Tackling AMR in Wastewater Systems with Sneaky Bacteria

生活污水处理是自维多利亚时代以来社区健康状况显著改善的主要原因之一。废物处理厂(WTP)有效地去除病原体、碳和氮，创造更健康的环境，减少水传播的传染病。然而，污水处理厂从来没有被设计来去除当代的污染物，如抗菌素耐药性细菌(ARB)或基因(ARG)。目前的废物处理场减少了废物中的许多ARB/ARG，但废物处理场中“最糟糕”的ARG亚组分增加了，特别是多ARG(MRG)，这可能会产生不可摧毁的病原体。研究人员一直在研究为什么多药耐药(MDR)会被选为WTP。然而，原因尚不清楚，它影响了我们的水基础设施的长期弹性。20世纪50年代，德国研究人员在污水处理厂的活性污泥(AS)中观察到一种奇怪的细菌形态，称为L细菌。L型细菌是暂时失去细胞壁的“正常”细菌。虽然很有趣，但这一观察结果并未被进一步探讨。然而，医学研究人员最近发现，L型细菌在耐多药尿路(U-道)感染中很常见，我推测L型细菌本质上是多药耐药菌，可能是污水处理厂出水中多药耐药的未知原因。为了验证这一聪明的想法，从英国的两个污水处理厂收集了大约40个样本，发现了非常高水平的L型菌株，特别是在AS絮凝物中。此外，所有L型菌株都是可能的“肠道”细菌，这意味着污水处理厂出水中的多药耐药可能是由于肠道起源的L型细菌的选择性生存，这些细菌“隐藏”在絮体中(处于伪休眠状态)，然后又“潜入”到污水处理厂的出水中，因为它们在L式的状态下在垃圾处理中存活下来。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；-量化污水处理厂中L-型细菌被选择和隐藏的环境条件，并确定是什么触发了它们的重新激活；-识别促进/抑制L-型状态的基因表达靶点，并确定在污水处理厂中可以选择性地摧毁L-型的特定位置；以及-进行试验台和中试规模的反应器工作，以开发新的处理策略来减少多药耐药性，特别是旨在减少L-型在污水处理厂流出物中的存活。我们已经有了。来自英国、西班牙和印度WTP的700株微生物多药耐药分离物，尽管很少有人对L形式的开发进行过测试。然而，早期数据表明，L-形式在AS中很常见，这与德国的观察结果一致。在此背景下，最初的工作将集中于详细描述我们目前的多药耐药分离株的特征，特别是对容易形成L的菌株进行分类，并识别(在我们的分离株中)是否存在关键的“L型触发基因”。目标基因将被提炼和检测，用于L型的诊断，以及它们的流行率和当地环境与多药耐药指标的关系(使用qPCR、NGS和耐药组学)。有了这些数据，将与三家产业合作伙伴对8个采用不同生物处理技术的全规模污水处理厂进行结构化抽样，找出L选型和生存的“热点”。现场数据将用于指导新技术和改造技术的实验室和试点规模测试，以降低污水处理厂出水中的MDR水平，这将为提高我们城市水基础设施的弹性，特别是减少AMR传播和保护社区健康提供战略依据。这项建议将为一个健康和有弹性的国家带来关键成果，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；旨在减少向环境释放MRG的设计。除了这一结果，还将在遗传学、生态学和L型菌株的选择方面取得超乎寻常的发现，这将向医学界通报耐多药感染，并提高临床和环境环境中的诊断水平。

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.