Table_1_Molecular Mechanisms of Colistin Resistance in Klebsiella pneumoniae in a Tertiary Care Teaching Hospital.docx

BackgroundOver the last two decades, the prevalence of colistin resistance among the members of Enterobacteriaceae has been increasing, particularly among Klebsiella pneumoniae isolates; this limits the potential use of colistin and leads to worsened clinical outcomes.MethodsWe investigated the prevalence and genetic characteristics of colistin-resistant K. pneumoniae (COLR-KP) in clinical isolates using genomic sequencing.ResultsIn total, 53 K. pneumoniae isolates (4.5%, 53/1,171) were confirmed as COLR-KP, of which eight isolates carried mobile colistin-resistant (mcr) gene. Although the overall prevalence rate (0.7%, 8/1,171) of mcr-like genes in clinical K. pneumoniae remained relatively low, the presence of mcr (15.1%, 8/53) among the COLR-KP isolates indicated that the mobile resistance gene was already widespread among K. pneumoniae isolates in hospital setting. We randomly selected 13 COLR-KP isolates (four mcr-bearing and nine non-mcr-bearing isolates) for whole-genome sequencing, including two pandrug-resistant and four sequence type 11 (ST11) isolates. Phylogenetic analysis revealed that all COLR-KP isolates were genetically diverse. Among the four mcr-bearing isolates, three (KP4, KP18, and KP30) were positive for mcr-1 and one (KP23) for mcr-8; none of the other mcr genes were detected. The mcr-1 in the KP4 and KP30 isolates were located in an IncX4 plasmid (approximately 33 kb) and could be successfully transferred to Escherichia coli J53AZR. In contrast, for the mcr-8-bearing plasmid in KP23 (IncFII), colistin resistance could not be transferred by conjugation. The mcr-1-producing isolate KP18 coexists a novel plasmid-carried tigecycline resistance gene tmexCD1-toprJ1. The most common chromosomal mutation associated with colistin resistance was a T246A amino acid substitution in PmrB, which was identified in most COLR-KP isolates (11/13, 84.6%). All ST11 isolates additionally had an R256G amino acid substitution. Critical virulence factors associated with hypervirulent K. pneumoniae were detected in four COLR-KP isolates; these virulence factors included aerobactin, salmochelin, and yersiniabactin.ConclusionWe found that mcr-bearing COLR-KP emerged in our hospital and was growing at an increasing rate. Simultaneous emergence of hypervirulence and colistin–tigecycline–carbapenem resistance in the epidemic clone ST11 K. pneumoniae was also observed; this highlights the significance of active and continuous surveillance.

背景：在过去二十年中，肠杆菌科细菌中粘菌素耐药性的普遍性呈上升趋势，尤其在肺炎克雷伯菌分离株中；这一现象限制了粘菌素的使用潜力，并导致临床结果恶化。方法：我们采用基因组测序技术，研究了临床分离株中粘菌素耐药肺炎克雷伯菌（COLR-KP）的流行率和遗传特征。结果：在总共的53株肺炎克雷伯菌分离株中（占4.5%，53/1,171），有8株分离株携带可移动的粘菌素耐药基因（mcr）。尽管在临床肺炎克雷伯菌中mcr类基因的整体流行率（0.7%，8/1,171）相对较低，但COLR-KP分离株中mcr（15.1%，8/53）的存在表明，可移动耐药基因已在医院环境中广泛传播。我们随机选取了13株COLR-KP分离株（包括4株携带mcr和9株不携带mcr的分离株）进行全基因组测序，其中包含2株广谱耐药株和4株序列型11（ST11）分离株。系统发育分析显示，所有COLR-KP分离株在遗传上呈现多样性。在携带mcr的4株分离株中，3株（KP4、KP18和KP30）为mcr-1阳性，1株（KP23）为mcr-8阳性；未检测到其他mcr基因。KP4和KP30分离株中的mcr-1位于IncX4质粒（约33 kb）中，并能成功转移到大肠杆菌J53AZR。相比之下，KP23（IncFII）携带的mcr-8质粒不能通过接合转移粘菌素耐药性。mcr-1产生株KP18同时携带一种新型质粒携带的替加环素耐药基因tmexCD1-toprJ1。与粘菌素耐药性相关最常见的染色体突变是PmrB中的T246A氨基酸替换，这一突变在大多数COLR-KP分离株（11/13，84.6%）中被发现。所有ST11分离株还额外存在R256G氨基酸替换。与高致病性肺炎克雷伯菌相关的关键致病因素在4株COLR-KP分离株中被检测到；这些致病因素包括铁载体、沙拉莫素和耶尔森菌素。结论：我们发现携带mcr的COLR-KP在我院出现，并且增长速度正在加快。在流行克隆ST11肺炎克雷伯菌中，同时出现高致病性、粘菌素-替加环素-碳青霉烯类耐药性的现象也被观察到；这突出了主动且持续的监测的重要性。