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Targeted next-generation sequencing of the 16S-23S rRNA region for culture-independent bacterial identification - increased discrimination of closely related species

机译：有针对性的下一代16S-23S rRNA区域测序，可进行与培养无关的细菌鉴定-增强对密切相关物种的区分

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The aim of this study was to develop an easy-to-use culture-free diagnostic method based on next generation sequencing (NGS) of PCR amplification products encompassing whole 16S-23S rRNA region to improve the resolution of bacterial species identification. To determine the resolution of the new method 67 isolates were subjected to four identification methods: Sanger sequencing of the 16S rRNA gene; NGS of the 16S-23S rRNA region using MiSeq (Illumina) sequencer; Microflex MS (Bruker) and VITEK MS (bioMerieux). To evaluate the performance of this new method when applied directly on clinical samples, we conducted a proof of principle study with 60 urine samples from patients suspected of urinary tract infections (UTIs), 23 BacT/ALERT (bioMerieux) positive blood culture bottles and 21 clinical orthopedic samples. The resolution power of NGS of the 16S-23S rRNA region was superior to other tested identification methods. Furthermore, the new method correctly identified pathogens established as the cause of UTIs and blood stream infections with conventional culture. NGS of the 16S-23S rRNA region also showed increased detection of bacterial microorganisms in clinical samples from orthopedic patients. Therefore, we conclude that our method has the potential to increase diagnostic yield for detection of bacterial pathogenic species compared to current methods.
机译：这项研究的目的是基于PCR扩增产物的下一代测序（NGS），开发一种易于使用的无培养物的诊断方法，该扩增产物涵盖整个16S-23S rRNA区域，以提高细菌种类鉴定的分辨率。为了确定新方法的分辨率，对67个分离株进行了四种鉴定方法：16S rRNA基因的Sanger测序；使用MiSeq（Illumina）测序仪的16S-23S rRNA区域的NGS； Microflex MS（布鲁克）和VITEK MS（bioMerieux）。为了评估这种新方法在直接应用于临床样品时的性能，我们进行了原理验证研究，对来自怀疑有尿路感染（UTIs）的患者的60个尿液样品，23个BacT / ALERT（bioMerieux）阳性血培养瓶和21个尿液样本进行了原理研究。临床骨科样本。 NGS对16S-23S rRNA区域的分辨能力优于其他测试的鉴定方法。此外，该新方法可以正确识别被确定为常规培养引起的UTI和血流感染的病原体。 16S-23S rRNA区域的NGS也显示出骨科患者临床样品中细菌微生物的检测增加。因此，我们得出的结论是，与当前方法相比，我们的方法具有提高检测细菌病原体的诊断率的潜力。

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