Detection and Identification of Mycobacteria in Clinical

通过常规程序检测和鉴定分枝杆菌种类的抗酸杆菌需要从患者样本中培养微生物，然后测试分离株的各种表型特征。这些方法可能需要几天到一个或几个月的时间。几种用于检测生长抗酸杆菌培养物的高度特异的分子探针的开发，大大缩短了一些分枝杆菌分离株的鉴定时间。近年来，聚合酶链式反应和等温核酸扩增技术已被用于检测临床标本中的结核分枝杆菌，具有高度的特异性和合理的敏感性。目前，还没有FDA批准的扩增分析系统能够检测多种分枝杆菌，同时排除临床样本中常见的其他细菌的交叉反应信号。蛋白质输出是细菌致病过程中的一个重要方面，因为大多数细菌毒力因子都是胞外蛋白。虽然对分枝杆菌中的蛋白质输出途径知之甚少，但在其他细菌中，特别是在大肠杆菌中，总分泌(SEC)途径已经得到了广泛的研究。SecA1是大肠杆菌SecA蛋白的分枝杆菌同源物，SecA蛋白是一种重要的前蛋白转位酶ATPase，为蛋白质跨细胞膜的输出提供动力。分枝杆菌SecA途径的不同寻常之处在于它有两种SecA蛋白：SecA1是必需的管家SecA蛋白，而SecA2是一种非必需的辅助分泌因子。本工作的目的是探索利用secA1基因序列来鉴定分枝杆菌属。我们决定将编码233个氨基酸残基的secA1的700个碱基片段定位在蛋白质的N末端，它包括底物特异性结构域或SSD，以及蛋白质易位所必需的连续序列。由于分枝杆菌细胞壁的特殊性质，在比较了5种分枝杆菌现有的secA1基因序列后，我们推测SecA1蛋白在分枝杆菌属中可能是保守的，但在不同物种之间表现出一些氨基酸差异，可能反映了与致病性、细胞内生存、环境适应和逆境耐受性有关的特殊分泌需求。通过对44株参考菌株的检测，初步研究表明利用secA1基因序列对分枝杆菌属进行鉴定的潜力。该方法已成功应用于69株临床分离株，验证了该方法的可行性。已经提交了一份描述这一初始工作的手稿。未来的研究计划与武装部队病理研究所的医生一起进行。

Detection and identification of acid-fast bacilli of Mycobacterium species by conventional procedures requires growing the organisms from patient specimens and then testing the isolates for various phenotypic characteristics. These methods may take days to one or more months. The development of a few highly specific molecular probes for testing cultures growing acid-fast bacilli has greatly reduced the time to identification of some mycobacterial isolates. Recently, the polymerase chain reaction (PCR) and isothermal nucleic acid amplification techniques have become utilized in assays that offer a high degree of specificity and reasonable sensitivity for detection of Mycobacterium tuberculosis in clinical samples. At present, there are no FDA approved amplification assay systems that are capable of detecting multiple Mycobacterium species while excluding cross-reactive signals from other bacteria commonly present in clinical samples. Protein export is an important aspect of bacterial pathogenesis since a majority of bacterial virulence factors are extracytoplasmic proteins. Although little is known about the protein export pathway in Mycobacteria, the general secretory (Sec) pathway has been extensively studied in other bacteria, in particular in E. coli. SecA1 is the mycobacterial homologue of the E. coli SecA protein, an essential preprotein translocase ATPase that provides the driving force for the export of proteins across the cytoplasmic membrane. The mycobacterial SecA pathway is unusual in that it has two SecA proteins: SecA1 is the essential housekeeping SecA protein, while SecA2 is a non-essential accessory secretion factor. The purpose of this work is to investigate the use of secA1 gene sequences for the identification of Mycobacterium spp. We decided to target a 700-base fragment of secA1 coding for 233 amino acid residues located on the N-terminal half of the protein, and which includes the substrate specificty domain, or SSD, and contiguous sequences essential for protein translocation. Because of the particular nature of mycobacterial cell walls and after comparing the available secA1 gene sequences from 5 mycobacterial species, we hypothesized that SecA1 protein would be conserved in the genus Mycobacterium while exhibiting some amino acid differences among the different species, probably reflecting particular secretion needs related to pathogenicity, intracellular survival, environmental adaptability and stress tolerance. A preliminary study has shown the potential of using secA1 gene sequences for the identification of Mycobacterium spp by testing 44 reference strains as a foundation. The procedure has also been successfully applied to 69 clinical isolates to demonstrate the feasibility of this identification method. A manuscript has been submitted describing this inital work. Future studies are planned with physicians at the Armed Forces Institute of Pathology.