定植的三管齐下的方法将为开发CDI的治疗方法开辟新的途径。

Gut bacteria can exchange DNA in many ways to resist antibiotics and cause untreatable infections. Our research on understanding how and when antibiotic resistance genes (ARGs) are exchanged with other bacteria, and finding new treatment agents should ultimately reduce these occurrences and make infections more easily treated. This research is focused on Clostridioides difficile, an important human pathogen that causes infection with high illness and death rates. C. difficile found in animals and the environment are capable of causing human disease. Antibiotic therapy that disrupts the balance of microbes in the gut is a major risk factor for C. difficile infection (CDI), hence antibiotic treatment of CDI often fails. Also C. difficile is constantly evolving to be antibiotic-resistant because of gene exchange events that involve contact with other cells, bacterial viruses (known as phages), and external DNA such as transposons. Some of these events occur more frequently than others probably because of environmental factors, and understanding these is important for controlling ARG exchange events. For this, we examine C. difficile cells in contact with other bacteria, phages, and external DNA under different environmental conditions that mimic gut conditions such as presence of antibiotics and changing pH, and measure differences in ARG being exchanged between cells. We are investigating new agents that kill or enhance antibiotic-killing of C. difficile as possible treatments for infection. Two such agents we investigate are phages and cationic peptides. Phages are natural enemies of bacteria, and many phages found so far can be genetically altered to efficiently kill C. difficile. We do this by removing phage genes that prevent efficient killing of bacteria, forcing the phage to replicate and break apart its bacterial host cell after infection. Cationic peptides are short pieces of positively-charged proteins that disrupt the cell wall or DNA of bacteria. In this research we will be testing a synthetic cationic peptide for its ability to enhance the activity of antibiotics against C. difficile by mixing them together and adding them to actively growing cells. We also look for agents that protect patients from recurrent CDI, which is a serious problem in about 20% of CDI patients. Probiotics are harmless bacteria that help our gut resist colonisation by pathogens. We are testing the ability of different types of probiotics to stop C. difficile from colonising a human gut model that mimics the natural microbial environment in humans suffering from CDI. This is done by growing bacteria from faecal samples of healthy humans in three flasks of pH, nutrient, and oxygen-free conditions similar to a human large intestine. Antibiotics and C. difficile are then added to the flasks to establish "infection", more antibiotics are added to remove C. difficile and simulate a recurring infection. Probiotics are then added to the system and checked to see if the probiotic prevents C. difficile. We also investigate the ability of harmless C. difficile (which lack the ability to produce toxins) to compete with toxin-producing C. difficile strains and prevent infection. So far we have found a harmless strain that prevents growth and toxin production by a superbug strain of C. difficile under a wide range of experimental conditions and this has not been shown before. This three-pronged approach to control C. difficile in acquiring ARG, growth, and re-colonisation will open new avenues for developing treatments for CDI.