Table 2_Unraveling the genome-wide repertoire of the novel chromosomally encoded mcr-8.6 gene variant in Klebsiella michiganensis isolated from manure.xlsx

The increasing rates of colistin resistance worldwide poses a significant threat to public health. While the most commonly described variant is mcr-1, other variants such as mcr-8 have been detected, typically associated with Klebsiella pneumoniae. However, little is known about the prevalence of mcr-8 in other bacterial species and environmental reservoirs. This study aimed to characterize a novel mcr-8 subvariant identified in a Klebsiella michiganensis strain isolated from manure in Portugal, collected during an annual longitudinal survey at an Open Air laboratory, as well as to depict its genomic context and potential mobility mechanisms. The strain was subjected to phenotypic susceptibility testing, whole-genome sequencing and hybrid genome assembly. In silico analysis included identification of resistance genes and mobile genetic element. The new gene variant mcr-8.6 and its genetic environment were characterized. The F731 strain presented susceptibility to colistin with a MIC = 0.25 mg/L, despite carrying a novel mcr-8 subvariant, mcr-8.6, which was located within a 61.6 kb chromosomal genomic island. This variant presented 23–24 amino acid substitutions compared to previous characterized MCR-8 proteins. The genomic island also harbored multiple insertion sequences (IS110, IS66, IS3), virulence factors, and metabolic and regulatory proteins, among others. Synteny analysis revealed high sequence identity between this genomic island and both chromosomal and plasmid regions from other bacterial strains isolated from different reservoirs worldwide, indicating prior mobility. Furthermore, other antimicrobial resistance genes were detected [e.g., aph(3′)-la, blaOXY–1–2], but no plasmid replicons were identified. This is the first report of a mcr-8 gene in a K. michiganensis, as well as the first occurrence in Portugal. Although F731 remains colistin-susceptible, the presence of a novel mcr-8.6 chromosomally encoded but located in a mobile genomic island underscores the risk of future horizontal gene transfer. These findings highlight the importance of further monitoring and continued surveillance in environmental and animal compartments in order to track the dissemination of antimicrobial resistance.

全球范围内粘菌素（colistin）耐药率持续攀升，对公共卫生构成严重威胁。当前研究最为广泛的变体为mcr-1基因，而其他变体如mcr-8则常与肺炎克雷伯菌（Klebsiella pneumoniae）相关联。然而，学界对mcr-8在其他细菌物种及环境储库中的流行情况仍知之甚少。本研究旨在对一株从葡萄牙粪便样本中分离得到的密歇根克雷伯菌（Klebsiella michiganensis）菌株所携带的新型mcr-8亚变体进行特征分析，该菌株采集自某露天实验室（Open Air laboratory）的年度纵向调查样本；同时本研究还对该变体的基因组背景及潜在移动机制进行了描述。研究人员对该菌株开展了表型药敏试验、全基因组测序（whole-genome sequencing）及混合基因组组装（hybrid genome assembly）。计算机模拟分析（in silico analysis）涵盖耐药基因与可移动遗传元件（mobile genetic element）的鉴定工作。最终对新型基因变体mcr-8.6及其遗传环境进行了特征解析。F731菌株虽携带新型mcr-8亚变体mcr-8.6，但其对粘菌素仍表现出敏感性，最低抑菌浓度（Minimum Inhibitory Concentration, MIC）为0.25 mg/L。该变体位于一段61.6 kb的染色体基因组岛中，与已表征的MCR-8蛋白相比，存在23至24个氨基酸替换。此基因组岛还携带有多种插入序列（IS110、IS66、IS3）、毒力因子以及代谢与调控蛋白等。共线性分析（synteny analysis）显示，该基因组岛与全球不同储库中分离得到的其他细菌菌株的染色体及质粒区域均具有高度序列同源性，提示其具备既往移动能力。此外，研究还检测到其他抗菌耐药基因，如aph(3′)-la、blaOXY–1–2，但未发现质粒复制子。本研究是mcr-8基因在密歇根克雷伯菌中的首次报道，同时也是葡萄牙境内的首次发现。尽管F731菌株仍对粘菌素敏感，但位于可移动基因组岛上的染色体编码型新型mcr-8.6的存在，凸显了未来发生水平基因转移的风险。本研究结果强调，对环境及动物相关样本开展进一步监测与持续追踪，对于掌握抗菌耐药性的传播态势具有重要意义。