Poultry Microbiological Safety and Processing Research Unit, U.S. National Poultry Research Center, Agricultural Research Service, United States Department of Agriculture, 950 College Station Road, Athens, GA, 30605-2720, USA.
Bacteriology, Mycology and Immunology Department, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt.
Curr Microbiol. 2020 Aug;77(8):1647-1652. doi: 10.1007/s00284-020-01965-w. Epub 2020 Apr 11.
Campylobacter jejuni is the leading bacterial foodborne pathogen that causes human acute gastrointestinal illness worldwide. Due to its genetic diversity, fastidious growth and sophisticated biochemical requirements, classification of Campylobacter by traditional techniques is problematic. Several molecular typing methods have been explored in this bacterium. One such method is to use clustered regularly interspaced short palindromic repeats (CRISPR). These CRISPRs consist of a direct repeat interspaced with nonrepetitive spacer sequences. In this study, we applied this genotyping method to explore the genetic diversity of C. jejuni isolated from poultry sources. Ninety-nine C. jejuni isolates from poultry environments in four different US states were used. Genomic DNA of the isolates were extracted from cultures using a commercial kit. PCR primers and conditions for CRISPR type 1 amplification were described previously. The amplicons were purified and sequenced by the Sanger dideoxy sequencing method. The direct repeats (DR) and spacers of the CRISPR sequences were identified using the CRISPRFinder. The results show there were 21% isolates no detectable, 30% isolates questionable, and 49% isolates confirmed CRISPR, respectively. The lengths of CRISPR range from 100 to 695 nucleotides. One type of DR was found in CRISPR of these isolates. The number of spacers in CRISPR ranges from 1 to 10 with various sequences. A total of 55 distinctive spacer sequences were identified in 78 isolates. Among them, 33 sequences were found unique in this study. In addition, the CRISPR genotyping had higher the Simpson's index of diversity value than that from flaA nucleotide typing. The results of our study show the CRISPR genotyping on C. jejuni may be complementary to the other genotyping methods.
空肠弯曲菌是导致全球人类急性胃肠道疾病的主要细菌性食源性病原体。由于其遗传多样性、苛刻的生长条件和复杂的生化需求，传统技术对弯曲菌的分类存在问题。已经探索了几种分子分型方法来用于该细菌。其中一种方法是使用成簇规律间隔短回文重复序列（CRISPR）。这些 CRISPR 由直接重复序列和非重复间隔序列组成。在这项研究中，我们应用这种基因分型方法来探索从禽类来源分离的空肠弯曲菌的遗传多样性。使用来自美国四个不同州的禽类环境的 99 株空肠弯曲菌分离株。使用商业试剂盒从培养物中提取分离株的基因组 DNA。先前描述了用于扩增 CRISPR 类型 1 的 PCR 引物和条件。通过 Sanger 双脱氧测序法纯化和测序扩增子。使用 CRISPRFinder 鉴定 CRISPR 序列的直接重复（DR）和间隔序列。结果显示，分别有 21%的分离株无法检测到、30%的分离株可疑、49%的分离株可确认 CRISPR。CRISPR 的长度范围为 100 到 695 个核苷酸。在这些分离株的 CRISPR 中发现了一种类型的 DR。CRISPR 中的间隔序列数范围为 1 到 10，具有各种序列。在 78 株分离株中总共鉴定出 55 个独特的间隔序列。其中，在本研究中发现 33 个序列是独特的。此外，CRISPR 基因分型的 Simpson 多样性指数值高于 flaA 核苷酸分型。我们的研究结果表明，CRISPR 基因分型可能对其他基因分型方法具有补充作用。
