感染性心内膜炎病原微生物的基因组特征与心脏病变严重程度的关联研究

Study of the association between genomic characteristics of microorganisms responsible for infective endocarditis and the severity of cardiac lesions

目的 本研究旨在通过全基因组测序和病理学评估,系统性地探讨感染性心内膜炎(Infective endocarditis, IE)患者的病原微生物基因组特征与宿主心脏组织病变严重程度之间的关联,分析病原微生物的基因变异对病变严重程度的影响。方法 本研究纳入2021年2月至2024年4月本院确诊的117例IE患者。采集病原微生物样本和心脏组织样本,使用Illumina NovaSeq 6000平台进行高通量全基因组测序,测序数据通过SPAdes组装和Prokka注释。利用GATK和FreeBayes检测单核苷酸多态性(SNPs)和插入缺失(INDELs),通过Mauve和MUMmer分析基因组结构变异(SVs)。对手术切除的瓣膜或心脏活检组织进行H&E染色和Masson三色染色,评估炎症、纤维化、组织坏死和血管增生等病理特征。使用qPCR验证关键毒力基因和耐药基因的表达水平。采用Spearman相关系数和多变量回归分析评估基因组变异与病变严重程度之间的关系。结果 基因组测序结果显示,117例样本的平均测序深度为(65.42±10.87)×,平均测序覆盖度为98.73%±1.25%。在病原微生物基因组中检测到的SNPs数量为5 471±1 157,INDELs数量为1 083±422。基因组结构变异的检测结果为重排12±5,倒位8±3,复制4±2,缺失6±2。主要毒力基因icaA、fnbA和clfA的检出率分别为72.65%、65.81%和58.12%;耐药基因mecA、vanA和blaZ的检出率分别为34.19%、23.93%和47.86%。qPCR验证结果显示,icaA基因在重度病变患者中表达水平显著升高(P<0.01),fnbA基因在中度和重度病变患者中显著升高(P<0.01),mecA基因在耐药性较强的病原菌中显著升高(P<0.01)。Spearman相关性分析显示,SNPs与病变严重程度评分的相关系数为0.432(P<0.01),INDELs为0.398(P<0.01)。多变量回归分析显示,SNPs(P=0.002)和INDELs(P<0.01)对病变严重程度具有独立影响,糖尿病也是显著影响因素(P=0.042)。结论 本研究发现病原微生物的基因组变异(SNPs和INDELs)与IE的病变严重程度呈显著正相关,且某些毒力基因和耐药基因在不同病变严重程度的患者中表达水平存在显著差异。

Objectivee To explore the association between the genomic characteristics of pathogenic microorganisms and the severity of host cardiac tissue lesions in patients with infective endocarditis(IE)through whole-genome sequencing and pathological evaluation,and to analyze the impact of genetic variation of pathogenic microorganisms on the severityof lesions.Methods This study included 117 patients with infective endocarditis diagnosed in the First Affiliated Hospital of Nanyang Medical College from February 2021 to April 2024.Pathogenic microorganism samples and cardiac tissue samples were collected,and high-throughput whole-genome sequencing was performed using the Ilumina NovaSeq 6000 platform.The sequencing data were assembled by SPAdes and annotated by Prokka.Single nucleotide polymorphisms(SNPs)and insertions and deletions(INDELs)were detected using GATK and FreeBayes,and genomic structural variations(SVs)were analyzed by Mauve and MUMmer.H&E staining and Masson trichrome staining were performed on surgically removed valves or cardiac biopsy tissues to evaluate pathological characteristics such as inflammation,fibrosis,tissue necrosis,and vascular proliferation.qPCR was used to verify the expression levels of key virulence genes and drug resistance genes.Spearman correlation coefficient and multivariate regression analysis were used to evaluate the relationship between genomic variation and lesion severity.ResultsThe genome sequencing results showed that the average sequencing depth of 117 samples was(65.42±10.87),and the average sequencing coverage was 98.73%±1.25%.The number of SNPs detected in the pathogen genome was 5471±1157,and the number of INDELs was 1083±422.The detection results of genomic structural variation were rearrangement 12±5,inversion 8±3,duplication 4±2,and deletion 6±2.The detection rates of major virulence genes icaA,fnbA,and clfA were 72.65%,65.81%,and 58.12%,respectively;the detection rates of drug resistance genes mecA,vanA,and blaZ were 34.19%,23.93%,and 47.86%,respectively.qPCR validation results showed that the expression level of icaA gene was significantly increased in patients with severe lesions(P<0.01),fnbA gene was significantly increased in patients with moderate and severe lesions(P<o.O1),and mecA gene was significantly increased in pathogens with strong drug resistance(P<o.O1).Spearman correlation analysis showed that the correlation coefficient between SNPs and lesion severity score was 0.432(P<0.01),and that of INDELs was 0.398(P<0.01).Multivariate regression analysis showed that SNPs(P=0.002)and INDELs(P<0.01)had independent effects on lesion severity,and diabetes was also a significant influencing factor(P=O.O42).Conclusion This study found that the genomic variations(SNPs and INDELs)of pathogenic microorganisms were significantly positively correlated with the severity of infective endocarditis,and the expression levels of some virulence genes and drug resistance genes were significantly different in patients with different lesion severity.