Yadav Jagjit S, Khan Izhar U H, Fakhari Farnaz, Soellner Mathew B
Molecular Toxicology Division, Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati, Ohio, USA.
Appl Occup Environ Hyg. 2003 Nov;18(11):966-75. doi: 10.1080/10473220390237700.
Mycobacteria and pseudomonads occurring in modern metalworking fluids (MWF) have been implicated in occupational health hazards as causal agents for hypersensitivity pneumonitis (HP) and other respiratory illnesses in machine workers exposed to these fluids and their aerosols. Unlike the conventional cultural and biochemical methods, which are often slow and ambiguous and detect only culturable cells, DNA-based methods offer a time-saving alternative for reliable detection and identification of both culturable and nonculturable bacteria in MWF and for selective quantification of individual genera of pathogens of interest in these fluids. This is the first report on DNA-based direct detection of mycobacteria and pseudomonads in MWF without culturing. Genus-specific PCR approach was successfully applied for screening of field MWF samples originating from different industrial users for detection of mycobacteria or pseudomonads including both culturable and nonculturable cells. PCR in combination with amplicon DNA sequencing led to the identification of Mycobacterium chelonae, Pseudomonas nitroreducens, and an undefined Pseudomonas species from these fluids. Genome fingerprinting by pulsed-field gel electrophoresis (PFGE) on Mycobacterium isolates further showed that the isolates represented three strains of M. chelonae although the possibility of one of the strains being clonal with M. immunogenum cannot be excluded. In parallel efforts, a quantitative competitive PCR method developed based on the Pseudomonas-specific PCR was applied to quantify total P. fluorescens cells in contaminated metalworking fluid and MWF aerosol without culturing. The DNA-based protocols developed in this study will allow rapid screening of field MWF samples for the presence of both culturable and nonculturable cells and thus facilitate effective fluid management and timely exposure assessment.
现代金属加工液（MWF）中出现的分枝杆菌和假单胞菌被认为是职业健康危害因素，是接触这些液体及其气雾剂的机器工人发生超敏性肺炎（HP）和其他呼吸道疾病的病原体。与传统的培养和生化方法不同，传统方法通常耗时且结果不明确，只能检测可培养的细胞，基于DNA的方法提供了一种省时的替代方法，可用于可靠地检测和鉴定MWMWF中的可培养和不可培养细菌，并选择性定量这些液体中感兴趣的单个病原体属。这是关于基于DNA直接检测MWF中分枝杆菌和假单胞菌而无需培养的首次报告。属特异性PCR方法成功应用于筛选来自不同工业用户的现场MWF样品，以检测分枝杆菌或假单胞菌，包括可培养和不可培养的细胞。PCR与扩增子DNA测序相结合，从这些液体中鉴定出龟分枝杆菌、硝基还原假单胞菌和一种未定义的假单胞菌属物种。对分枝杆菌分离株进行脉冲场凝胶电泳（PFGE）基因组指纹分析进一步表明，这些分离株代表了三株龟分枝杆菌，尽管不能排除其中一株与免疫原性分枝杆菌克隆的可能性。同时，基于假单胞菌特异性PCR开发的定量竞争PCR方法用于定量污染金属加工液和MWF气雾剂中总荧光假单胞菌细胞，无需培养。本研究中开发的基于DNA的方案将允许快速筛选现场MWF样品中可培养和不可培养细胞的存在，从而有助于有效的液体管理和及时的暴露评估。
