摘要
      抗生素耐药性是当今全球健康、粮食安全和发展面临的最大威胁之一，导致越来越多的难以治疗的感染和经济负担。它可以影响任何年龄和任何国家的任何人。抗生素的滥用和滥用、卫生条件差以及缺乏卫生基础设施，都加速了这一进程。从“一个健康”的概念来看，水是连接抗生素耐药性主要产生的所有隔间（人类、动物和自然环境）的主要纽带。它携带微生物、抗生素等药物、以移动遗传元件（MGE）形式存在的浮动遗传信息以及赋予抗生素耐药性的基因。人们认为，在废水和饮用水处理厂等人为屏障中发现的细菌可能在将耐药细菌转移和传播到自然环境中发挥作用。然而，细菌可以通过水平基因转移（HGT）进行交换以在这些区室中进一步传播抗生素抗性基因（ARGs）的机制尚不清楚。自然转化是主管细菌将细胞外DNA拉入细胞质的主要HGT现象之一。尽管如此，目前还不清楚哪些细菌可以在什么情况下使用这种机制。在废水等复杂系统中解开这种自由漂浮的细胞外DNA（exDNA）部分的组成对于鉴定促进基因转移的环境条件至关重要。本文旨在进一步了解exDNA在复杂系统中抗生素耐药性细菌（ARBs）转移和发展中的作用。第2章评估了不同灭菌程序后不同模型微生物释放的DNA的状态。结果表明，目前的杀菌方法对微生物灭活是有效的。然而，稳定的DNA从微生物培养物中释放出来，并最终进入污水流，其中含有来自人类和动物排泄物的微生物的遗传信息。在第3章中，开发了一种使用色谱法从复杂的废水基质中分离和富集exDNA而不引起细胞裂解的方法，如进水（从1L中获得9μg exDNA）、活性污泥（从1LL中获得5.6μg）和处理过的出水（从5L中获得4.3μg）。因此，这对于描述其基因组成是必要的。令人惊讶的是，研究结果强调，exDNA主要由MGE组成（65%），这带来了风险，因为MGE在细胞外部分的流行主要通过自然转化间接促进抗生素耐药性的发展。在两个实地调查章节（第4章和第5章）中，评估了ARGs和MGE的转移及其在全尺寸Nereda®反应器中的去除能力，该反应器用好氧颗粒污泥去除营养物质，并在无氯饮用水处理厂中进行了评估。这两章总结了抗生素耐药性细菌走向水卫生的历程。抗性决定簇降低了它们到达废水（1.1 log基因拷贝数mL−1）和饮用水处理厂（2.5 log基因拷贝量mL−1。关于exDNA还不太清楚，因为处理过程涉及细胞衰变和裂解，从而将exDNA释放到环境中。在实验室规模和全尺寸实验中对exDNA进行分析后，在第6章中评估了环境因素（如抗生素浓度增加）对活性污泥浓缩中exDNA转化的影响。我们展示了当施加强环境压力（≥50 mg L−1）时，远距离相关微生物用于DNA摄取的可行性。因此，它表明，在环境抗生素浓度下的自然转化可能不是细菌在复杂系统中吸收exDNA的驱动力。然而，重点应该放在其他方面，如研究设施和制药工业排放。最后，第7章对废水中ARGs（细胞内和细胞外）和ARBs的修复策略进行了评估。我们展示了废水和饮用水处理厂的副产品，如污泥生物炭和氧化铁涂层砂，如何有效地去除废水中的ARBs和exDNA。总之，这篇论文表明，来自水基质的exDNA部分是一个被忽视的含有MGE和ARGs的遗传片段库。因此，这些可以作为遗传物质转化有能力的细菌并开发ARBs。然而，在环境抗生素浓度下的exDNA转化并不是细菌在混合培养中进化和适应的主要机制。重要的是要强调，人为屏障在修复ARBs方面是有效的，这应该将重点从废水处理厂转移到多个隔间，同时解决抗生素耐药性问题。
Abstract
Antibiotic resistance is one of the biggest threats to global health, food security, and development today, leading to a growing number of difficult-to-treat infections and an economic burden. It can affect anyone of any age and in any country. It is mainly accelerated by the misuse and abuse of antibiotics, poor hygiene, and a lack of sanitation infrastructure. From the One Health concept, water is the main link connecting all the compartments where antibiotic resistance has primarily developed (human, animal, and natural environments). It carries microorganisms, pharmaceuticals such as antibiotics, floating genetic information in the form of mobile genetic elements (MGEs), and genes conferring antibiotic resistance. It is thought that bacteria found in anthropogenic barriers such as wastewater and drinking water treatment plants could play a role in transferring and disseminating resistant bacteria into the natural environment. However, the mechanisms by which bacteria can exchange via horizontal gene transfer (HGT) to further disseminate antibiotic resistance genes (ARGs) in such compartments are unknown. Natural transformation is one of the main HGT phenomena by which competent bacteria pull extracellular DNA into their cytoplasm. Still, it remains widely unknown which bacteria can use such a mechanism and under which circumstances. Unraveling the composition of such free-floating extracellular DNA (exDNA) fraction in complex systems such as wastewater is crucial to identify the environmental conditions promoting gene transfer. This thesis aims to understand further the role of exDNA in the transfer and development of antibiotic-resistant bacteria (ARBs) from complex systems. The status of released DNA from different model microorganisms after different sterilization procedures was evaluated in Chapter 2. The results showed that current sterilization methods are effective in microorganism inactivation. However, stable DNA is released from microbial cultures and ends up in sewage streams with genetic information from microorganisms originating from human and animal discharges. In Chapter 3, a method using chromatography to isolate and enrich exDNA without causing cell lysis from complex wastewater matrices like influent (9 μg exDNA was obtained out of 1 L), activated sludge (5.6 μg out of 1 L), and treated effluent (4.3 μg out of 1 L) was developed. Thus, this was necessary to profile its genetic composition. Surprisingly, results highlighted that exDNA is mainly comprised of MGEs (65%), posing a risk as the prevalence of MGEs in the extracellular fraction can indirectly promote antibiotic resistance development mainly via natural transformation. In the two field investigation chapters (Chapters 4 and 5), the transfer of ARGs and MGEs and their removal capacity in a full-scale Nereda® reactor removing nutrients with aerobic granular sludge and in chlorine-free drinking water treatment plants were evaluated. These two chapters summarize the journey that antibiotic-resistant bacteria follow toward water sanitation. Resistance determinants decreased their load reaching effluents from wastewater (1.1 log gene copies mL−1) and drinking water treatment plants (2.5 log gene copies mL−1), at least when inside active bacterial cells. It is less clear regarding exDNA since the treatment process involves cell decay and lysis that releases exDNA into the environment. After profiling the exDNA both in lab-scale and full-scale experiments, the effect of environmental factors such as increasing antibiotic concentrations was evaluated on exDNA transformation in an activated sludge enrichment in Chapter 6. We showed the feasibility of distantly-related microorganisms for DNA uptake when strong environmental pressures (≥50 mg L−1) were applied. Thus, it shows that natural transformation under environmental antibiotic concentrations may not be the driving force by which bacteria take up exDNA in complex systems. However, the focus should be on other compartments such as research facilities and pharmaceutical industrial discharges. Finally, strategies to remediate ARGs (intracellular and extracellular) and ARBs from wastewater effluents were evaluated in Chapter 7. We showed how byproducts from wastewater and drinking water treatment plants, such as sewage-sludge biochar and iron-oxide coated sands, were effective at removing ARBs and exDNA from effluent waters. Collectively, this thesis shows that the exDNA fraction from water matrices is an overlooked pool of genetic fragments containing MGEs and ARGs. Thus, these could be used as genetic material to transform competent bacteria and develop ARBs. However, exDNA transformation under environmental antibiotic concentrations is not the main mechanism by which bacteria evolve and adapt in mixed cultures. It is important to highlight that anthropogenic barriers are effective at remediating ARBs, which should redirect the focus from wastewater treatment plants and tackle the antibiotic resistance issue from multiple compartments simultaneously.

https://research.tudelft.nl/en/publications/transfer-dynamics-of-antibiotic-resistance-determinants-across-ur