DeForge L E, Billeci K L, Kramer S M
Department of BioAnalytical Technology, Genentech, Inc., Mailstop 98, 1 DNA Way, South San Francisco, CA, 94080-4990, USA.
J Immunol Methods. 2000 Nov 1;245(1-2):79-89. doi: 10.1016/s0022-1759(00)00279-9.
Given the increasing incidence of methicillin resistant Staphylococcus aureus (MRSA) and the recent emergence of MRSA with a reduced susceptibility to vancomycin, alternative approaches to the treatment of infection are of increasing relevance. The purpose of these studies was to evaluate the effect of IFN-gamma on the ability of white blood cells to kill S. aureus and to develop a simpler, higher throughput bacterial killing assay. Using a methicillin sensitive clinical isolate of S. aureus, a clinical isolate of MRSA, and a commercially available strain of MRSA, studies were conducted using a killing assay in which the bacteria were added directly into whole blood. The viability of the bacteria in samples harvested at various time points was then evaluated both by the classic CFU assay and by a new assay using alamarBlue. In the latter method, serially diluted samples and a standard curve containing known concentrations of bacteria were placed on 96-well plates, and alamarBlue was added. Fluorescence readings were taken, and the viability of the bacteria in the samples was calculated using the standard curve. The results of these studies demonstrated that the CFU and alamarBlue methods yielded equivalent detection of bacteria diluted in buffer. For samples incubated in whole blood, however, the alamarBlue method tended to yield lower viabilities than the CFU method due to the emergence of a slower growing subpopulation of S. aureus upon incubation in the blood matrix. A significant increase in bacterial killing was observed upon pretreatment of whole blood for 24 h with 5 or 25 ng/ml IFN-gamma. This increase in killing was detected equivalently by the CFU and alamarBlue methods. In summary, these studies describe a method that allows for the higher throughput analysis of the effects of immunomodulators on bacterial killing.
鉴于耐甲氧西林金黄色葡萄球菌（MRSA）的发病率不断上升，以及最近出现了对万古霉素敏感性降低的MRSA，感染治疗的替代方法变得越来越重要。这些研究的目的是评估γ干扰素对白细胞杀死金黄色葡萄球菌能力的影响，并开发一种更简单、高通量的细菌杀伤试验。使用金黄色葡萄球菌的甲氧西林敏感临床分离株、MRSA的临床分离株和市售的MRSA菌株，采用将细菌直接加入全血的杀伤试验进行研究。然后通过经典的菌落形成单位（CFU）试验和使用alamarBlue的新试验评估在不同时间点采集的样本中细菌的活力。在后一种方法中，将系列稀释的样本和含有已知浓度细菌的标准曲线置于96孔板上，并加入alamarBlue。读取荧光读数，并使用标准曲线计算样本中细菌的活力。这些研究结果表明，CFU法和alamarBlue法对缓冲液中稀释的细菌检测效果相当。然而，对于在全血中孵育的样本，由于在血液基质中孵育后出现生长较慢的金黄色葡萄球菌亚群，alamarBlue法的活力往往低于CFU法。在用5或25 ng/mlγ干扰素对全血进行24小时预处理后，观察到细菌杀伤显著增加。CFU法和alamarBlue法对这种杀伤增加的检测效果相当。总之，这些研究描述了一种方法，该方法允许对免疫调节剂对细菌杀伤的影响进行高通量分析。
