摘要
目的 探讨携带不同耐药基因对耐碳青霉烯类肺炎克雷伯菌(CRKP)的生长适应性及毒力作用的影响。方法 通过PCR方法扩增碳青霉烯类耐药基因(bla_(KPC)、bla_(NDM)、bla_(VIM)、bla_(OXA)、bla_(IMP))及β-内酰胺类耐药基因(bla_(TEM)、bla_(SHV)、bla_(CTX)、bla_(LAP)),测序确定基因型。扩增7个管家基因rpoB、gapA、mdh、pgi、phoE、infB和tonB,测序确定基因型并获得菌株ST类型。根据所携带的碳青霉烯类耐药基因不同,将菌株进行分组,在不同亚胺培南抗生素浓度(0、2、8 mg/mL)下培养细菌,绘制生长曲线,比较各组细菌的生长适应性变化;通过线虫侵染模型用CRKP感染线虫,绘制线虫生存曲线,比较各组细菌对线虫的毒力作用。结果 无抗生素压力下各组菌株生长适应性相似;在2 mg/mL亚胺培南培养基中,双抗性基因组中bla_(KPC)+bla_(NDM)组、bla_(IMP)+bla_(OXA)组生长适应性最强,单抗性基因组中bla_(IMP)组生长适应性最低,差异具有统计学意义(P<0.05)。在8 mg/mL亚胺培南培养基中,双抗性基因组中bla_(KPC)+bla_(NDM)组和bla_(NDM)+bla_(OXA)组生长适应性最强,且分别强于单抗性基因组bla_(KPC)组和bla_(OXA)组;单抗性基因组中bla_(OXA)组生长适应性最弱,bla_(KPC)和bla_(NDM)生长最快,差异具有统计学意义(P<0.05)。在无抗生素压力下,各组菌株对线虫毒力作用有所不同,bla_(KPC)组、bla_(KPC)+bla_(NDM)组和bla_(IMP)+bla_(OXA)组生存曲线右移,线虫中位生存时间延长,菌株毒力减弱;bla_(IMP)+bla_(NDM)组生存曲线左移,线虫中位生存时间缩短,菌株毒力增强,差异具有统计学意义(P<0.05)。结论 携带不同的耐药基因以及携带单个或多个耐药基因均会对菌株的生长适应性和毒力产生不同的影响。
Objective To investigate the effects of different drug resistance genes on the growth adaptability and virulence of carbapenem-resistant Klebsiella pneumoniae.Methods The carbapenem resistance genes(bla_(KPC),bla_(NDM),bla_(VIM),bla_(OXA),bla_(IMP)) and β-lactam resistance genes(bla_(TEM),bla_(SHV),bla_(CTX),bla_(LAP)) were amplified by PCR,and the genotypes were determined by sequencing.Seven housekeeping genes,including rpoB,gapA,mdh,pgi,phoE,infB and tonB,were amplified and sequenced to determine the genotypes and obtain the ST types of strains.According to the different carbapenem resistance genes,the strains were divided into different groups,and then cultured at different concentrations of imipenem(0 mg/mL,2 mg/mL and 8 mg/mL),the growth curve was drawn to compare the growth adaptability of the strains in each group.According to the C.elegans infection model,infect C.elegans with carbapenem-resistant Klebsiella pneumoniae,the survival curve of C.elegans was drawn to compare the virulence of each group of strain.Results The growth adaptability of strains in all groups was similar without antibiotic pressure.In 2 mg/mL imipenem medium,the growth adaptability of bla_(KPC)+bla_(NDM) group and bla_(IMP)+bla_(OXA) group in the dual resistance gene groups was the strongest,while that of bla_(IMP) group in the single resistance gene groups was the lowest,and the difference was statistically significant.In 8 mg/mL imipenem medium,bla_(KPC)+bla_(NDM) group and bla_(NDM)+bla_(OXA) group had the strongest growth adaptability,which was stronger than bla_(KPC) group and bla_(OXA )group,respectively.Among the single resistance gene groups,bla_(OXA )group had the weakest growth adaptability,while bla_(KPC) and bla_(NDM) had the fastest growth,and the differences were statistically significant.In the absence of antibiotic pressure,the virulence of each group was different.The survival curve of bla_(KPC) group,bla_(KPC)+bla_(NDM) group and bla_(IMP)+bla_(OXA) group shifted to the right,the median survival time of C.elegans was prolonged,means that the virulence of strains was weakened.The survival curve of bla_(IMP)+bla_(NDM) group shifted to the left,the median survival time of C.elegans was shortened,suggested that the virulence of strain was enhanced,and the difference was statistically significant.Conclusion Carrying different drug resistance genes and carrying single or multiple drug resistance genes have different effects on the growth adaptability and virulence of strains.
作者
瞿巧莉
李霄
王鹏飞
胡莹
单斌
毛俊
陈如才
QU Qiaoli;LI Xiao;WANG Pengfei;HU Ying;SHAN Bin;MAO Jun;CHEN Rucai(Department of Clinical Laboratory,The Second Affiliated Hospital of Kunming Medical University,Kunming 650101,China;State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan,Key Laboratory for Southwest Microbial Diversity of the Ministry of Education,Yunnan University,Kunming 650032,China;Department of Key Laboratory,The Second Affiliated Hospital of Kunming Medical University,Kunming 650101,China;Department of Clinical Laboratory,The First Affiliated Hospital of Kunming Medical University,Kunming 650001,China)
出处
《西部医学》
2024年第3期325-332,共8页
Medical Journal of West China
基金
国家自然科学基金项目(31960029)
云南省科技厅科技计划项目[2019FE001(-229)]
云南省卫生健康委员医学学科带头人培养计划(D-2018041)
云南省卫生健康委员会医学后备人才培养计划(H-2019062)。
关键词
耐碳青霉烯类肺炎克雷伯菌
质粒
适应性
毒力
耐药性
Carbapenem-resistant Klebsiella pneumoniae
Plasmid
Adaptability
Virulence
Drug resistance
