高毒力碳青霉烯类耐药肺炎克雷伯菌流行病学和分子生物学分析

目的 对沈阳市第四人民医院碳青霉烯类耐药肺炎克雷伯菌（CRKP）的流行病学分析,并对其中高毒力碳青霉烯类耐药肺炎克雷伯菌（CR-hvKP）的表型、耐药基因、荚膜血清和毒力因子进行探究,为深入了解CR-hvKP的耐药机制、进化途径以及鉴别依据提供参考。方法 收集2017—2019年我院住院患者临床样本中分离出的肺炎克雷伯菌（KP）,使用全自动微生物分析仪鉴定和药敏实验,再利用改良碳青霉烯灭活实验（mCIM）和EDTA-改良碳青霉烯灭活实验（eCIM）方法进行菌株表型检测,PCR方法进行碳青霉烯耐药基因检测。通过拉丝实验筛选CR-hvKP,采用PCR进行荚膜血清型和毒力因子检测。结果 本研究共收集到KP 1 531株,其中CRKP 160株（10.45%）,主要来源于痰液标本,占比53.13%。对头孢菌素类、青霉素类、美罗培南、亚胺培南完全耐药,对复方磺胺甲噁唑和替加环素敏感,耐药率分别为45%和10%。CRKP携带blaKPC 基因127株,blaNDM基因26株,同时携带2种基因3株,携带blaOXA-48基因2株,未检测出任何基因2株。mCIM对碳青霉烯酶检出率为95.56%, eCIM对丝氨酸酶检出率为93.18%,对金属β-内酰胺酶检出率为96.55%。拉丝实验初步筛选得到6株CR-hvKP,分离率3.75%,低于其他已报道医院检出率。除1株产K2型荚膜血清菌株来自血液标本外,其余5株来自痰标本,产K1型荚膜血清。6株CR-hvKP全部携带耐药基因blaKPC和4种毒力因子rmpA、rmpA2、iutA、iroN。结论 沈阳市第四人民医院CRKP检出率位于全国平均水平,CRKP对多种抗菌药物耐药,对替加环素敏感,主要产KPC类碳青霉烯酶。CR-hvKP检出率低于其他已报道医院,具有产K1型荚膜血清,携带blaKPC耐药基因和多种毒力因子的特点。当K1型产KPC类碳青霉烯酶CRKP同时携带毒力因子rmpA、rmpA2、iutA、iroN,可进化成为CR-hvKP,提示临床应注意鉴别并进一步加强监测,以防止高毒力耐药菌株的传播和流行。

Epidemiology and molecular biology of carbapenem-resistant hypervirulent Klebsiella pneumoniae

Objective To investigate the epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) in our hospital, and to explore the phenotype, resistance gene, capsule serotype and virulence factor of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP), so as to provide reference for further understanding the drug resistance mechanism, evolutionary pathway and identification of CR-hvKP. Methods A total of 1 531 strains of Klebsiella pneumoniae (KP) isolated from clinical samples of inpatients in the Fourth Hospital of People of Shenyang were collected from 2017 to 2019. VITEK 2 Compact automatic microbial analyzer was used for bacterial identification and antimicrobial susceptibility analysis. The phenotypes of the strains were detected by modified carbapenem inactivation method (mCIM) and EDTA-CIM (eCIM) methods, and the carbapenem resistance gene was detected by PCR method. CR-hvKP was screened by string test, and the capsular serotype and virulence factor were detected by PCR. Results A total of 1 531 Klebsiella pneumoniae strains were detected, including 160 (10.45%) CRKP strains, mainly from sputum specimens, accounting for 53.13%. It was completely resistant to cephalosporins, penicillins, meropenem and imipenem, and sensitive to cotrimoxazole and tigecycline, with resistance rates of 45% and 10%. There were 127 strains carrying blaKPC gene, 26 strains carried blaNDM gene, 3 strains carried both two genes, 2 strains carried blaOXA-48 gene, and 2 strains carried no target drug-resistance genes. The positive rate of carbapenemases detected by mCIM was 95.56%, and by eCIM that of serinase was 93.18%, and of metal β-lactamase was 96.55%. 6 CRKP strains were positive for string test, with a separation rate of 3.75%, which was lower than that reported in other hospitals. The other 5 CR-hvKP strains produced K2 serotype were isolated from sputum samples, except one CR-hvKP strain produced K1 serotype were isolated from blood samples. All the 6 strains of CR-hvKP carried the drug resistance gene blaKPC and four virulence factors rmpA, rmpA2, iutA, iroN. Conclusion The detection rate of CRKP in our hospital is at the national average level. CRKP is resistant to a variety of antimicrobial agents and sensitive to tigecycline, and mainly produce KPC carbapenases. The detection rate of CR-hvKP is lower than that of other reported hospitals, and CR-hvKP has the characteristics of producing K1 capsular serum, carrying blaKPC resistance gene and a variety of virulence factors. When CRKP of K1 type produce KPC type carbapenemase, which also carry virulence factors rmpA, rmpA2, iutA and iroN, it can evolve into CR-hvKP, suggesting that clinical attention should be paid to identification and further strengthening of monitoring to prevent the spread and prevalence of hypervirulent resistant strains.