碳青霉烯类耐药肺炎克雷伯菌对奥马环素的耐药规律及机制研究

Study on regularity and mechanisms of omadacycline resistance in carbapenem-resistant Klebsiella pneumoniae

目的研究碳青霉烯类耐药肺炎克雷伯菌(CRKP)对新型四环素类药物奥马环素的耐药规律及机制,为CRKP感染的临床用药提供理论参考。方法收集2019年1月-2021年12月期间阳江市人民医院临床分离的非重复CRKP菌株306株,利用微量肉汤稀释法检测奥马环素对CRKP临床分离株的最低抑菌浓度(MIC)和检测奥马环素耐药菌株对其他临床常用药物的MIC,通过PCR扩增结合测序方法确定耐药菌株的多位点序列分型(MLST)和碳青霉烯酶型,使用琼脂平板稀释法检测外排泵抑制剂对耐药菌株奥马环素MIC的影响,运用qRT-PCR比较奥马环素敏感与耐药菌株中耐药结节分化细胞(RND)型外排泵基因和调控基因的表达水平差异。结果CRKP菌株对奥马环素耐药率为9.5%(29/306),29株奥马环素耐药菌株主要来源于痰液(51.7%)、血液(24.1%)和尿液(17.2%)。耐药菌株的MLST型主要为ST11[65.5%(19/29)]和ST15[24.1%(7/29)],携带的碳青霉烯酶主要为KPC-2[62.1%(18/29)]和KPC-3[13.8%(4/29)]。奥马环素在CRKP中与米诺环素、多西环素及四环素存在交叉耐药现象。加入外排泵抑制剂苯丙氨酸-精氨酸-β-萘酰胺(PaβN)后,58.6%(17/29)耐药菌株的奥马环素MIC值降低4倍以上。奥马环素耐药菌株中外排泵编码基因acrA和调控基因ramA、rarA的表达水平显著高于奥马环素敏感菌株,差异均有统计学意义(t=1.613、1.257、1.290,P均<0.05)。结论奥马环素在CRKP中耐药率高于替加环素,其耐药机制的形成可能与外排泵AcrAB和调控蛋白RamA、RarA的高表达有关。

Objective To investigate the regularity and mechanisms of omadacycline resistance in carbapenem-resistant Klebsiella pneumoniae(CRKP),and provide a theoretical evidence for the clinical treatment of CRKP.Methods A total of 306 non-repetitive CRKP isolates in People's Hospital of Yangjiang from January 2019 to December 2021 were collected and subjected to minimum inhibitory concentration(MIC)of omadacycline using the broth microdilution method;omadacycline-resistant strains tested for MIC against commonly used antibiotics were using the same method.The multilocus sequence typing(MLST)and the carbapenemase gene were determined by PCR amplification and sequencing.The effect of efflux-pump inhibitors on omadacycline MIC in resistant strains was determined by agar dilution.The expression levels of efflux pumps which in resistance-nodulation-cell-division family(RND)and their regulators were compared between omadacycline-sensitive and omadacycline-resistant strains by qRT-PCR.Results The resistance rate of omadacycline was 9.5%(29/306)in CRKP strains;the 29 strains were mainly from sputum(51.7%),blood(24.1%)and urine(17.2%).The ST-types of resistant strains were predominantly ST11[65.5%(19/29)]and ST15[24.1%(7/29)],and their carbapenemases were KPC-2[62.1%(18/29)]and KPC-3[13.8%(4/29)].Omadacycline was cross-resistant with minocycline,doxycycline,and tetracycline in CRKP.Furthermore,the addition of the efflux-pump inhibitor Phe-Arg-β-naphthylamide(PaβN)resulted in more than 4-fold reduction in omadacycline MIC in 58.6%(17/29)of resistant strains.The expression levels of efflux pump coding gene acrA and regulatory genes ramA and rarA in omacycline-resistant strains were significantly higher than those in omacycline-sensitive strains;the differences were significant(t=1.613,1.257,1.290;all P<0.05).Conclusion The resistance rate of omadacycline in CRKP was higher than that of tigecycline,and the mechanism of resistance might be related to the high expression of the efflux pump AcrAB and the regulatory proteins RamA and RarA.