Table_1_Characterization of Klebsiella pneumoniae carrying the blaNDM-1 gene in IncX3 plasmids and the rare In1765 in an IncFIB-IncHI1B plasmid.docx

BackgroundToday, the blaNDM gene is widely distributed on several plasmids from a variety of Gram-negative bacteria, primarily in transposons and gene cassettes within their multidrug-resistant (MDR) regions. This has led to the global dissemination of the blaNDM gene.
MethodsThe determination of class A beta-lactamase, class B and D carbapenemases was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI). Antimicrobial susceptibility testing was performed using both the BioMerieux VITEK2 system and antibiotic paper diffusion methods. Plasmid transfer was then evaluated by conjugation experiments and plasmid electroporation assays. To comprehensively analyze the complete genome of K. pneumoniae strain F11 and to investigate the presence of mobile genetic elements associated with antibiotic resistance and virulence genes, Nanopore and Illumina sequencing platforms were used, and bioinformatics methods were applied to analyze the obtained data.
ResultsOur findings revealed that K. pneumoniae strain F11 carried class A beta-lactamase and classes B+D carbapenemases, and exhibited resistance to commonly used antibiotics, particularly tigecycline and ceftazidime/avibactam, due to the presence of relevant resistance genes. Plasmid transfer assays demonstrated successful recovery of plasmids pA_F11 and pB_F11, with average conjugation frequencies of 2.91×10-4 and 1.56×10-4, respectively. However, plasmids pC_F11 and pD_F11 failed in both conjugation and electroporation experiments. The MDR region of plasmid pA_F11 contained rare In1765, TnAs2, and TnAs3 elements. The MDR2 region of plasmid pB_F11 functioned as a mobile genetic “island” and lacked the blaNDM-1 gene, serving as a “bridge” connecting the early composite structure of bleMBL and blaNDM-1 to the recent composite structure. Additionally, the MDR1 region of plasmid pB_F11 comprised In27, TnAs1, TnAs3, and Tn2; and plasmid pC_F11 harbored the recent composite structure of bleMBL and blaNDM-1 within Tn3000 which partially contained partial Tn125.
ConclusionThis study demonstrated that complex combinations of transposons and integron overlaps, along with the synergistic effects of different drug resistance and virulence genes, led to a lack of effective therapeutic agents for strain F11, therefore its dissemination and prevalence should be strictly controlled.

背景 当前，blaNDM基因广泛分布于多种革兰阴性菌的多种质粒之上，主要定位于其多重耐药（Multidrug-resistant, MDR）区域内的转座子与基因盒中，这导致了blaNDM基因的全球播散。
方法 依照临床与实验室标准化协会（Clinical and Laboratory Standards Institute, CLSI）的推荐方案，完成了A类β-内酰胺酶、B类及D类碳青霉烯酶的检测。采用梅里埃VITEK2系统（BioMerieux VITEK2）与纸片扩散法进行抗菌药物敏感性试验。随后通过接合实验与质粒电转化实验评估质粒的转移能力。为全面解析肺炎克雷伯菌（K. pneumoniae）F11的完整基因组，并探究与抗生素耐药及毒力基因相关的移动遗传元件的存在情况，本研究使用纳米孔测序平台（Nanopore）与因美纳测序平台（Illumina）进行测序，并采用生物信息学方法对所得数据进行分析。
结果 本研究结果显示，肺炎克雷伯菌F11携带A类β-内酰胺酶以及B类与D类碳青霉烯酶，由于相关耐药基因的存在，该菌株对常用抗菌药物呈现耐药表型，尤其是替加环素与头孢他啶/阿维巴坦。质粒转移实验成功回收了pA_F11与pB_F11质粒，其平均接合频率分别为2.91×10^-4与1.56×10^-4。然而，pC_F11与pD_F11质粒在接合实验与电转化实验中均未实现转移。pA_F11的多重耐药区域包含罕见的In1765、TnAs2与TnAs3元件。pB_F11的MDR2区域作为移动遗传‘岛’发挥功能，且不含blaNDM-1基因，可作为连接bleMBL与blaNDM-1早期复合结构与近期复合结构的‘桥梁’。此外，pB_F11的MDR1区域包含In27、TnAs1、TnAs3与Tn2元件；pC_F11在Tn3000（部分包含Tn125片段）中携带bleMBL与blaNDM-1的近期复合结构。
结论 本研究证实，转座子与整合子的复杂重叠组合，加之不同耐药基因与毒力基因的协同效应，导致F11菌株缺乏有效的治疗药物，因此需对其传播与流行进行严格管控。