Mycobacteria in metalworking fluids

描述：非结核分枝杆菌（NTM）经常定植在现代金属加工液（MWFs）中，并且越来越多地与机械工人的职业性呼吸系统疾病（如过敏性肺炎（HP））有关。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策略。我们的第一笔资金已经用于开发基于PCR的方案，用于水基MWF中NTM的培养独立检测和定量，以及从MWF中分离和基因型鉴定几个NTM菌株，它们属于M. immunogenum和M. chelonae两个物种。我们提出的未来目标是开发下一代简化的基于dna的方法，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)制定有效的NTM回收方案和基于dna的简单检测和定量不同MWF类型的NTM；(2)通过优化和使用高通量方案，确定MWF中现有菌株和新菌株的相对杀菌剂敏感性；(3)研究现有NTM菌株和hp连锁NTM菌株的抗原性。将开发分枝杆菌特异性多克隆抗体的免疫磁分离技术，用于选择性细胞回收，以提高NTM的检测和培养分离。我们目前的PCR方案将适用于NTM的比色检测。将开发一种基于荧光原位杂交（FISH）的新方案，用于显微镜辅助培养独立的mwf特异性分枝杆菌种类的检测和定量。基于细胞内ATP的高通量方案将被优化并用于测量杀菌剂敏感性。MWF流行菌株/基因型的相对抗原潜力将根据HP患者或MWF暴露工人致敏血细胞的细胞免疫反应来确定。将免疫原分枝杆菌多克隆抗体或HP患者血清作为探针，在鉴定的抗原菌株的细胞蛋白裂解物和培养滤液中鉴定推定的蛋白抗原。由此产生的信息将有助于NORA的目标，为分枝杆菌污染和暴露评估提供一套实用的方法和菌株特征数据库，以促进与MWF暴露有关的干预策略的发展。

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.