利福平降低hvKP毒力的機(jī)制及聯(lián)合CRO的體內(nèi)外抗菌活性研究

摘要：目的 探索利福平（RFP）降低高毒力肺炎克雷伯菌（hvKP）莢膜合成的抗毒力能力及其與頭孢曲松（CRO）聯(lián)合用藥的體內(nèi)外抗菌效果。方法 在2021年承德醫(yī)學(xué)院附屬醫(yī)院分離的肺炎克雷伯菌中篩選出4株符合hvKP判定標(biāo)準(zhǔn)的臨床菌株，使用PCR方法測(cè)定菌株的莢膜血清型，并使用多位點(diǎn)序列分型方法對(duì)菌株進(jìn)行基因分型。采用莢膜多糖定量、墨汁染色、熒光定量PCR等方法分析RFP作用前后hvKP菌株莢膜含量、莢膜直徑以及莢膜相關(guān)基因表達(dá)量的變化。在此基礎(chǔ)上檢測(cè)RFP與CRO聯(lián)合用藥的體外抗菌效果，并建立大蠟螟感染模型，分析二者聯(lián)合使用的體內(nèi)抗菌療效。結(jié)果 在篩選的4個(gè)hvKP菌株中，10號(hào)和35號(hào)菌株均為K1-ST23型，12號(hào)菌株為K57-ST592型，96號(hào)菌株為K5-ST485型。1/4 MIC濃度的RFP可以在不明顯影響細(xì)菌生長(zhǎng)的情況下顯著降低莢膜多糖含量（Plt;0.05），減小莢膜直徑（Plt;0.001）并下調(diào)莢膜合成相關(guān)基因的轉(zhuǎn)錄水平（Plt;0.001）。4個(gè)菌株RFP和CRO聯(lián)合藥敏試驗(yàn)結(jié)果FIC指數(shù)均表現(xiàn)為協(xié)同或部分協(xié)同效應(yīng)，且hvKP感染后的大蠟螟幼蟲(chóng)經(jīng)CRO+1/4MIC RFP治療后，生存率明顯高于CRO單藥治療時(shí)的生存率（Plt;0.05）。結(jié)論 利福平具有抑制hvKP莢膜合成的抗毒力能力并可通過(guò)減少黏液生成增強(qiáng)藥物滲透性而提高抗菌藥物療效。

關(guān)鍵詞：高毒力肺炎克雷伯菌；利福平；抗毒力；聯(lián)合用藥

中圖分類(lèi)號(hào)：R978.3 文獻(xiàn)標(biāo)志碼：A

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

Abstract 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 （Plt;0.05）， decreased capsule diameters （Plt;0.001） and down-regulated the transcription levels of genes involved in capsule synthesis （Plt;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 （Plt;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.

Key words HvKP; Rifampicin; Anti-virulence; Drug combinations

肺炎克雷伯菌（Klebsiella pneumoniae， KP）是臨床常見(jiàn)的條件致病菌，根據(jù)毒力特點(diǎn)可將其分為普通型肺炎克雷伯菌（classic" Klebsiella pneumoniae，cKP）和高毒力肺炎克雷伯菌（hypervirulent" Klebsiella pneumoniae，hvKP）[1]。HvKP具有高毒力、高侵襲力以及高致死率和致殘率等特點(diǎn)，并且流行率逐年增高[2]，對(duì)人類(lèi)健康造成巨大威脅。……