背景：艰难梭菌感染（CDI）在医疗保健环境中很普遍。超毒力和抗生素抗性菌株的出现导致CDI发病率增加和频繁爆发。虽然主要的毒力因子是TcdA和TcdB毒素，但抗生素抗性被认为在艰难梭菌的感染和传播中起关键作用。方法：在里斯本的一家三级医院检测到12例患者的CDI暴发，该医院从1月持续到7月，2016年2月达到高峰。从粪便样本的厌氧培养中获得的艰难梭菌分离株是用Etest®strips对11种抗生素进行抗菌药敏试验，测定毒素基因谱，PCR-核糖分型，多位点可变数目串联重复序列分析（MLVA）和全基因组测序（WGS）。结果：在检测到的12例CDI病例中，11例患者中有11例被鉴定出来。所有菌株均为tcdA  -  / tcdB +，属于核糖核型017，对克林霉素，红霉素，庆大霉素，亚胺培南，莫西沙星，利福平和四环素均有较高水平的耐药性。分离株属于四种遗传相关的MLVA类型，六种分离株形成克隆簇。为WGS选择了三个来自不同MLVA类型的爆发分离株。生物信息学分析显示存在几种抗生素抗性决定因子，包括gyrA中的Thr82Ile取代，赋予莫西沙星抗性，rpoB中替代His502Asn和Arg505Lys对利福平抗性，tetM基因与四环素抗性相关，以及两个编码推定氨基糖苷类修饰的基因酶，aadE和aac（6'） -  aph（2“）。此外，鉴定了先前未描述的61.3kb推定的移动元件，其呈现镶嵌结构并含有与大环内酯，林可酰胺和链阳性菌素抗性相关的基因ermG，mefA / msrD和vat。在B类青霉素结合蛋白Cys721Ser中发现的取代被认为有助于亚胺培南抗性。结论：我们描述了一种流行病，tcdA  -  / tcdB +，来自与医院爆发相关的核糖体017的艰难梭菌的多药耐药性克隆，提供了进一步证据表明缺乏TcdA不会损害这些菌株的感染潜力。我们确定了抗菌素耐药性的几个决定因素，包括位于移动元件中的新抗体，突出了水平基因转移在艰难梭菌的致病性和流行病学成功中的重要性。

Background: Clostridium difficile infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of C. difficile. Methods: A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The C. difficile isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest®strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS). Results: Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were tcdA -/tcdB + and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in gyrA, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in rpoB for rifampicin resistance, the tetM gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, aadE and aac(6')-aph(2″). Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes ermG, mefA/msrD and vat, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance. Conclusion: We describe an epidemic, tcdA -/tcdB +, multidrug resistant clone of C. difficile from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of C. difficile.

https://www.ncbi.nlm.nih.gov/pubmed/30574133