Tackling AMR in Wastewater Systems with Sneaky Bacteria

家庭污水处理是自维多利亚时代以来社区卫生得到显著改善的主要原因之一。废物处理厂（WTPs）有效地去除病原体、碳和氮，创造一个更健康的环境，减少水传播的传染病。然而，wtp从未被设计用于去除当代污染物，如抗微生物耐药性细菌（ARB）或基因（ARG）。目前的wtp从废物中减少了许多arb /ARGs，但“最糟糕”的ARGs亚部分在wtp中增加，特别是多ARGs (mrg)，这可能产生不可摧毁的病原体。研究人员一直在研究为何在wtp中选择耐多药（MDR）。然而，原因尚不清楚，这影响了我们水基础设施的长期恢复能力。在20世纪50年代，德国研究人员在wtp的活性污泥（AS）中观察到一种奇怪的细菌形式，称为l型细菌。l型细菌是暂时失去细胞壁的“正常”细菌。虽然很有趣，但这一观察结果并没有得到进一步的研究。然而，医学研究人员最近发现l型细菌在耐多药尿路感染中很常见，我的猜测是l型细菌，本质上是耐多药，可能是WTP流出物中耐多药的“未知”原因。为了验证这一聪明的想法，从两个英国wtp收集了约40个样本，发现了非常高水平的l型菌株，特别是在AS絮凝体中。此外，所有l型菌株都是假定的“肠道”细菌，这意味着WTP废水中的耐多药耐药可能是由于肠道源l型细菌的选择性生存，这些细菌“隐藏”在凝菌中（处于伪休眠状态），然后“潜入”到WTP废水中，因为它们以l型状态在废物处理中存活下来。该项目做了以下工作：-开发更好的方法来检测废水中的l型；-量化wtp中l型细菌被选择和隐藏的环境条件，并确定触发它们重新激活的因素；-确定促进/抑制l型状态的基因表达靶点，并确定wtp中l型可以选择性破坏的特定位置；开展试验台和中试规模的反应器工作，制定新的处理策略，以减少耐多药耐药性，特别是减少污水处理厂废水中的l型存活。在英国、西班牙和印度，我们已经从wtp中分离出了700株耐多药微生物菌株，尽管很少有l型菌株被检测出来。然而，早期数据表明l型在AS中很常见，这与德国的观察结果一致。在这种背景下，最初的工作将集中在详细描述我们目前的MDR分离株，特别是对易于l型形成的菌株进行分类，并确定关键的“l型触发基因”的存在和缺失（在我们的分离株中）。将对目标基因进行改进和测试，以诊断l型，以及它们的流行程度和当地环境与耐多药指标的关系（使用qPCR、NGS和抗性组学）。根据这些数据，将与三个工业合作伙伴在8个采用不同生物处理技术的全尺寸wtp上进行结构化采样，以确定l型选择和存活的“热点”。现场数据将用于指导实验室和试点规模的新技术和改造技术测试，以降低污水处理厂污水中的耐多药耐药性水平，这将为提高城市水基础设施抵御能力的战略提供信息，特别是减少耐药耐药性的传播和保护社区健康。该提案将为“健康和有弹性的国家”提供关键成果，特别是H2， H3， R2和R3，因为它将产生影响未来所有WTP设计的基本和实用数据；减少mrg释放到环境中的设计。除了这一结果，l型菌株的遗传学、生态学和选择方面也将取得突破性的发现，这将使医学界了解耐多药感染，并改善临床和环境环境中的诊断。

Domestic wastewater treatment is among the main reasons why community health has improved dramatically since Victorian Times. Waste treatment plants (WTPs) effectively remove pathogens, carbon, and nitrogen, creating a healthier environment and reducing the waterborne infectious disease. However, WTPs were never designed to remove contemporary contaminants, such as antimicrobial resistant bacteria (ARB) or genes (ARG). Current WTPs reduce many ARBs/ARGs from wastes, but the "worst" sub-fraction of ARGs increase in WTPs, especially multi-ARGs (MRGs) that create the potential for indestructible pathogens. Researchers have been studying why multidrug resistance (MDR) is selected in WTPs. However, the cause is unknown, which impacts the long-term resilience of our water infrastructure.In the 1950s, German researchers observed a strange bacterial form in activated sludge (AS) in WTPs, called L-form bacteria. L-forms are "normal" bacteria that temporarily lose their cell wall. Although interesting, this observation was not pursued further. However, medical researchers recently discovered that L-form bacteria are common in MDR urinary tract (u-tract) infections, and my speculation is that L-form bacteria, which are intrinsically MDR, might be the "unknown" cause of MDR in WTP effluents. To test this bright idea, ~40 samples were collected from two UK WTPs and very high levels of L-form strains were found, especially in AS floc. Further, all L-form strains were putative "gut" bacteria, implying MDR in WTP effluents may be due to the selective survival of gut-originated L-form bacteria that "hide" in floc (in pseudo-dormant state) and then "sneak" back into WTP effluents because they survive waste treatment in their L-form state. The project do the following: - Develop better methods for detecting L-forms in wastewater;- Quantify environmental conditions in WTPs where L-form bacteria are selected and hide, and determine what triggers their reactivation;- Identify gene expression targets that promote/repress the L-form state and identify specific locations in WTPs where L-forms can be selectively destroyed; and- Perform bench- and pilot-scale reactor work to develop new treatment strategies to reduce MDR, especially aimed at reducing L-form survival in WTPs effluents.We already have >700 microbial MDR isolates from WTPs in the UK, Spain and India, although few have been tested for L-form development. However, early data suggest L-forms are common in AS, consistent with German observations. Within this context, work initially will focus on characterising our current MDR isolates in detail, especially categorising strains prone to L-formation and also identifying the presence and absence of key "L-form trigger genes" (in our isolates). Target genes will be refined and tested for diagnostics of L-forms, and also how their prevalence and local environments relate to MDR indictors (using qPCR, NGS and resistomics). With these data, structured sampling will be performed with three industrial partners on eight full-scale WTPs with different biological treatment technologies to identify "hot spots" of L-form selection and survival. Locale data will be used to guide lab- and pilot-scale testing of new and retrofit technologies to reduce MDR levels in WTP effluents, which will inform strategies for increasing resilience in our urban water infrastructure, especially reducing AMR spread and protecting community health. This proposal will deliver key outcomes for A Healthy and Resilient Nation, specifically H2, H3, R2 and R3, because it will generate basic and practical data that impacts all WTP designs in future; designs to reduce MRGs released to the environment. Beyond this outcome, transcendent discoveries will be made on the genetics, ecology and selection of L-form strains, which will inform the medical community on MDR infections and also improve diagnostics in both clinical and environmental settings.