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)

形成艰难梭状芽胞杆菌的革兰氏阳性厌氧内孢子是引起人畜医院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，当肠道中的自然菌群被抗生素扰乱时发生。艰难梭菌是一种超强毒力的医院内致病菌，是全世界卫生保健系统的负担，作为一种新出现的病原体，艰难梭菌是感染预防和控制中的一个主要讨论问题。艰难梭菌的内孢子阻碍了CDI的治疗，这种内孢子可以在肠道中持续进行抗生素治疗，迅速使肠道中的营养细胞重新填充，并导致感染复发。艰难梭菌作为肠道的定殖者，可以在人和动物的粪便中排泄出细胞和孢子。出于这个原因，艰难梭菌显然与粪便有关。预计艰难梭菌也可以在污水、污水污泥和动物粪便中发现，以及在可能被地表水或饲料等粪便污染的其他来源中发现。由于厌氧消化对污水污泥和动物粪便处理的重要性日益增加，应关注艰难梭菌在厌氧消化过程中的生存和繁殖，以及厌氧粪便和废水处理系统增加向环境中的释放，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。打算对环境艰难梭菌进行计数和分离，并通过分析例如产生毒素A或B和二元毒素CDT的基因来进一步表征产毒潜力。除了核糖体分型外，用于确定最低抑菌浓度和确定分离环境菌株中各自的抗生素耐药基因和接合转座子的抗生素敏感性试验应提供有关流行病学行为的信息。通过建立艰难梭菌细胞和Tn5397接合转座子的定量聚合酶链式反应，可以量化粪便相关样本中艰难梭菌的细胞计数和致病性。由于过去几年大量的厌氧消化器用于污泥和粪便的处理，因此将在实验室反应器中模拟污泥和牛粪的AD，以考察艰难梭菌的存活甚至生长或减少/消除困难梭菌的细胞数量和细胞外的接合质粒。这些测试将包括粪便的长期储存和臭氧和紫外线消毒程序，这是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，并使其能够就传播途径得出结论，并评估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.