Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients
Culture-independent identification of pathogenic bacteria and polymicrobial infections in the genitourinary tract of renal transplant recipients
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DOI：
10.1128/jcm.41.12.5500-5510.2003
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发表时间：
2003-12-01
影响因子：
9.4
通讯作者：
Chakraborty, T
中科院分区：
文献类型：
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作者：
Domann, E;Hong, G;Chakraborty, T
肾移植受者易发生由常见尿路病原体和机会性细菌引起的尿路感染，经常导致显著的多种微生物感染。在这项研究中，建立了一种不依赖于培养的基于16 S rRNA的方法来鉴定不寻常的，苛养的或厌氧的细菌，并调查尿路标本中的细菌多样性。用变性高效液相色谱法（DHPLC）（WAVE System）分析包含16 S rRNA的维斯至V8区域的相似大小的扩增子。从常见的尿路致病性细菌的单一扩增子的人工混合物产生不同的峰谱，其身份通过测序单独收集的峰产物来确认。我们评估了该方法在109例肾移植受者尿路标本中的应用;发现培养阳性标本的相关性为100%，DHPLC产生峰型。然而，对于培养阴性标本，DHPLC有助于检测新的峰形。这些单个峰的DNA测序用于鉴定所涉及的细菌。因此，在PCR阳性但培养阴性的样品中，该方法允许检测先前已知的泌尿病原体，如溶脲棒状杆菌和迷走加德纳菌，但也检测不常见的病原体，包括溶粪厌氧球菌、普通拟杆菌、小类杆菌、具核梭杆菌、惰性乳杆菌、Leptotrichia cavonii、口腔普雷沃氏菌、瘤胃普雷沃氏菌、水生拉恩氏菌、和中间链球菌被检测为单一病原体或作为多种微生物感染的组分。所描述的方法是可重复的和快速的，并且能够对微生物群落和多微生物感染进行基于DHPLC的分析和基于序列的调查。对肾移植受者感染的详细了解将指导抗生素治疗方案，并为降低移植物排斥反应的风险提供新的视角。
Renal transplant recipients are predisposed to urinary tract infections caused by both common uropathogens and opportunistic bacteria resulting frequently in significant polymicrobial infections. In this study, a culture-independent 16S rRNA-based approach was established to identify unusual, fastidious, or anaerobic bacteria and to investigate bacterial diversity in urinary tract specimens. Similarly sized amplicons encompassing the VIS to V8 region of the 16S rRNA were analyzed with denaturing high-performance liquid chromatography (DHPLC) (WAVE System). Artificial mixtures of single amplicons from commonly encountered uropathogenic bacteria produced distinct peak profiles whose identities were confirmed by sequencing individually collected peak products. We evaluated the application of the method on 109 urinary tract specimens from renal transplant recipients; 100% correlation was found for culture-positive specimens, and DHPLC generated peak profiles. However, for culture-negative specimens, DHPLC facilitated the detection of novel peak profiles. DNA sequencing of these individual peaks was used to identify the bacteria involved. Thus, in PCR-positive but culture-negative samples the method allowed detection of previously known uropathogens such as Corynebacterium urealyticum and Gardnerella vaginalis, but also unusual agents including Anaerococcus Zactolyticus, Bacteroides vulgatus, Dialister invisus, Fusobacterium nucleatum, Lactobacillus iners, Leptotrichia amnionii, Prevotella buccalis, Prevotella ruminicola, Rahnella aquatilis, and Streptococcus intermedius were detected as single pathogens or as constituents of polymicrobial infections. The method described is reproducible and rapidly and enables both DHPLC-based profiling and sequence-based investigation of microbial communities and polymicrobial infections. A detailed understanding of infections found in recipients of renal transplants will guide antibiotic therapy regimens and provide new perspectives for decreasing the risk of graft rejection.