Objectives

Clostridioides difficile (CD) is widely reported as one of the most prevalent multi-drug resistant (MDR) organisms. Assessment of temporally disparate isolate collections can give valuable epidemiological data to further the understanding of antimicrobial resistance progression.

Methods

A collection of 75 CD isolates (1980–86) was characterised by PCR ribotyping, cell cytotoxicity assay and susceptibility testing with a panel of 16 antimicrobials and compared to a modern surveillance collection consisting of 416 UK isolates (2012–2016). Agar-incorporation was performed to ascertain susceptibility data for vancomycin, metronidazole, rifampicin, fidaxomicin, moxifloxacin, clindamycin, imipenem, chloramphenicol, tigecycline, linezolid, ciprofloxacin, piperacillin/tazobactam, ceftriaxone, amoxicillin, tetracycline and erythromycin. Genomes were obtained using Illumina HiSeq3000 sequencing and assembled using CLC Genomics Workbench. Resistance genes were identified using the Comprehensive Antibiotic Research Database's Resistance Gene Identifier and ResFinder3.0.

Results

Twenty-six known and one previously unobserved ribotype (RT) were detected. RT015 and RT020 dominated; 21.3% and 17.3%, respectively. Three moxifloxacin resistant (16–32 mg/L) RT027 isolates were recovered, pre-dating the earliest reports of this phenotype/genotype. Phenotypic resistance was observed to moxifloxacin (9.3% of isolates), ciprofloxacin (100%), erythromycin (17.3%), tetracycline (9.3%), linezolid and chloramphenicol (4.0%). Phenotypic comparisons with modern strains revealed increasing minimum inhibitory concentrations (MIC), with MIC₅₀ elevations of one doubling-dilution for the majority of compounds, excluding clindamycin and imipenem. Moxifloxacin MIC₉₀ comparisons revealed a two doubling-dilution increase between temporal isolate collections. Historical genomes revealed twenty different resistance determinants, including ermB (8.0% of isolates), tetM (9.3%), cfr (5.3%) and gyrA substitution Thr-82→Ile (9.3%). Seventeen isolates (22.7%) were resistant to ≥3 compounds (MDR), demonstrating ten different combinations. Intra-RT diversity was observed.

Conclusions

Antibiotic resistance in CD has increased since the early 1980s, across the majority of classes. Moxifloxacin resistance determinants may pre-date its introduction.

中文翻译：

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英国的抗菌素耐药性进展：艰难梭菌抗菌素敏感性的时间比较

目标

艰难梭菌(CD) 被广泛报道为最普遍的多重耐药 (MDR) 生物之一。对时间上不同的分离物收集的评估可以提供有价值的流行病学数据，以进一步了解抗菌素耐药性的进展。

方法

一组 75 个 CD 分离株（1980-86 年）通过 PCR 核糖分型、细胞毒性测定和药敏试验对一组 16 种抗菌药物进行了表征，并与由 416 个英国分离株（2012-2016 年）组成的现代监测收集进行了比较。进行琼脂掺入以确定万古霉素、甲硝唑、利福平、非达霉素、莫西沙星、克林霉素、亚胺培南、氯霉素、替加环素、利奈唑胺、环丙沙星、哌拉西林/他唑巴坦、头孢曲松、阿莫西林、四环素和红霉素的敏感性数据。使用 Illumina HiSeq3000 测序获得基因组，并使用 CLC Genomics Workbench 进行组装。使用综合抗生素研究数据库的抗性基因标识符和 ResFinder3.0 鉴定抗性基因。

结果

检测到 26 种已知和一种以前未观察到的核型 (RT)。RT015和RT020为主；分别为 21.3% 和 17.3%。回收了三个莫西沙星耐药 (16–32 mg/L) RT027 分离株，早于该表型/基因型的最早报道。观察到对莫西沙星（9.3% 的分离株）、环丙沙星（100%）、红霉素（17.3%）、四环素（9.3%）、利奈唑胺和氯霉素（4.0%）的表型耐药性。与现代菌株的表型比较显示最小抑制浓度 (MIC) 增加，大多数化合物的 MIC ₅₀升高一倍稀释，不包括克林霉素和亚胺培南。莫西沙星 MIC ₉₀比较显示时间隔离集合之间有两个加倍稀释增加。历史基因组揭示了 20 个不同的耐药决定因素，包括ermB（8.0% 的分离株）、tetM（9.3%）、cfr（5.3%）和gyrA替代 Thr-82→Ile（9.3%）。十七株 (22.7%) 对≥3 种化合物 (MDR) 具有耐药性，表明有十种不同的组合。观察到RT内多样性。

结论

自 1980 年代初以来，CD 中的抗生素耐药性在大多数类别中都有所增加。莫西沙星耐药决定因素可能早于它的引入。