Mycobacteria in metalworking fluids

金属加工液中的分枝杆菌

• 批准号: 6857936
• 负责人: Jagjit S Yadav
• 金额: $ 26.01万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-09-30 至 2008-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6857936
• 关键词: Mycobacterium; allergic pneumonitis; clinical research; environmental exposure; fluorescent in situ hybridization; genetic strain; human subject; metal working fluid; method development; occupational hazard; polymerase chain reaction

DESCRIPTION: Non-tuberculous mycobacteria (NTM) frequently colonize the modem metalworking fluids (MWFs) and are increasingly being implicated in occupational respiratory illnesses such as hypersensitivity pneumonitis (HP) in machine workers. Their early detection/recovery and characterization will help take pre-emptive measures and develop intervention strategies. Our first funding has led to the development of PCR based protocols for culture-independent detection and quantification of NTM in water based MWF, coupled with isolation and genotypic identification of several NTM strains from MWF, which belonged to two species M. immunogenum and M. chelonae. Our proposed future goals are to develop next generation of simplified DNA-based approaches that can be adapted in in-house laboratories and to investigate the relative biocide susceptibility and antigenic potential of these MWF isolated NTM strains (genotypes). The specific aims are (1). to develop protocols for efficient NTM recovery and simple DNA-based detection and quantification of NTM from different MWF types; (2). to determine the relative biocide susceptibility of the available and new NTM strains from MWF by optimization and use of a high throughput protocol; (3). to investigate antigenic characteristics of the available and HP-linked NTM strains. Immunomagnetic separation using Mycobacterium-specific polyclonal antibodies will be developed for selective cell recovery for improved NTM detection and cultural isolation. Our current PCR protocol will be adapted for colorimetric detection of NTM. A new protocol based on Fluorescence in-situ hybridization (FISH) will be developed for microscopeassisted culture-independent detection and quantification of the MWF-specific mycobacterial species. A high throughput protocol based on intracellular ATP will be optimized and used for measurement of biocide susceptibility. Relative antigenic potential of the MWF prevalent strains/genotypes will be determined based on cellular immune response in the sensitized blood cells from HP patients or MWF exposed workers. M. immunogenum polyclonal antibodies or HP patient sera will be applied as probes to identify the putative protein antigens in the cell protein lysates and culture filtrates of the identified antigenic strain. The resulting information will help NORA's objectives by providing a set of practical methods for mycobacterial contamination- and exposure- assessment and a strain characteristics database, for facilitating development of intervention strategies related to MWF exposures.

描述：非结核分枝杆菌(NTM)经常在现代金属加工液(MWF)中定植，并越来越多地与职业呼吸系统疾病有关，如机械工人的过敏性肺炎(HP)。它们的早期发现/恢复和特征将有助于采取先发制人的措施和制定干预战略。我们的第一笔资金导致了基于PCR的水基MWF中NTM独立培养检测和定量的方法的发展，并从MWF中分离和鉴定了几株NTM菌株，它们属于两个物种：免疫分枝杆菌和龟分枝杆菌。我们提出的未来目标是开发下一代可在内部实验室采用的基于DNA的简化方法，并调查这些MWF分离的NTM菌株(基因型)的相对杀生剂敏感性和抗原性潜力。具体目标是(1)。建立有效回收不同MWF类型的NTM的方法和基于DNA的简便检测和定量方法；通过优化和使用高通量方案，确定MWF现有的和新的NTM菌株对杀菌剂的相对敏感性；目的研究现有的与幽门螺杆菌相关的NTM株的抗原性。使用分枝杆菌特异性多克隆抗体的免疫磁性分离将被开发用于选择性细胞回收，以改进NTM检测和培养分离。我们目前的聚合酶链式反应方法将适用于NTM的比色检测。一种基于荧光原位杂交(FISH)的新方法将被开发用于MWF特定分枝杆菌种类的显微镜易于培养独立的检测和定量。将优化一种基于细胞内ATP的高通量检测方法，并将其用于杀菌剂敏感性的检测。根据HP患者或接触MWF工人致敏血细胞的细胞免疫反应，将确定MWF流行株/基因型别的相对抗原性。免疫原性支原体多克隆抗体或幽门螺杆菌患者血清将作为探针，在已鉴定的抗原菌株的细胞蛋白裂解物和培养滤液中鉴定可能的蛋白抗原。由此产生的信息将通过提供一套实用的分枝杆菌污染和暴露评估方法和菌株特性数据库来帮助Nora实现目标，以促进与MWF暴露相关的干预策略的发展。