变栖克雷伯菌全基因组测序及毒力特征分析

职业与健康 ›› 2024, Vol. 40 ›› Issue (6): 732-738.

变栖克雷伯菌全基因组测序及毒力特征分析

沈兰凤¹, 胡仁静¹, 万林¹, 黄骏²

1.江南大学附属中心医院检验科,江苏无锡 214002;
2.无锡市锡山区疾病预防控制中心检验科,江苏无锡 214101

收稿日期:2023-08-19 修回日期:2023-10-09 出版日期:2024-03-15 发布日期:2026-03-16
通信作者: 黄骏,主管技师,E-mail：253397076@qq.com
作者简介:沈兰凤,女,主管技师,主要从事临床病原菌耐药机制及毒力研究工作。
基金资助:
无锡市卫健委科研项目（T202134）; 青海省海东市科技局科研项目（2021-HDKJ-Y1）

Whole-genome sequencing and virulence characteristics of Klebsiella variicola

SHEN Lanfeng¹, HU Renjing¹, WAN Lin¹, HUANG Jun²

1. Laboratory Department,Affiliated Central Hospital of Jiangnan University,Wuxi Jiangsu,214002, China;
2. Laboratory Department,Center for Disease Control and Prevention in Xishan District of Wuxi,Wuxi Jiangsu,214101, China

Received:2023-08-19 Revised:2023-10-09 Online:2024-03-15 Published:2026-03-16
Contact: HUANG Jun,Technician in charge,E-mail：253397076@qq.com

摘要/Abstract

摘要： 目的对变栖克雷伯菌进行全基因组测序及大蜡螟毒力实验,为变栖克雷伯菌分子毒力特征研究提供参考。方法收集江南大学附属中心医院2021年1—12月从患者血液中分离的变栖克雷伯菌共 3 株,进行菌种鉴定及全基因组测序;通过大蜡螟感染模型中大蜡螟幼虫lgLD₅₀的差异来进行变栖克雷伯菌毒力分析。结果 3株变栖克雷伯菌均可通过Vitek MS V3.2完成菌种鉴定,质谱图谱的8 250 m/z处均有特征峰。黏液丝实验显示wxkv2055黏液丝实验阳性。wxkv2055和wxkv2031仅对氨苄西林耐药,对其他抗生素均敏感,wxkv2096对氨苄西林、环丙沙星、左氧氟沙星、阿米卡星、庆大霉素、甲氧苄啶-磺胺甲噁唑耐药。全基因组测序显示,3株菌的GC含量为56.8%~57.2%,荚膜血清型包括KL14、KL16、KL34,O抗原分型包括O5、O3/O3a,MLST分型包括1169、595、718,wxkv2055携带1个23.81kb的IncHI1B型毒力质粒。大蜡螟毒力模型分析显示,wxkv2055的lgLD₅₀低于wxkv2031和wxkv2096,差异均有统计学意义（均P<0.05）,wxkv2055的lgLD₅₀与对照高毒力菌NTUH-K2044比较,差异无统计学意义（P>0.05）。结论血液中分离的变栖克雷伯菌的wxkv2055携带毒力质粒,大蜡螟毒力模型提示其毒力较高,3株变栖克雷伯菌分子流行病学与肺炎克雷伯菌存在一定差异。目前基层实验室需提升对变栖克雷伯菌的鉴别能力,对变栖克雷伯菌的致病力和菌株特征进行深入研究,为临床诊疗提供指导。

关键词: 变栖克雷伯菌, 全基因组测序, 毒力, 比较基因组, 大蜡螟

Abstract: Objective To perform whole-genome sequencing of Klebsiella variicola and virulence experiments of Galleria mellonella,in order to provide reference for the study of molecular virulence characteristics of Klebsiella variicola. Methods A total of three strains of Klebsiella variicola isolated from patients blood in the Affiliated Central Hospital of Jiangnan University from January to December 2021 were collected,to perform the species identification and whole-genome sequencing. The difference of lgLD₅₀ of Galleria mellonella infection model was used to analyze the virulence of Klebsiella variicola. Results The three strains of Klebsiella variicola could be identified by Vitek MS V3.2,and the characteristic peak was found at 8 250 m/z in all the mass spectra. The string test showed that the wxkv2055 was positive. The dug susceptibility test showed that wxkv2055 and wxkv2031 were only resistant to ampicillin and sensitive to other antibiotics,while wxkv2096 was resistant to ampicillin,ciprofloxacin,levofloxacin,amikacin,gentamicin,and trimethoprim/sulfamethoxazole. The whole genome sequencing showed that the GC contents of the three strains were 56.8%-57.2%,the capsular serotypes included KL14,KL16,KL34,the O antigen typing included O5,O3/O3a,and the MLST typing included 1169,595,and 718. The wxkv2055 carries a 23.81 kb IncHI1B virulence plasmid. The virulence model analysis of Galleria mellonella showed that the lgLD₅₀ of wxkv2055 was significantly lower than that of wxkv2031 and wxkv2096（both P<0.05）,the lgLD₅₀ of wxkv2055 was not statistically different from that of the control hypervirulent bacteria NTUH-K2044（P>0.05）. Conclusions The Klebsiella variicola wxkv2055 isolated from blood carries virulence plasmid. The Galleria mellonella virulence model indicated that the virulence of wxkv2055 is higher. The molecular epidemiology of three strains of Klebsiella variicola is different from Klebsiella pneumoniae. At present,the grassroots laboratories need to improve the ability to identify Klebsiella variicola,conduct in-depth research on the pathogenicity and strain characteristics of Klebsiella variicola,and provide guidance for clinical diagnosis and treatment.

Key words: Klebsiella variicola, Whole-genome sequencing, Virulence, Comparative genome analysis, Galleria mellonella

中图分类号:

R117

沈兰凤, 胡仁静, 万林, 黄骏. 变栖克雷伯菌全基因组测序及毒力特征分析. [J]职业与健康, 2024, 40(6): 732-738.

SHEN Lanfeng, HU Renjing, WAN Lin, HUANG Jun. Whole-genome sequencing and virulence characteristics of Klebsiella variicola. [J]OCCUPATION AND HEALTH, 2024, 40(6): 732-738.

参考文献

[1] MARTÍNEZ-ROMERO E,RODRÍGUEZ-MEDINA N,BELTRÁN-ROJEL M,et al.Genome misclassification of Klebsiella variicola and Klebsiella quasipneumoniae isolated from plants,animals and humans[J].Salud Publica Mex,2018,60(1):56-62
[2] PEI N,LI Y,LIU C,et al.Large-scale genomic epidemiology of Klebsiella pneumoniae identified clone divergence with hypervirulent plus antimicrobial-resistant characteristics causing within-ward strain transmissions[J].Microbiol Spectr,2022,10(2):e0269821.
[3] MAATALLAH M,VADING M,KABIR M H,et al.Klebsiella variicola is a frequent cause of bloodstream infection in the stockholm area,and associated with higher mortality compared to K. pneumoniae[J].PLoS One,2014,9(11):e113539.
[4] IMAI K,ISHIBASHI N,KODANA M,et al.Clinical characteristics in blood stream infections caused by Klebsiella pneumoniae,Klebsiella variicola,and Klebsiella quasipneumoniae:A comparative study,Japan,2014-2017[J].BMC Infect Dis,2019,19(1):946.
[5] GIRARD V,MONNIN V,GIRAUD D,et al.Multicenter evaluation of the VITEK MS matrix-assisted laser desorption/ionization-time of flight mass spectrometry system for identification of bacteria,including Brucella,and yeasts[J].Eur J Clin Microbiol Infect Dis,2021,40(9):1909-1917.
[6] ALIKHAN N F,PETTY N K,BEN ZAKOUR N L,et al.BLAST ring image generator(BRIG):Simple prokaryote genome comparisons[J].BMC Genomics,2011,12:402.
[7] ROSENBLUETH M,MARTíNEZ L,SILVA J,et al.Klebsiella variicola,a novel species with clinical and plant-associated isolates[J].Syst Appl Microbiol,2004,27(1):27-35.
[8] RODRIGUES C,PASSET V,RAKOTONDRASOA A,et al.Identification of Klebsiella pneumoniae,Klebsiella quasipneumoniae,Klebsiella variicola and Related Phylogroups by MALDI-TOF Mass Spectrometry[J].Front Microbiol,2018,9:3000.
[9] LEUNG L M,FONDRIE W E,DOI Y,et al.Identification of the ESKAPE pathogens by mass spectrometric analysis of microbial membrane glycolipids[J].Sci Rep,2017,7(1):6403.
[10] BARRIOS-CAMACHO H,AGUILAR-VERA A,BELTRAN-ROJEL M,et al.Molecular epidemiology of Klebsiella variicola obtained from different sources[J].Sci Rep,2019,9(1):10610.
[11] SAXENBORN P,BAXTER J,TILEVIK A,et al.Genotypic characterization of clinical Klebsiella spp.isolates collected from patients with suspected community-onset sepsis,sweden[J].Front Microbiol,2021, 12:640408.
[12] 蒋晓圆. IncHI5、Inc(pRBL16)、Inc(pCT14)和Inc(pND6-2)型耐药质粒遗传特性研究[D].北京:军事科学院,2020.
[13] 董会玥. pLVPK-like毒力质粒在碳青霉烯耐药肺炎克雷伯菌(CRKP)中变异和进化的分子机制研究[D].郑州:郑州大学,2021.
[14] SHADKAM S,GOLI H R,MIRZAEI B,et al.Correlation between antimicrobial resistance and biofilm formation capability among Klebsiella pneumoniae strains isolated from hospitalized patients in Iran[J].Ann Clin Microbiol Antimicrob,2021,20(1):13.
[15] WU K M,LI L H,YAN J J,et al.Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044,a strain causing liver abscess and meningitis[J].J Bacteriol,2009,191(14):4492-4501.
[16] LAM M,WYRES K L,JUDD L M,et al.Tracking key virulence loci encoding aerobactin and salmochelin siderophore synthesis in Klebsiella pneumoniae[J].Genome Med,2018,10(1):77.
[17] LIU C,DU P,XIAO N,et al.Hypervirulent Klebsiella pneumoniae is emerging as an increasingly prevalent K. pneumoniae pathotype responsible for nosocomial and healthcare-associated infections in Beijing,China[J].Virulence,2020,11(1):1215-1224.
[18] BRISSE S,VERHOEF J.Phylogenetic diversity of Klebsiella pneumoniae and Klebsiella oxytoca clinical isolates revealed by randomly amplified polymorphic DNA,gyrA and parC genes sequencing and automated ribotyping[J].Int J Syst Evol Microbiol,2001,51(Pt 3):915-924.
[19] FONSECA E L,RAMOS N D,ANDRADE B G,et al.A one-step multiplex PCR to identify Klebsiella pneumoniae,Klebsiella variicola,and Klebsiella quasipneumoniae in the clinical routine[J].Diagn Microbiol Infect Dis,2017,87(4):315-317.
[20] MBELLE N M,FELDMAN C,SEKYERE J O,et al.Pathogenomics and evolutionary epidemiology of multi-drug resistant clinical klebsiella pneumoniae isolated from pretoria,South Africa[J].Sci Rep,2020,10(1):1232.
[21] DI D Y,JANG J,UNNO T,et al.Emergence of Klebsiella variicola positive for NDM-9,a variant of new delhi metallo-β-lactamase,in an urban river in South Korea[J].J Antimicrob Chemother,2017,72(4):1063-1067.
[22] POTTER R F,LAINHART W,TWENTYMAN J,et al.Population structure,antibiotic resistance,and uropathogenicity of Klebsiella variicola[J].mBio,2018,9(6):e02481-18.
[23] FORDE B M,DE OLIVEIRA D,FALCONER C,et al.Strengths and caveats of identifying resistance genes from whole genome sequencing data[J].Expert Rev Anti Infect Ther,2022,20(4):533-547.