Wisittipanit Nuttachat, Pulsrikarn Chaiwat, Srisong Sudarat, Srimora Rungthiwa, Kittiwan Nattinee, Poonchareon Kritchai
Department of Material Engineering, School of Science, Mae Fah Luang University, Chiang Rai, Thailand.
Department of Medical Sciences, WHO National Salmonella and Shigella Center, National Institute of Health, Ministry of Public Health, Nonthaburi, Thailand.
PeerJ. 2020 Jun 16;8:e9113. doi: 10.7717/peerj.9113. eCollection 2020.
Nontyphoidal spp. constitute a major bacterial cause of food poisoning. Each serotype causes distinct virulence to humans.
A small cohort study was conducted to characterize several aspects of isolates obtained from stool of diarrheal patients ( = 26) admitted to Phayao Ram Hospital, Phayao province, Thailand. A simple CRISPR 2 molecular analysis was developed to rapidly type isolates employing both uniplex and high resolution melting (HRM) curve analysis.
CRISPR 2 monoplex PCR generated a single serotype-specific amplicon, showing 4,[5],12:i:- with highest frequency (42%), Enteritidis (15%) and Stanley (11%); Typhimurium was not detected. CRISPR 2 HRM-PCR allowed further classification of 4,[5],12:i:- isolates based on their specific CRISPR 2 signature sequences. The highest prevalence of infection was during the summer season (April to August). Additional studies were conducted using standard multiplex HRM-PCR typing, which confirmed CRISPR 2 PCR results and, using a machine-learning algorithm, clustered the majority of serotypes into six clades; repetitive element-based (ERIC) PCR, which clustered the serotypes into three clades only; antibiogram profiling, which revealed the majority resistant to ampicillin (69%); and test for extended spectrum -lactamase production (two isolates) and PCR-based detection of alleles.
CRISPR 2 PCR provided a simple assay for detection and identification of clinically-relevant serotypes. In conjunction with antibiogram profiling and rapid assay for -lactamase producers, this approach should facilitate detection and appropriate treatment of Salmonellosis in a local hospital setting. In addition, CRISPR 2 HRM-PCR profiling enabled clustering of 4,[5],12:i:-isolates according to CRISPR 2 locus signature sequences, indicative of their different evolutionary trajectories, thereby providing a powerful tool for future epidemiological studies of virulent serotypes.
非伤寒沙门氏菌属是食物中毒的主要细菌病因。每种血清型对人类造成不同程度的毒力。
开展了一项小型队列研究，以表征从泰国帕尧府帕尧拉姆医院收治的腹泻患者（n = 26）粪便中分离出的菌株的几个方面特征。开发了一种简单的CRISPR 2分子分析方法，采用单重和高分辨率熔解（HRM）曲线分析对分离菌株进行快速分型。
CRISPR 2单重PCR产生了单一的血清型特异性扩增子，显示4,[5],12:i:-的频率最高（42%），肠炎沙门氏菌（15%）和斯坦利沙门氏菌（11%）；未检测到鼠伤寒沙门氏菌。CRISPR 2 HRM-PCR允许根据其特定的CRISPR 2特征序列对4,[5],12:i:-分离株进行进一步分类。沙门氏菌感染的最高患病率出现在夏季（4月至8月）。使用标准多重HRM-PCR分型进行了额外研究，证实了CRISPR 2 PCR结果，并使用机器学习算法将大多数血清型聚类为六个进化枝；基于重复元件的（ERIC）PCR仅将血清型聚类为三个进化枝；抗菌谱分析显示大多数对氨苄西林耐药（69%）；以及超广谱β-内酰胺酶产生检测（两株分离株）和基于PCR的等位基因检测。
CRISPR 2 PCR为临床相关沙门氏菌血清型的检测和鉴定提供了一种简单的检测方法。结合抗菌谱分析和β-内酰胺酶产生菌的快速检测，这种方法应有助于在当地医院环境中检测和适当治疗沙门氏菌病。此外，CRISPR 2 HRM-PCR分析能够根据CRISPR 2基因座特征序列对4,[5],12:i:-分离株进行聚类，表明它们不同的进化轨迹，从而为未来毒力沙门氏菌血清型的流行病学研究提供了一个强大的工具。
