Department of Infectious Diseases and Microbiology, Faculty of Veterinary Medicine, University of Veterinary Sciences Brno, Brno, Czech Republic.
Department of Zoology, Fisheries, Hydrobiology and Apiculture, Faculty of AgriSciences, Mendel University, Brno, Czech Republic.
Microbiol Spectr. 2022 Aug 31;10(4):e0080622. doi: 10.1128/spectrum.00806-22. Epub 2022 Aug 11.
Clostridioides difficile is one of the most important human pathogens. The identification of its possible sources is important for the understanding of C. difficile infection (CDI) epidemiology. A total of 16 water samples from wastewater and surface water in South Moravia in the Czech Republic and 82 samples of fish and gulls were collected between May and July 2019. C. difficile isolates were cultured by direct plating and after enrichment on chromogenic media. Susceptibility testing to eight antimicrobials was performed by Etest. C. difficile isolates were characterized by ribotyping, multilocus sequence typing, multilocus tandem repeats analysis, and toxin gene detection. Samples from fish and gulls were C. difficile negative; a total of 15 C. difficile isolates from 8 out of 16 water samples were cultured (6 out of 14 surface water samples yielded 6 isolates, and 2 out of 2 wastewater samples yielded 9 isolates). Direct plating was culture positive in 6 out of 16 samples (12 isolates), and enrichment culture was positive in an additional 2 out of 16 samples (3 isolates). Twelve different ribotyping profiles and 14 sequence types of clades 1, 4, and 5 were identified. Five isolates did not carry genes for toxins, and eight isolates carried genes for toxins A and B; the remaining two isolates (RT078) carried the genes for toxins A, B, and binary. All C. difficile isolates were susceptible to amoxicillin, moxifloxacin, tetracycline, and vancomycin and resistant to ciprofloxacin. A high level of erythromycin resistance (>256 mg/L) was detected in eight isolates. Clindamycin resistance was found in 14 isolates, 6 of which showed a high level of resistance (>256 mg/L) and carried . Surprisingly, one isolate (RT010, ST15) showed resistance to metronidazole (12 mg/L) with the presence of the plasmid pCD-METRO. In conclusion, a diverse spectrum of C. difficile strains was found in wastewater and surface water. A recently discovered plasmid-bound resistance to metronidazole was detected in C. difficile from the surface water sample. The combination of direct plating and culture after enrichment was used in order to gain a spectrum of C. difficile ribotypes present in the water samples. Toxigenic C. difficile ribotypes detected in surface water and in wastewater treatment plants overlapped with those derived from patients with CDI and/or animals. Importantly, a recently discovered plasmid-mediated resistance to metronidazole, a drug used for the treatment of CDI, was detected in C. difficile from river water.
艰难梭菌是最重要的人类病原体之一。确定其可能的来源对于了解艰难梭菌感染（CDI）的流行病学非常重要。本研究于 2019 年 5 月至 7 月间在捷克共和国南摩拉维亚采集了 16 份来自废水和地表水的水样以及 82 份鱼类和海鸥样本。通过直接平板培养和在显色培养基上富集后培养艰难梭菌分离株。通过 Etest 进行了对 8 种抗菌药物的药敏试验。通过核糖体分型、多位点序列分型、多位点串联重复分析和毒素基因检测对艰难梭菌分离株进行了特征分析。鱼类和海鸥样本均为艰难梭菌阴性；从 16 个水样中的 8 个水样中培养出 15 株艰难梭菌（14 个地表水样本中有 6 个样本培养出 6 株，2 个废水样本培养出 9 株）。直接平板培养阳性的有 16 个样本中的 6 个（12 株），另外 2 个样本通过富集培养阳性（3 株）。鉴定出 12 种不同的核糖体分型谱和 14 种属于 1 型、4 型和 5 型分支的序列型。5 株分离株不携带毒素基因，8 株分离株携带毒素 A 和 B 基因；其余 2 株（RT078）携带毒素 A、B 和二元毒素基因。所有艰难梭菌分离株均对阿莫西林、莫西沙星、四环素和万古霉素敏感，对环丙沙星耐药。8 株分离株中检测到高水平的红霉素耐药性（>256 mg/L）。14 株分离株对克林霉素耐药，其中 6 株显示高水平耐药（>256 mg/L），并携带.令人惊讶的是，一株分离株（RT010，ST15）对甲硝唑（12 mg/L）表现出耐药性，同时存在质粒 pCD-METRO。综上所述，在废水和地表水样本中发现了多种艰难梭菌菌株。在来自地表水样本的艰难梭菌中检测到一种最近发现的与质粒结合的甲硝唑耐药性。通过直接平板培养和富集后培养的组合，获得了水样中存在的艰难梭菌核糖体类型谱。在地表水和污水处理厂中检测到的产毒艰难梭菌核糖体型与来自 CDI 患者和/或动物的核糖体型重叠。重要的是，在河水中的艰难梭菌中检测到一种最近发现的与质粒相关的甲硝唑耐药性，甲硝唑是治疗 CDI 的药物。
