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.

产品说明：非结核分枝杆菌（NTM）经常定植在现代金属加工液（MWF）中，并越来越多地与机械工人的职业性呼吸道疾病如过敏性肺炎（HP）有关。早期发现/恢复和定性将有助于采取预防措施和制定干预战略。我们的第一笔资金已经导致开发了基于PCR的方案，用于在水基MWF中非培养检测和定量NTM，以及从MWF中分离和基因型鉴定几种NTM菌株，其属于两个物种M。免疫原菌和M.龟类。我们提出的未来目标是开发下一代简化的基于DNA的方法，这些方法可以在内部实验室中进行调整，并研究这些MWF分离的NTM菌株（基因型）的相对杀菌剂敏感性和抗原潜力。具体目标是（1）。开发有效的NTM回收和简单的基于DNA的检测和定量的NTM从不同的MWF类型的协议;（2）。通过优化和使用高通量方案来确定来自MWF的可用的和新的NTM菌株的相对杀生物剂敏感性;（3）.研究可用的和HP连锁的NTM菌株的抗原特性。将开发使用分枝杆菌特异性多克隆抗体的免疫磁性分离，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PCR方案将适用于NTM的比色检测。一个新的协议的基础上荧光原位杂交（FISH）将开发显微镜辅助培养的独立检测和量化的MWF特异性分枝杆菌物种。基于细胞内ATP的高通量方案将被优化并用于测量杀生物剂敏感性。将根据HP患者或MWF暴露工人致敏血细胞中的细胞免疫应答，确定MWF流行菌株/基因型的相对抗原潜力。M.免疫原多克隆抗体或HP患者血清将被用作探针，以鉴定细胞蛋白裂解物和经鉴定的抗原菌株的培养物中的推定蛋白抗原。由此产生的信息将有助于诺拉的目标，提供一套实用的方法，分枝杆菌污染和暴露评估和菌株特征数据库，以促进制定有关MWF暴露的干预战略。