Sadeghifard Nourkhoda, Gürtler Volker, Beer Michael, Seviour Robert J
Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3552, Australia.
Appl Environ Microbiol. 2006 Nov;72(11):7311-23. doi: 10.1128/AEM.01179-06. Epub 2006 Sep 15.
Clostridium difficile is a major spore-forming environmental pathogen that causes serious health problems in patients undergoing antibiotic therapy. Consequently, reliable and sensitive methods for typing individual strains are required for epidemiological and environmental studies. Ribotyping is generally considered the best method, but it fails to account for sequence diversity which might exist in intergenic 16S-23S rRNA spacer regions (ISRs) within and among strains of this organism. Therefore, this study was undertaken to compare the sequence of each individual ISR in five strains of C. difficile to explore the extent of this diversity and see whether such information might provide the basis for more sensitive and discriminatory strain typing methods. After targeted PCR amplification, cloning, and sequencing, the diversity of the ISRs was used as a measure of rRNA operon copy number. In C. difficile strains 630, ATCC 43593, A, and B, 11, 11, 7, and 8 ISR length variants, respectively, were found (containing different combinations of sequence groups [i to xiii]), suggesting 11, 11, 7, and 8 rrn copies in the respective strains. Many ISRs of the same length differed markedly in their sequences, and some of these were restricted in occurrence to a single strain. Most of these ISRs did not contain any tRNA genes, and only single copies of the tRNA(Ala) gene were found in those that did. The presence of ISR sequence groups (i to xiii) varied between strains, with some found in one, two, three, four, or all five strains. We conclude that the intergenic 16S-23S rRNA spacer regions showed a high degree of diversity, not only among the rrn operons in different strains and different rrn copies in a single strain but also among ISRs of the same length. It appears that C. difficile ISRs vary more at the inter- and intragenic levels than those of other species as determined by empirical comparison of sequences. The precise characterization of these sequences has demonstrated a high level of mosaic sequence block rearrangements that are present or absent in multiple strain-variable rrn copies within and between five different strains of C. difficile.
艰难梭菌是一种主要的形成孢子的环境病原体，会给接受抗生素治疗的患者带来严重的健康问题。因此，流行病学和环境研究需要可靠且灵敏的方法来对单个菌株进行分型。核糖体分型通常被认为是最佳方法，但它无法考虑到该生物体菌株内部和之间的基因间16S - 23S rRNA间隔区（ISRs）中可能存在的序列多样性。因此，本研究旨在比较五株艰难梭菌中每个单独ISR的序列，以探究这种多样性的程度，并查看此类信息是否可为更灵敏和更具区分性的菌株分型方法提供依据。经过靶向PCR扩增、克隆和测序后，ISRs的多样性被用作rRNA操纵子拷贝数的衡量指标。在艰难梭菌菌株630、ATCC 43593、A和B中，分别发现了11、11、7和8种ISR长度变体（包含序列组[i至xiii]的不同组合），这表明相应菌株中分别有11、11、7和8个rrn拷贝。许多长度相同的ISRs在序列上有显著差异，其中一些仅在单个菌株中出现。这些ISRs中的大多数不包含任何tRNA基因，只有那些包含tRNA（Ala）基因的ISRs中发现了单拷贝。ISR序列组（i至xiii）的存在因菌株而异，有些在一株、两株、三株、四株或所有五株菌株中都有发现。我们得出结论，基因间16S - 23S rRNA间隔区显示出高度的多样性，不仅在不同菌株的rrn操纵子之间以及单个菌株的不同rrn拷贝之间，而且在长度相同的ISRs之间也是如此。通过序列的实证比较确定，艰难梭菌的ISRs在基因间和基因内水平上似乎比其他物种的ISRs变化更大。这些序列的精确表征表明，在五株不同的艰难梭菌菌株内部和之间的多个菌株可变rrn拷贝中，存在或不存在高水平镶嵌序列块重排。
