降压药左旋氨氯地平胁迫下肺炎克雷伯菌产生多种抗生素耐药性的机制研究

Investigation of Emergence and Underlying Mechanisms of Multidrug R esistance in Klebsiella pneumoniae Induced by S-amlodipine Stress

左旋氨氯地平(S-amlodipine,S-AM)是一种降压药,在高血压患者中被广泛使用,部分患者更是长期使用。然而,作为一种非抗生素药物,在其长期胁迫下,细菌是否会进化出抗生素抗性,我们对此一无所知。为了探讨S-AM暴露对细菌抗生素耐药性的影响,本研究针对肠道固有的条件致病菌肺炎克雷伯菌进行了为期60 d的2 mg·L^(-1)S-AM胁迫下体外进化实验。通过抗生素敏感性测试确定了进化菌株对6种抗生素的耐药性;运用基因组测序技术、荧光定量PCR分析及生物信息学分析,深入探究了S-AM诱导细菌抗生素耐药性的机制。结果显示,在S-AM暴露下肺炎克雷伯菌的突变频率从10^(-7)增加到1.5×10^(-1);进化菌株的胞内活性氧(reactive oxygen species,ROS)水平显著提高;同时对多黏菌素、卡那霉素、氯霉素、红霉素和莫西沙星表现出更高的耐药性。基因组学分析结果显示,在进化菌株中发现了3个非同义单核苷酸多态性(single nucleotide polymorphism,SNP)突变位点、1个同义SNP突变位点、2个基因间的SNP和1个基因间的插入位点。其中,突变的crrB基因协同crrA基因提高了进化菌株细胞膜脂多糖(lipopolysaccharide,LPS)脂质A组分的修饰,增加了细菌细胞膜表面的正电荷并阻止多黏菌素与细胞相互作用,从而提高了肺炎克雷伯菌对多黏菌素的耐药性。荧光定量PCR的结果显示,S-AM暴露下双组分调节系统基因(如crrAB、phoPQ、pmrAB和pmrDFIK)、多药外排泵基因(如emrD和oqxAB)以及固有耐药基因(如aadA 2)的表达水平升高,这可能是导致细菌多种抗生素耐药性增加的原因。总之,我们的研究首次发现S-AM胁迫下肺炎克雷伯菌对多种抗生素产生耐药性,并阐明了潜在的分子机制,为长期用药患者的健康风险提出预警,并为进一步研究S-AM长期胁迫下的广泛肠道细菌耐药性奠定基础。

S-amlodipine(S-AM)is a commonly prescribed antihypertensive drug for patients with hypertension,and it is used for an extended duration in most hypertensive patients.However,it remains unclear whether long-term exposure to non-antibiotic drug S-amlodipine induces bacterial antibiotic resistance.To investigate the potential impact of S-AM on bacterial antibiotic resistance,we conducted a 60-day in vitro evolution experiment in which intestinal pathogen K.pneumoniae was exposed to 2 mg·L^(-1)S-AM.We determined the resistance of the e volved strains to six antibiotics through antibiotic susceptibility testing.Furthermore,we delved into the potential resistance mechanisms of K.pneumoniae via genome sequencing,fluorescence quantitative PCR,and bioinformatics analysis.This study observed that the mutation frequency of K.pneumoniae increased from 10^(-7)to 1.5×10^(-1)under S-AM stress,and the intracellular reactive oxygen species(ROS)levels of the evolved strains significantly in creased compared to that of the control strains.Additionally,the evolved strains exhibited heightened resistance to polymyxin,kanamycin,chloramphenicol,erythromycin,and moxifloxacin.Whole genome sequencing analysis r esults showed the presence of three non-synonymous single nucleotide polymorphism(SNP)mutation sites,one synonymous SNP mutation site,two intergenic SNPs,and one intergenic insertion site in the evolved strains.These non-synonymous gene mutations include the HAMP domain-containing histidine kinase crrB gene,sensor domain-containing diguanylate cyclase,and type 3 fimbria adhesin subunit mrkD gene.Notably,the mutated crrB gene,in collaboration with the crrA gene,enhanced the modification of the lipid A component of the cell membrane lipopoly-saccharide(LPS)in the evolved strains.This modification increased the positive charge on the bacterial cell m embrane surface,hindering polymyxin-cell interactions and contributing to increased polymyxin resistance in K.pneumoniae.Fluorescence quantitative PCR results revealed that exposure to S-AM enhanced the mRNA e xpressions of two-component regulatory system genes(e.g.,crrAB,phoPQ,pmrAB,and pmrDFIK),multidrug e fflux pump genes(e.g.,emrD and oqxAB),and the intrinsic resistance gene aadA2,which might be responsible for increased bacterial resistance to multiple antibiotics.In summary,this study is the first to demonstrate that S-AM stress triggers multiple antibiotic resistance in K.pneumoniae and elucidates the underlying molecular m echanisms,serving as an early warning for potential health risks associated with long-term medication in h ypertensive patients.It lays a foundation for further exploration of antibiotic resistance in diverse intestinal bacteria under prolonged S-AM stress.