基于基因组学CRKP的RND外排泵分布及生物膜研究

Distribution of RND efflux pumps and biofilm studies based on genomic CRKP*

目的探讨耐药结节细胞分化(RND)外排泵在耐碳青霉烯类肺炎克雷伯菌(CRKP)中的分布情况及与生物膜形成的作用。方法收集该院临床分离的CRKP菌株,采用Whonet5.6软件分析其标本来源、科室分布等;采用生物膜形成试验观察不同培养时间生物膜形成情况;采用二代测序数据分析RND外排泵基因、耐药基因、分子分型等;采用PCR扩增RND外排泵基因。结果63株CRKP中87.3%来源于痰液,77.5%分离自重症监护室(ICU)。二代测序显示,多数菌株携带10余种耐药基因,55.6%菌株耐药基因与表型匹配;携带RND外排泵基因的61株菌株中,PCR扩增阳性23株(37.7%),荚膜血清型主要为KL64(95.7%);PCR扩增阴性38株(62.3%),荚膜血清型较分散,包括KL64(77.5%)、KL47(10.0%)、K50(7.5%)和K20(5.0%)这4种;24 h生物膜阳性31株(49.2%),36 h生物膜阳性48株(76.2%);RND阳性与阴性菌株的生物膜形成能力差异无统计学意义(P>0.05)。结论CRKP主要分离自ICU老年患者,多数菌株存在RND外排泵基因但未表达,与生物膜形成无明显相关,其在多重耐药中的作用有待进一步探讨。

Objective To investigate the distribution of drug-resistant nodular cell differentiation(RND)efflux pump in carbapenem-resistant Klebsiella pneumoniae(CRKP)and its role in biofilm formation.Methods The clinical isolates of CRKP were collected from the hospital.Whonet5.6 software was used to analyze the specimen source and department distribution.Biofilm formation test was used to observe the biofilm formation at different culture times.The next generation sequencing data were used to analyze the RND efflux pump genes,drug resistance genes,molecular typing,etc.The RND efflux pump gene was amplified by PCR.Results Among the 63 CRKP strains,87.3%were isolated from sputum,and 77.5%were isolated from intensive care unit(ICU).The results of next generation sequencing showed that most of the strains carried more than 10 drug resistance genes,and 55.6%of the strains had drug resistance genes matched with the phenotype.Among the 61 strains carrying RND efflux pump gene,23 strains(37.7%)were positive by PCR,and the predominant capsule serotype was KL64(95.7%).38 strains(62.3%)were negative for PCR amplification,and the capsule serotypes were dispersed,including KL64(77.5%),KL47(10.0%),K50(7.5%)and K20(5.0%).Totally 31 strains(49.2%)were positive for biofilm at 24 h and 48 strains(76.2%)at 36 h.There was no significant difference in biofilm formation ability between RND positive and negative strains(P>0.05).Conclusion CRKP is mainly isolated from elderly patients in ICU.Most of the CRKP strains have RND efflux pump genes but do not express them.There is no significant correlation between RND efflux pump genes and biofilm formation,the role in multiple drug resistance remains to be further discussed.