新生儿重症监护病房患儿粪便肺炎克雷伯菌分离株毒力特征分析

Virulence characteristics of Klebsiella pneumoniae isolates from the feces of children in neonatal intensive care unit

摘要:

【背景】肺炎克雷伯菌(Klebsiella pneumoniae)常存在于人体上呼吸道和肠道，属于条件性致病菌。目前由于临床上多重耐药菌株的出现及细菌毒力的不断增强使其受到了广泛关注。新生儿重症监护病房(neonatal intensive care unit,NICU)患儿免疫系统不完善，高毒力肺炎克雷伯菌的存在具有院内传播的潜在风险。【目的】探究2021年北京妇产医院/北京妇幼保健院NICU患儿粪便肺炎克雷伯菌存在情况与分离株毒力特征。【方法】对采集自北京妇产医院/北京妇幼保健院NICU患儿421份粪便样本进行肺炎克雷伯菌分离培养、全基因组测序及毒力基因分析。利用毒力因子数据库(virulence factor database,VFDB)和Kleborate数据库进行肺炎克雷伯菌毒力基因注释与多位点序列分型(multilocus sequence typing,MLST)，并通过拉丝试验、大蜡螟生存试验及细胞毒力试验验证各菌株毒力。【结果】在421份粪便样品中共检出13株肺炎克雷伯菌，检出率为3.1%，肺炎克雷伯菌携带者主要集中于ABO溶血、低出生体重、新生儿肺炎和新生儿高胆红素血症患者。肺炎克雷伯菌分离株基因组大小为5 Mb，基因组组装后序列长度中位数N₅₀大于20 kb，G+C含量为57%。13株菌分别具有13种ST型别、13种K血清型与7种O血清型。利用VFDB注释肺炎克雷伯菌分离株的846个毒力基因，菌株Kp50具有最多的毒力蛋白和毒力结构基因，Kleborate毒力因子评分中菌株Kp50评分最高。表型试验发现在13株肺炎克雷伯菌中，仅有菌株Kp50具有高黏表型和更强的细胞毒力，具有铁载体表达能力的菌株Kp50、Kp260、Kp185和Kp273在大蜡螟幼虫试验中呈现较高的毒力。【结论】北京妇产医院/北京妇幼保健院NICU患儿粪便样本中肺炎克雷伯菌检出率较低，分离得到的肺炎克雷伯菌分型呈现高度多样性，存在携带多种毒力基因和表型的高毒力菌株，需引起临床重视，做好高毒力菌株在院内传播的预防与控制。

关键词:肺炎克雷伯菌;新生儿重症监护病房;全基因组测序;毒力基因;毒力表型

Abstract:

[Background] Klebsiella pneumoniae, a conditional pathogen, often colonizes the upper respiratory tract and intestinal tract of the human body. Due to the emergence of clinically multi-drug resistant strains and the continuous enhancement of strain virulence, this pathogen has received extensive attention. Children in the neonatal intensive care unit (NICU) have an imperfect immune system and are exposed to the risk of nosocomial transmission of highly virulent strains of K. pneumoniae. [Objective] To investigate the presence and virulence characteristics of K. pneumoniae isolates from feces of children in the NICU of Beijing Obstetrics and Gynecology Hospital/Beijing Maternal and Child Health Care Hospital in 2021. [Methods] The strain isolation, whole genome sequencing, and virulence gene analysis of K. pneumoniae were carried out on 421 fecal samples collected from children in the NICU of Beijing Obstetrics and Gynecology Hospital/Beijing Maternal and Child Health Care Hospital. The virulence factor database (VFDB) and Kleborate were used for annotation of the virulence genes and the multilocus sequence typing (MLST) of K. pneumoniae, and the virulence of each strain was examined by the string test, the Galleria mellonella survival test, and the cytotoxicity test. [Results] A total of 13 strains of K. pneumoniae were detected in the 421 fecal samples, with a detection rate of 3.1%. K. pneumoniae was mainly carried by the patients with ABO hemolytic, low birth weight, neonatal pneumonia, and neonatal hyperbilirubinemia. The genome of K. pneumoniae isolates was 5 Mb, with median sequence length after genome assembly N₅₀>20 kb and G+C content of 57%. The 13 strains presented 13 ST types, 13 K serotypes, and 7 O serotypes. Among the 846 virulence genes annotated by the VFDB, strain Kp50 had the largest number of virulence proteins and virulence genes, and this strain had the highest virulence score determined by Kleborate. Phenotypic tests revealed that among the 13 K. pneumoniae strains, only strain Kp50 had a high mucoid phenotype and stronger cytotoxicity. Strains Kp50, Kp260, Kp185, and Kp273, which had siderophore expression ability, presented stronger virulence in the larvae of Galleria mellonella. [Conclusion] K. pneumoniae isolates from the fecal samples of children in the NICU of Beijing Obstetrics and Gynecology Hospital/Beijing Maternal and Child Health Care Hospital showed a low detection rate and high diversity. There are highly virulent strains carrying multiple virulence genes and phenotypes. High attention should be paid to the prevention and control of the spread of highly virulent strains in hospitals.

Key words:Klebsiella pneumoniae;neonatal intensive care unit (NICU);whole genome sequencing;virulence gene;virulence phenotype