利福平降低hvKP毒力的机制及联合CRO的体内外抗菌活性研究

Study on the mechanism of rifampicin in reducing the virulence of hvKP and its antimicrobial activity in combination with CRO in vitro and in vivo

目的 探索利福平(RFP)降低高毒力肺炎克雷伯菌(hvKP)荚膜合成的抗毒力能力及其与头孢曲松(CRO)联合用药的体内外抗菌效果。方法 在2021年承德医学院附属医院分离的肺炎克雷伯菌中筛选出4株符合hvKP判定标准的临床菌株,使用PCR方法测定菌株的荚膜血清型,并使用多位点序列分型方法对菌株进行基因分型。采用荚膜多糖定量、墨汁染色、荧光定量PCR等方法分析RFP作用前后hvKP菌株荚膜含量、荚膜直径以及荚膜相关基因表达量的变化。在此基础上检测RFP与CRO联合用药的体外抗菌效果,并建立大蜡螟感染模型,分析二者联合使用的体内抗菌疗效。结果 在筛选的4个hvKP菌株中,10号和35号菌株均为K1-ST23型,12号菌株为K57-ST592型,96号菌株为K5-ST485型。1/4 MIC浓度的RFP可以在不明显影响细菌生长的情况下显著降低荚膜多糖含量(P<0.05),减小荚膜直径(P<0.001)并下调荚膜合成相关基因的转录水平(P<0.001)。4个菌株RFP和CRO联合药敏试验结果FIC指数均表现为协同或部分协同效应,且hvKP感染后的大蜡螟幼虫经CRO+1/4MIC RFP治疗后,生存率明显高于CRO单药治疗时的生存率(P<0.05)。结论 利福平具有抑制hvKP荚膜合成的抗毒力能力并可通过减少黏液生成增强药物渗透性而提高抗菌药物疗效。

Objective To explore the ability of rifampicin(RFP)to reduce the capsule synthesis of hypervirulent Klebsiella pneumoniae(hvKP)and the antibacterial effect of RFP combined with ceftriaxone(CRO)in vitro and in vivo.Methods Four clinical strains that conformed to the criteria for hvKP were screened from Klebsiella pneumoniae isolated from the Affiliated Hospital of Chengde Medical University in 2021.The capsular serotypes of the strains were determined by PCR,and the genotypes of the strains were determined by multilocus sequence typing.Capsular polysaccharide quantification,India ink staining,and fluorescence quantitative PCR were used to analyze the changes in capsule content,capsule diameters,and expression of capsule-related genes of hvKP strains before and after RFP treatment.On this basis,the antibacterial effect of RFP combined with CRO in vitro was detected,and a Galleria mellonella infection model was established to analyze the antibacterial effect of the combination of RFP and CRO in vivo.Results Among the four hvKP strains screened,strains 10 and 35 were K1-ST23 type,strain 12 was K57-ST592 type,and strain 96 was K5-ST485 type.RFP at 1/4 MIC concentration significantly decreased capsular polysaccharide content(P<0.05),decreased capsule diameters(P<0.001)and down-regulated the transcription levels of genes involved in capsule synthesis(P<0.001)without significantly affecting bacterial growth.The FIC index of RFP and CRO combined drug sensitivity test results of 4 strains showed synergistic or partial synergy effects,and the survival rate of Galleria mellonella larvae after hvKP infection treated with CRO+1/4MIC RFP was significantly higher than that of CRO monotherapy(P<0.05).Conclusion Rifampicin has the anti-virulence ability to inhibit hvKP capsule synthesis and can improve the efficacy of antibacterial drugs by reducing mucus production and enhancing drug permeability.