The University of Texas Graduate School of Biomedical Sciences, Houston, TX, USA.
J Clin Microbiol. 2011 Dec;49(12):4219-24. doi: 10.1128/JCM.01033-11. Epub 2011 Oct 5.
The alarming emergence of hypervirulent strains of Clostridium difficile with increased toxin production, severity of disease, morbidity, and mortality emphasizes the need for a culture method that permits simultaneous isolation and detection of virulent strains. The C. difficile toxins A and B are critical virulence factors, and strains can either be toxin-producing (virulent) or non-toxin-producing (nonvirulent). Strains that are isolated from human infections generally produce either toxin A or toxin B or both. The methods currently available for culturing C. difficile do not differentiate strains that produce active toxins from strains that do not produce toxins or produce inactive toxins. As a result, the identification and isolation of toxin-producing strains from stool is currently a two-step process. First, the stool is plated on a selective medium, and then suspected colonies are analyzed for toxin production or the presence of the toxin genes. We describe here a novel selective and differential culture method, the Cdifftox plate assay, which combines in a single step the specific isolation of C. difficile strains and the detection of active toxin. This assay was developed based on our recent finding that the A and B toxins of C. difficile cleave chromogenic substrates that have stereochemical characteristics similar to their natural substrate, UDP-glucose. The Cdifftox plate assay is shown here to be extremely accurate (99.8% effective) in detecting toxin-producing strains through the analysis of 528 C. difficile isolates selected from 50 tissue culture cytotoxicity assay-positive clinical stool samples. The Cdifftox plate assay advances and improves the culture approach such that only C. difficile strains will grow on this agar, and virulent strains producing active toxins can be differentiated from nonvirulent strains, which do not produce active toxins. This new method reduces the time and effort required to isolate and confirm toxin-producing C. difficile strains.
产毒力更强的艰难梭菌（Clostridium difficile）菌株的惊人出现，增加了毒素的产生、疾病的严重程度、发病率和死亡率，这强调了需要一种能够同时分离和检测毒力菌株的培养方法。艰难梭菌毒素 A 和 B 是关键的毒力因子，菌株可以是产毒（毒力）的，也可以是非产毒（非毒力）的。从人类感染中分离的菌株通常产生毒素 A 或毒素 B 或两者兼有。目前用于培养艰难梭菌的方法无法区分产生活性毒素的菌株与不产生毒素或产生非活性毒素的菌株。因此，从粪便中鉴定和分离产毒菌株目前是一个两步的过程。首先，将粪便接种在选择性培养基上，然后分析可疑菌落是否产毒或是否存在毒素基因。我们在这里描述了一种新的选择性和差异培养方法，即 Cdifftox 平板检测法，它将艰难梭菌菌株的特异性分离和活性毒素的检测结合在一个步骤中。该检测法是基于我们最近的发现，即艰难梭菌的 A 和 B 毒素切割具有与天然底物 UDP-葡萄糖相似立体化学特征的显色底物。通过对从 50 份组织培养细胞毒性测定阳性的临床粪便样本中选择的 528 株艰难梭菌分离株的分析，显示 Cdifftox 平板检测法在检测产毒菌株方面非常准确（有效率为 99.8%）。Cdifftox 平板检测法推进并改进了培养方法，使得只有艰难梭菌菌株能够在这种琼脂上生长，并且能够区分产生活性毒素的毒力菌株和不产生活性毒素的非毒力菌株。这种新方法减少了分离和确认产毒艰难梭菌菌株所需的时间和精力。
