Survival and pathogenicity of Clostridioides difficile in sewage, sewage sludge, surface water, animal manure, fodder, crops and silage -Treatment requirements to minimize health risks" (SUPER safe)

革兰氏阳性、形成厌氧内孢子的艰难梭菌是人类和牲畜院内感染性腹泻的主要原因。艰难梭菌感染（CDI）是一种毒素介导的肠道疾病，当肠道中的天然菌群被抗生素破坏时发生。难辨梭菌是世界范围内卫生保健系统的负担，作为一种新出现的病原体，难辨梭菌是感染预防和控制中讨论的主要问题。CDI的治疗受到艰难梭菌内孢子的阻碍，它们可以在肠道中持续使用抗生素治疗，迅速用营养细胞重新填充肠道并导致感染复发。作为肠道的定植者，艰难梭菌的细胞和孢子可以在人类和动物的粪便中排泄和发现。因此，艰难梭菌与粪便明显相关。预计在污水、污水污泥和动物粪便以及其他可能被粪便污染的来源（如地表水或饲料）中也可以发现艰难梭菌。由于厌氧消化AD在污水污泥和动物粪便处理中的重要性日益增加，因此应关注艰难梭菌在AD期间的生存和繁殖，以及厌氧粪便和废水处理系统向环境中释放的增加以及残留物作为肥料的利用。因此，拟议的研究项目的目的是确定艰难梭菌高流行的环境来源。目的是枚举和分离环境中的艰难梭菌，并通过分析产生毒素A或B和二元毒素CDT的基因来进一步表征其产毒潜力。除了核糖分型外，抗生素敏感性试验（用于测定MIC和分离环境菌株中各自的抗生素耐药基因和共轭转座子）应提供流行病学行为的信息。通过建立定量pcr法对艰难梭菌细胞和共轭转座子Tn5397进行计数，定量粪便相关样品中艰难梭菌的细胞计数和致病性。由于过去几年安装了大量的厌氧消化器来处理污泥和粪便，因此将在实验室反应器中模拟污水污泥和牛粪的AD，从细胞数量和细胞外的共轭质粒的角度来研究艰难梭菌的存活甚至生长或减少/消除。这些测试将包括长期的粪便储存、臭氧化和紫外线消毒程序，以及污水的最终处理步骤。通过加强微生物学和环境工程领域的研究，预期的工作方案应取得成果，并扩大关于艰难梭菌在“粪便相关”样本中的生存和致病潜力的知识，并可得出关于传播途径的结论，并评估对人类和动物的健康风险。

The Gram-positive, anaerobic endospore forming Clostridioides difficile is the major cause of nosocomial infectious diarrhoea in humans and livestock. C. difficile infection (CDI) is a toxin-mediated intestinal disease and occurs when the natural flora in the guts is disrupted by antibiotics. The hypervirulent nosocomial pathogen C. difficile is a burden for health care systems worldwide and, as an emerging pathogen C. difficile is a major matter of discussion in infection prevention and control. Treatment of CDI is hampered by C. difficile endospores which can persist antibiotic treatment in the guts, rapidly repopulate guts with vegetative cells and lead to recur of an infection. As a colonizer of the intestine C. difficile cells and spores could be excreted and found in feces of humans and animals. For this reason C. difficile can be clearly associated with feces. It is expected that C. difficile can be found in sewage, sewage sludge and animal manures as well and in other sources which could be contaminated with feces like surface water or fodder. Since there is a rising importance of anaerobic digestion AD for sewage sludge and animal manure treatment, attention should focus on survival and multiplication of C. difficile during AD and increasing release into the environment by anaerobic manure and wastewater treatment systems and utilization of residues as fertilizers.The aim of the proposed research project is thus to identify environmental sources with a high prevalence of C. difficile. Enumeration and isolation of environmental C. difficile and further characterization of the toxigenic potential by analyzing e.g. genes for production of Toxin A or B and the binary toxin CDT is intended. Beside ribotyping, antibiotic susceptibility testing for determination of MIC and determination of respective antibiotic resistance genes and conjugative transposons present in isolated environmental strains should give information about epidemiological behavior. By establishing qPCR for enumeration of C. difficile cells and the conjugative transposon Tn5397 cell counts and pathogenicity of C. difficile in fecal-associated samples will be quantified. Since a large number of anaerobic digestors were installed in past years for treatment of sludge and manure, AD of sewage sludge and cattle manure will be simulated in lab-reactors to investigate survival or even growth or the reduction/elimination of C. difficile in terms of cell numbers and the conjugative plasmid outside of the cells. Such tests will include long term manure storage and ozonation and UV disinfection procedures, final treatment steps for sewage. By strengthening research in the field of microbiology and environmental engineering the anticipated work program should deliver results and extend knowledge on survival and the pathogenic potential of C. difficile in “fecal-associated” samples and allow to draw conclusions on transmission ways and to assess the health risk for human and animals.