Antimicrobial resistance (AMR) is recognized as a global threat. AMR bacteria accumulate in sewage sludge however, knowledge on the persistence of human pathogens and AMR in the sludge line of the wastewater treatment is limited. Sludge can be used, with or without additional treatment, as fertilizer in agricultural fields. The aim of this study is to obtain knowledge about presence of human pathogens and AMR in the sewage sludge, before and after the anaerobic digestion (AD) applying innovative combinations of methods. Fifty sludge samples were collected. Cultivation methods combined with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and Antibiotic Susceptibility Test (AST) were used obtaining knowledge about the microbial community, pathogens, and antibiotic resistant bacteria while the droplet digital Polymerase Chain Reaction (ddPCR) was performed to detect most common AMR genes. In total, 231 different bacterial species were identified in the samples. The most abundant species were spore-forming facultative anaerobic bacteria belonging to Bacillus and Clostridium genera. The AD causes a shift in the microbial composition of the sludge (p = 0.04). Seven pathogenic bacterial species constituting 188 colonies were isolated and tested for susceptibility to Clindamycin, Meropenem, Norfloxacin, Penicillin G, and Tigecycline. Of the Clostridium perfringens and Bacillus cereus isolates 67 and 50 %, respectively, were resistant to Clindamycin. Two B. cereus and two C. perfringens isolates were also resistant to other antibiotics showing multidrug resistance. ARGs (blaOXA, blaTEM, ermB, qnrB, tet(A)-(W), sulI-II) were present at 7–8 Log gene copies/kg of sludge. AD is the main driver of a reduction of some ARGs (1 Log) but resistant bacteria were still present. The results showed the usefulness of the integration of the proposed analytical methods and suggest a decrease in the risk of presence of cultivable pathogens including resistant isolates after AD but a persistent risk of ARGs’ horizontal transmission.

中文翻译：

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厌氧消化对污泥中病原菌及耐药性的影响


抗菌素耐药性（AMR）被认为是全球性威胁。抗菌素耐药性细菌在污水污泥中积聚，然而，关于废水处理污泥管线中人类病原体和抗菌素耐药性的持久性的知识有限。无论是否经过额外处理，污泥均可用作农田肥料。本研究的目的是了解应用创新方法组合进行厌氧消化 (AD) 前后污水污泥中人类病原体和 AMR 的存在情况。收集了 50 个污泥样品。采用培养方法结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和抗生素敏感性测试（AST），获得有关微生物群落、病原体和抗生素耐药细菌的知识，同时液滴数字聚合酶链进行反应 (ddPCR) 来检测最常见的 AMR 基因。样本中总共鉴定出 231 种不同的细菌种类。最丰富的物种是属于芽孢杆菌属和梭菌属的产芽孢兼性厌氧细菌。 AD 会导致污泥微生物组成发生变化 (p = 0.04)。分离了 7 种致病菌，组成 188 个菌落，并测试了对克林霉素、美罗培南、诺氟沙星、青霉素 G 和替加环素的敏感性。产气荚膜梭菌和蜡状芽孢杆菌分离株中分别有 67% 和 50% 对克林霉素具有抗药性。两种蜡样芽孢杆菌和两种产气荚膜梭菌分离株也对其他抗生素具有耐药性，表现出多重耐药性。 ARG（blaOXA、blaTEM、ermB、qnrB、tet(A)-(W)、sulI-II）的含量为 7-8 Log 基因拷贝/kg 污泥。 AD 是某些 ARG 减少的主要驱动力 (1 Log)，但耐药细菌仍然存在。结果显示了所提出的分析方法整合的有用性，并表明 AD 后可培养病原体（包括耐药菌株）存在的风险降低，但 ARG 水平传播的风险持续存在。