耐碳青霉烯类肺炎克雷伯菌对喹诺酮类药物的耐药特性及机制研究

宋曼雅; 刘长鑫; 张侃; 马林; 王博; 郭桦; 丁俊谕; 赵慧珺; 周治同; 管希周

doi:10.12435/j.issn.2095-5227.2023.008

耐碳青霉烯类肺炎克雷伯菌对喹诺酮类药物的耐药特性及机制研究

Resistance characteristics and mechanism of carbapenem-resistant Klebsiella pneumoniae to quinolones

摘要

摘要:
背景耐碳青霉烯类肺炎克雷伯(carbapenem-resistant Klebsiella pneumoniae，CRKP)对喹诺酮类药物的耐药机制及对新一代喹诺酮类药物西他沙星的耐药特性目前国内未见相关研究。
目的研究CRKP对喹诺酮类药物的耐药特性及耐药机制。
方法收集解放军总医院临床分离非重复耐碳青霉烯类肺炎克雷伯菌29株，采用微量肉汤稀释法检测CRKP对喹诺酮类药物的敏感度及外排泵抑制剂对CRKP耐药性的影响；通过棋盘法检测西他沙星联合其他抗菌药物对CRKP的体外抗菌活性；采用Sanger测序检测喹诺酮耐药决定区(quinolone resistance determining region，QRDR)突变；全基因组测序检测质粒介导的喹诺酮耐药基因(plasmid mediated quinolone resistance，PMQR)、外排泵、多位点序列分型等分子病原学特征。
结果 29株CRKP对西他沙星、莫西沙星、环丙沙星的耐药率分别为90%、93%、93%，西他沙星MIC₅₀值是莫西沙星的4倍、环丙沙星的8倍。西他沙星对bla_KPC、bla_NDM、bla_OXA-48阳性的CRKP的敏感度分别为4.7%、66.6%、0。联合药敏结果表明西他沙星与黏菌素、依拉环素、替加环素联合用药对CRKP的协同、部分协同及相加作用分别为70%、20%、6%。bla_KPC、bla_NDM、bla_OXA-48阳性的CRKP的QRDR突变率分别为95%、0、100%；质粒介导的PMQR携带率为83%；RND家族外排泵在CRKP菌株中普遍携带，外排泵抑制剂使CRKP对西他沙星的敏感度由10%上升至83%，耐药率由90%降至7%。解放军总医院优势传播菌株为ST11型bla_KPC-2阳性菌株。
结论 CRKP对喹诺酮类药物耐药率高，西他沙星与黏菌素的协同效果最好。QRDR突变和外排泵过表达是CRKP对喹诺酮类药物主要的耐药机制，产NDM酶CRKP的QRDR未见突变并且对喹诺酮类药物敏感性较好；ST11型bla_KPC-2阳性菌株是解放军总医院优势流行菌株。

Abstract:
Background There is a lack of research about Carbapenem-resistant Klebsiae pneumoniae (CRKP) resistance to quinolones and citafloxacin (a new generation of quinolones) in China.
Objective To investigate the resistance characteristics and mechanisms of carbapenem-resistant Klebsiella pneumoniae (CRKP) to quinolones.
Methods Twenty-nine clinical isolates of non-repetitive carbapenem-resistant Klebsiella pneumoniae were collected from Chinese PLA General Hospital. The sensitivity of CRKP to quinolones and the effect of efflux pump inhibitors on the resistance of CRKP were detected by broth microdilution method. The in vitro antibacterial activity of sitafloxacin combined with other antibacterial agents against CRKP was detected by checkerboard method. QRDR (quinolone resistance-determining region) mutations were detected by Sanger sequencing. Plasmid-mediated quinolone resistance gene (PMQR), efflux pump, and multilocus sequence typing (MLSP) were detected by whole genome sequencing.
Results The resistance rates of 29 CRKP strains to sitafloxacin, moxifloxacin, and ciprofloxacin were 90%, 3%, and 93%, respectively. The MIC₅₀ of sitafloxacin was 4 times that of moxifloxacin and 8 times that of ciprofloxacin. The susceptibility rates of sitafloxacin to bla_KPC, bla_NDM, and bla_OXA-48 positive CRKP were 4.7%, 66.6%, and 0, respectively. The results of antimicrobial synergy study showed that synergistic, partial synergistic and additive effects of sitafloxacin combined with colistin, eravacycline, and tigecycline to the CRKP were 70%, 20%, and 6%, respectively. The QRDR mutation rates of bla_KPC, bla_NDM, and bla_OXA-48 positive CRKP were 95%, 0, and 100%, respectively. The rate of plasmid-mediated quinolone resistance gene (PMQR) was 83%. RND efflux pumps were commonly carried in CRKP strains. Efflux pump inhibitors increased the susceptibility rate of CRKP to sitafloxacin from 10% to 83%, and reduced the resistance rate from 90% to 7%. All the dominant ST types were bla_KPC-2 positive strains.
Conclusion CRKP has a high resistance rate to quinolones. Sitafloxacin and colistin have the best synergistic effect. QRDR mutation and overexpression of efflux pump are the main resistance mechanisms of CRKP to quinolones, while the NDM-producing CRKP has no mutation in QRDR and is sensitive to quinolones. ST11 bla_KPC-2 positive strains are the predominant strains in our hospital.

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