Abstract：
OBJECTIVE: Polymyxins are currently the last-resort treatment against multi-drug resistant Gram-negative bacterial infections, but plasmid-mediated mobile polymyxin resistance genes (mcr) threaten its efficacy, especially in carbapenem-resistant Enterobacter cloacae complex (CRECC). The objective of this study was to provide insights into the mechanism of polymyxin-induced bacterial resistance and the effect of overexpression of mcr-9.
METHODS: The clinical strain CRECC414 carrying the mcr-9 gene was treated with a gradient concentration of polymyxin. Subsequently, the broth microdilution was used to determine the minimum inhibitory concentration (MIC) and RT-qPCR was utilized to assess mcr-9 expression. Transcriptome sequencing and whole genome sequencing (WGS) was utilized to identify alterations in strains resulting from increased polymyxin resistance, and significant transcriptomic differences were analysed alongside a comprehensive examination of metabolic networks at the genomic level.
RESULTS: Polymyxin treatment induced the upregulation of mcr-9 expression and significantly elevated the MIC of the strain. Furthermore, the WGS and transcriptomic results revealed a remarkable up-regulation of arnBCADTEF gene cassette, indicating that the Arn/PhoPQ system-mediated L-Ara4N modification is the preferred mechanism for achieving high levels of resistance. Additionally, significant alterations in bacterial gene expression were observed with regards to multidrug efflux pumps, oxidative stress and repair mechanisms, cell membrane biosynthesis, as well as carbohydrate metabolic pathways.
CONCLUSIONS: Polymyxin greatly disrupts the transcription of vital cellular pathways. A complete PhoPQ two-component system is a prerequisite for polymyxin resistance of Enterobacter cloacae, even though mcr-9 is highly expressed. These findings provide novel and important information for further investigation of polymyxin resistance of CRECC.
METHODS: The clinical strain CRECC414 carrying the mcr-9 gene was treated with a gradient concentration of polymyxin. Subsequently, the broth microdilution was used to determine the minimum inhibitory concentration (MIC) and RT-qPCR was utilized to assess mcr-9 expression. Transcriptome sequencing and whole genome sequencing (WGS) was utilized to identify alterations in strains resulting from increased polymyxin resistance, and significant transcriptomic differences were analysed alongside a comprehensive examination of metabolic networks at the genomic level.
RESULTS: Polymyxin treatment induced the upregulation of mcr-9 expression and significantly elevated the MIC of the strain. Furthermore, the WGS and transcriptomic results revealed a remarkable up-regulation of arnBCADTEF gene cassette, indicating that the Arn/PhoPQ system-mediated L-Ara4N modification is the preferred mechanism for achieving high levels of resistance. Additionally, significant alterations in bacterial gene expression were observed with regards to multidrug efflux pumps, oxidative stress and repair mechanisms, cell membrane biosynthesis, as well as carbohydrate metabolic pathways.
CONCLUSIONS: Polymyxin greatly disrupts the transcription of vital cellular pathways. A complete PhoPQ two-component system is a prerequisite for polymyxin resistance of Enterobacter cloacae, even though mcr-9 is highly expressed. These findings provide novel and important information for further investigation of polymyxin resistance of CRECC.
摘要：
目的：多粘菌素目前是治疗多药耐药革兰阴性菌感染的最后手段，但是质粒介导的可移动多粘菌素抗性基因（mcr）威胁其功效，尤其是在耐碳青霉烯类阴沟肠杆菌复合体（CRECC）中。这项研究的目的是提供对多粘菌素诱导的细菌耐药性的机制和mcr-9过表达的影响的见解。
方法:用梯度浓度的多粘菌素处理携带mcr-9基因的临床菌株CRECC414。随后，肉汤微量稀释用于确定最小抑制浓度（MIC），RT-qPCR用于评估mcr-9表达。转录组测序和全基因组测序（WGS）用于鉴定由多粘菌素抗性增加引起的菌株的变化。在基因组水平上对代谢网络进行全面检查的同时，分析了显着的转录组差异。
结果：多粘菌素处理诱导mcr-9表达上调，并显着提高菌株的MIC。此外，WGS和转录组结果显示arnBCADTEF基因盒显著上调，表明Arn/PhoPQ系统介导的L-Ara4N修饰是实现高水平抗性的优选机制。此外,关于多药外排泵，观察到细菌基因表达的显著变化，氧化应激和修复机制，细胞膜生物合成，以及碳水化合物代谢途径。
结论：多粘菌素极大地破坏了重要细胞通路的转录。完整的PhoPQ双组分系统是阴沟肠杆菌多粘菌素耐药性的先决条件，尽管mcr-9的表达很高。这些发现为进一步研究CRECC的多粘菌素抗性提供了新的重要信息。
方法:用梯度浓度的多粘菌素处理携带mcr-9基因的临床菌株CRECC414。随后，肉汤微量稀释用于确定最小抑制浓度（MIC），RT-qPCR用于评估mcr-9表达。转录组测序和全基因组测序（WGS）用于鉴定由多粘菌素抗性增加引起的菌株的变化。在基因组水平上对代谢网络进行全面检查的同时，分析了显着的转录组差异。
结果：多粘菌素处理诱导mcr-9表达上调，并显着提高菌株的MIC。此外，WGS和转录组结果显示arnBCADTEF基因盒显著上调，表明Arn/PhoPQ系统介导的L-Ara4N修饰是实现高水平抗性的优选机制。此外,关于多药外排泵，观察到细菌基因表达的显著变化，氧化应激和修复机制，细胞膜生物合成，以及碳水化合物代谢途径。
结论：多粘菌素极大地破坏了重要细胞通路的转录。完整的PhoPQ双组分系统是阴沟肠杆菌多粘菌素耐药性的先决条件，尽管mcr-9的表达很高。这些发现为进一步研究CRECC的多粘菌素抗性提供了新的重要信息。
