Objective: To investigate the molecular mechanism underlying inherent fosfomycin resistance of Klebsiella pneumoniae (K. pneumoniae).
Methods: The draft genomic sequences of 14 clinical hypervirulent/hypermucoviscous K. pneumoniae (HvKP/ HmKP) isolates were obtained using the next-generation sequencing technology. The genomic sequences were analyzed using the Resistance Gene Identifier (RGI) software for predicting the resistome based on homology and SNP models in the Comprehensive Antibiotic Resistance Database (CARD) and for identification of the presence of phosphomycin resistancerelated genes uhpt and fosA and their mutations in the bacterial genomes. The results were verified by analyzing a total of 521 full-length genomic sequences of K. pneumonia strains obtained from GenBank.
Results: All the 14 clinical isolates of HvKP/ HmKP carried hexose phosphate transporter (UhpT) gene mutation, in which the glutamic acid was mutated to glutamine at 350aa (UhpTE350Q mutation); the presence of fosA6 gene was detected in 12 (85.71%) of the isolates and fosA5 gene was detected in the other 2 (14.29%) isolates. Analysis of the genomic sequences of 521 K. pneumonia strains from GenBank showed that 508 (97.50%) strains carried UhpTE350Q mutation, 439 (84.26%) strains harbored fosA6, and 80 (15.36%) strains harbored fosA5; 507 (97.31%) strains were found to have both UhpTE350Q mutation and fosA6/5 genes in the genome. Only 12 (2.30%) strains carried fosA6/5 genes without UhpTE350Q mutation; 1 (0.19%) strain had only UhpTE350Q mutation without fosA6/5 genes, and another strain contained neither UhpTE350Q mutation nor fosA6/5 genes.
Conclusion: UhpTE350Q mutation with the presence of fosA6/5 genes are ubiquitous in K. pneumonia genomes, indicating a possible intrinsic mechanism of fosfomycin resistance in the bacterium to limit the use of fosfomycin against infections caused by K. pneumoniae, especially the multi-resistant HvKP/HmKP strains.
目的: 分析肺炎克雷伯菌磷霉素耐药基因的分布，探讨磷霉素老药新用在该菌的限制机制。
方法: 使用二代测序技术对14株临床高毒性/高粘性肺炎克雷伯菌分离株(HvKP/HmKP)测序绘制基因组草图，基于综合抗生素耐药性数据库CARD中的同源性和SNP模型，通过抗性基因识别软件分析预测磷霉素耐药相关基因uhpT、fosA及其突变；并对GenBank中获得的521条肺炎克雷伯菌全基因组序列进行同步分析验证。
结果: 本研究中14株HvKP/HmKP临床分离株均具有己糖磷酸盐转运蛋白基因(UhpT)突变，其在350aa处谷氨酸突变为谷氨酰胺(UhpTE350Q)。12株菌(12/14，85.71%)基因组携带fosA6基因，其余2株菌(14.29%)中携带fosA5基因。GenBank的521株肺炎克雷伯菌基因组序列中，携带UhpTE350Q突变的菌株508株(97.50%)，携带fosA6基因的菌株439株(84.26%)，携带fosA5基因的菌株80株(15.36%)；同时携带UhpTE350Q突变和fosA6/5基因的菌株507株(97.31%)；无UhpTE350Q突变、仅fosA6/5基因的菌株12株(2.30%)；仅携带UhpTE350Q突变、无fosA6/5基因的菌株1株(0.19%)；无UhpTE350Q突变、无fosA6/5基因的菌株1株(0.19%)。
结论: 染色体UhpTE350Q突变与fosA6/5基因在肺炎克雷伯菌染色体中的普遍存在意味着出现转运蛋白突变和产生磷霉素修饰酶，这可能是该菌对磷霉素天然耐药的内在机制，会限制磷霉素在肺炎克雷伯菌，特别是多重耐药的HvKP/HmKP相关抗感染中的临床应用。
Keywords: Klebsiella pneumoniae; UhpTE350Q mutation; drug resistance mechanisms; fosA gene; fosfomycin.