整合子在碳青霉烯耐药及敏感肺炎克雷伯菌中的分布特征及耐药相关性分析

The analysis of the distribution characteristics and drug resistance correlation of integrons in carbapenem-resistant and carbapenem-sensitive Klebsiella pneumoniae

目的分析碳青霉烯类耐药肺炎克雷伯菌(CRKP)与碳青霉烯类敏感肺炎克雷伯菌(CSKP)中整合子的分布差异和可变区耐药基因盒类型及耐药相关性。方法收集宁波地区2021年1月至2022年12月临床分离CRKP 96株及同期宁波大学附属李惠利医院非重复分离CSKP 96株,采用聚合酶链式反应(PCR)对其第1、2、3类整合子筛查,获得阳性整合子,再对其可变区及可变区启动子扩增后测序,分析其在实验菌株中的耐药相关性;采用多位点序列分型(MLST)分析菌株基因分型和同源性。结果192株肺炎克雷伯菌(KP)共检出第1类整合子84株,其中72株来自CRKP,12株来自CSKP,检出第2类整合子12株,均来自CRKP,未检出第3类整合子;第1类整合子可变区扩增后,其产物检测出6种不同耐药基因盒,以耐氨基糖苷类(aadA2,aadA1)为主(57.1%),第2类整合子阳性可变区基因盒仅dfrA27-arr-3-aadA11种;KP整合子阳性菌株对常用抗菌药物的耐药率明显高于整合子阴性菌株(P<0.05);第1类整合子可变区启动子各型均有分布,杂合子PcH2占比最高(40.3%),4株第2类整合子阳性菌株可变区启动子检测结果显示均为Pc2D-Pc2A-Pc2B-Pc2C型;CSKP中检出ST分型38种,ST23型最多,共11株(11.5%),CRKP中检出ST分型13种,ST11型最多,共36株(37.5%)。结论CRKP中整合子分布明显高于CSKP,携带耐药基因主要介导氨基糖苷类和甲氧苄啶类药物耐药,与耐药性明显相关,启动子类型呈多样性,整合子阳性菌株以ST11型为主,具有较高同源性,存在院内克隆传播可能。

Objective To analyze the differences in the distribution of integrons and the types of variable resistance gene boxes and the correlation with drug resistance between carbapenem-resistant Klebsiella pneumoniae(CRKP)and carbapenem-sensitive Klebsiella pneumoniae(CSKP).Methods 96 clinical isolates of CRKP and 96 non-repetitive isolates of CSKP from Ningbo Li Huili Hospital from January 2021 to December 2022 were collected.At the same time,polymerase chain reaction(PCR)was used to screen the integrons of categories 1,2 and 3.The positive integrons obtained were amplified and sequenced after their variable region and variable region promoter.The relationship between drug resistance and experimental strains was analyzed.Finally,multi-site sequence typing(MLST)was used to analyze the genotyping and homology of the strains.Results A total of 84 Class 1 integrons were detected from 192 Klebsiella pneumoniae(KP),of which 72 were from CRKP and 12 were from CSKP.12 class 2 integrons were all from CRKP,but no Class 3 integrons were detected.A total of 6 drug resistance gene boxes were detected in Class 1 variable region amplification products,mainly aminoglycoside resistance(aadA2,aadA1)(57.1%),and only one type of class 2 integron-positive variable region gene box was dfrA27-arr-3-aadA1.The drug resistance rate of KP integron-positive strain to common antibiotics was significantly higher than that of KP integron-negative strain(P<0.05).The variable region promoters of Class 1 integron were distributed in all types,and heterozygote PcH2 accounted for the highest proportion(40.3%).The variable region promoters of 4 Class 2 integron-positive strains were Pc2D-Pc2A-Pc2B-Pc2C types.38 ST types were detected in CSKP,with a maximum of 11 ST23 strains(11.5%),and 13 ST types were detected in CRKP,with a maximum of 36 ST11 strains(37.5%).Conclusion The distribution of integrons in CRKP is significantly higher than that of CSKP,and the gene carrying drug resistance mainly mediates aminoglycoside and trimethoprim drug resistance,which is significantly related to drug resistance.The promoter types are diverse,and the integron-positive strains are mainly ST11 type,which has high homology and the possibility of hospital-based cloning transmission.