Green G L, Brostoff J, Hudspith B, Michael M, Mylonaki M, Rayment N, Staines N, Sanderson J, Rampton D S, Bruce K D
Life Sciences, King's College London, London, UK.
J Appl Microbiol. 2006 Mar;100(3):460-9. doi: 10.1111/j.1365-2672.2005.02783.x.
To study large intestinal mucosal bacterial communities by Denaturing Gradient Gel Electrophoresis (DGGE) profiling and sequencing of 16S rRNA gene polymerase chain reaction (PCR) products amplified from DNA extracted from colorectal biopsies taken from healthy individuals. The specific aims were to determine how similar the mucosa-associated bacterial communities are within and between individuals and also to characterize the phylogenetic origin of isolated DGGE bands.
Human colorectal biopsies were taken at routine colonoscopy from 33 patients with normal looking mucosa. The DNA was extracted directly from single biopsies and the bacterial 16S rDNA PCR amplified. The PCR products were profiled using DGGE to generate a fingerprint of the dominant members of the bacterial community associated with the biopsy. The reproducibility of this method was high (>98%). Washed and unwashed biopsies gave similar DGGE banding patterns (Median Similarity Coefficient - MSC 96%, InterQuartile Range - IQR 3.0%, n = 5). Adjacent biopsies sampled from the same patient using different forceps gave similar DGGE profiles (MSC 94%, n = 2). Two colorectal biopsies sampled at locations 2-5 cm apart, from each of 18 patients, resulted in very similar profiles (MSC 100%, IQR 2.8%). Biopsies sampled from different locations within the large intestine of the same patient also gave similar DGGE profiles (MSC 98% IQR 3.3%n = 6). Although all patients (n = 33) gave different DGGE profiles, some similarity (c. 34%) was observed between profiles obtained from 15 patients arbitrarily selected. 35 DGGE bands were excised and sequenced. Many were found to be most closely related to uncultured bacterial sequence entries in the Genbank database. Others belonged to typical gut bacterial genera including Bacteroides, Ruminococcus, Faecalibacterium and Clostridium.
Bacterial communities adherent to colorectal mucosa within a normal patient show little variation; in contrast, mucosal bacterial communities sampled from different patients with normal colorectal mucosa show a high degree of variation.
This research demonstrates that DGGE profiling of 16S rRNA gene PCR products amplified from DNA extracted directly from mucosal samples offers fresh insight into the bacterial communities that are adherent to colorectal mucosa. These findings are important with respect to further studies on the gastrointestinal tract in health and disease.
通过变性梯度凝胶电泳（DGGE）分析以及对从健康个体的结肠直肠活检组织提取的DNA扩增的16S rRNA基因聚合酶链反应（PCR）产物进行测序，研究大肠黏膜细菌群落。具体目标是确定个体内部和个体之间黏膜相关细菌群落的相似程度，并对分离出的DGGE条带的系统发育起源进行表征。
在常规结肠镜检查时从33例黏膜外观正常的患者获取人类结肠直肠活检组织。直接从单个活检组织中提取DNA，并对细菌16S rDNA进行PCR扩增。使用DGGE对PCR产物进行分析，以生成与活检组织相关的细菌群落主要成员的指纹图谱。该方法的重现性很高（>98%）。洗涤和未洗涤的活检组织产生相似的DGGE条带模式（中位相似系数-MSC 96%，四分位间距-IQR 3.0%，n = 5）。使用不同镊子从同一患者采集的相邻活检组织产生相似的DGGE图谱（MSC 94%，n = 2）。从18例患者中的每例患者相距2 - 5 cm的位置采集的两块结肠直肠活检组织，结果显示出非常相似的图谱（MSC 100%，IQR 2.8%）。从同一患者大肠内不同位置采集的活检组织也产生相似的DGGE图谱（MSC 98%，IQR 3.3%，n = 6）。尽管所有患者（n = 33）都给出了不同的DGGE图谱，但在任意选择的15例患者获得的图谱之间观察到了一些相似性（约34%）。切除并测序了35条DGGE条带。发现许多条带与Genbank数据库中未培养细菌序列条目关系最为密切。其他条带属于典型的肠道细菌属，包括拟杆菌属、瘤胃球菌属、粪杆菌属和梭菌属。
正常患者结肠直肠黏膜上附着的细菌群落变化很小；相比之下，从不同的结肠直肠黏膜正常患者采集的黏膜细菌群落显示出高度的变异性。
本研究表明，对直接从黏膜样本提取的DNA扩增的16S rRNA基因PCR产物进行DGGE分析，为附着在结肠直肠黏膜上的细菌群落提供了新的见解。这些发现对于进一步研究胃肠道的健康和疾病具有重要意义。
