P14 Optimization of real-time sequencing for rapid detection of bacterial species and antimicrobial resistance in blood and urine infections
P14 Optimization of real-time sequencing for rapid detection of bacterial species and antimicrobial resistance in blood and urine infections
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DOI：
10.1093/jacamr/dlad077.018
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发表时间：
2023-08-02
影响因子：
3.4
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中科院分区：
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优化直接从人类尿液和血液样本中获取细菌 DNA 的过程，以使用长读长 Nanopore 测序仪生成高质量的基因组数据。从微生物学诊断实验室收集了总共 22 份血培养物和 24 份尿样，其大肠杆菌或肺炎克雷伯菌及相应分离株呈阳性。 DNA 分离优化通过测试三种不同的样品体积和七种 DNA 提取试剂盒进行。使用 Qubit 荧光计对 DNA 浓度进行定量。使用快速条形码试剂盒在 R9.4.1 流动池中的 MinION Mk1C 设备上进行纳米孔测序。使用 EPI2ME ARMA 管道确定细菌种类和抗菌素耐药性 (AMR) 基因。基因组用 Flye 组装，用 Medaka 打磨，并用 Krakentools 去除剩余的人类重叠群。还使用 Illumina 技术对同一样本中的细菌分离株进行了测序。使用 Quast 对 Illumina 和 Nanopore 组件进行质量检查，使用 Kraken 来识别细菌种类，使用 MLST 进行多位点序列分型，使用 ResFinder 数据库进行 ABRicate 来检测 AMR 基因。使用不同的试剂盒从 24 份尿液和 22 份血液样本中总共提取了 72 次 DNA，以找到每种样本类型的最佳提取方案。尿液样本中的 DNA 是从 5 mL 中分离出来的，而 1 mL 最适合血培养。我们发现，使用 MolYsis basic 5 Kit (VH-Bio) 提取 DNA 之前的宿主去除步骤至关重要。使用 GenElut 细菌基因组 DNA 试剂盒 (Merck) 提取尿液样本中的 DNA，而 QIAamp Biostic 菌血症试剂盒 (Qiagen) 最适合对血液样本进行成功的细菌基因组测序。从 11 份尿液样本和 1 份血液样本（已测序的 54 份样本中）中分离出的细菌已成功测序。在所有 12 个病例中，细菌读数与常规微生物学诊断过程中发现的致病微生物（大肠杆菌）相匹配的比例最高。检测到多种临床相关的 AMR 基因，例如 dfrA 和 bla CTX-M 基因，与表型抗菌药物敏感性检测结果相匹配。通过 10 个尿液样本的 Nanopore 测序获得的基因组组装与相应临床分离株的 Illumina 测序进行比较，显示出高度匹配，验证了 Nanopore 测序作为支持诊断和治疗选择的替代选择。我们已经成功优化了直接从血液和尿液样本中提取致病细菌 DNA 并对其进行测序的方案。短读长测序证实了纳米孔测序仪获得的结果，为临床环境中的长读长测序提供了支持，以支持及时诊断和治疗。
To optimize the process to obtain bacterial DNA directly from human urine and blood samples to produce high-quality genomic data using long-read Nanopore sequencer. A total of 22 blood cultures and 24 urine samples positive for Escherichia coli or Klebsiella pneumoniae and corresponding isolates were collected from the microbiology diagnostics laboratory. DNA isolation optimization was performed testing three different sample volumes and seven DNA extraction kits. DNA concentration was quantified with Qubit fluorometer. Nanopore sequencing was performed on a MinION Mk1C device in R9.4.1 flow cells using the Rapid Barcoding Kit. Bacterial species and antimicrobial resistance (AMR) genes were determined with EPI2ME ARMA pipeline. Genomes were assembled with Flye, polished with Medaka and remaining human contigs removed with Krakentools. Bacterial isolates from the same samples were also sequenced using Illumina technology. Illumina and Nanopore assemblies were quality checked with Quast, Kraken was used to identify bacterial species, MLST for multilocus sequence typing and ABRicate with ResFinder database to detect AMR genes. A total of 72 DNA extractions from 24 urine and 22 blood samples with different kits were performed to find the optimal extraction protocol for each sample type. DNA from urine samples was isolated from 5 mL, while 1 mL was best for blood cultures. We found that the host depletion step prior to DNA extraction using MolYsis basic 5 Kit (VH-Bio) was essential. DNA from urine samples was extracted with GenElute Bacterial Genomic DNA Kit (Merck) and QIAamp Biostic bacteremia Kit (Qiagen) was best to allow successful bacterial genome sequencing for blood samples. Bacterial isolates from 11 urine and 1 blood sample (out of 54 sequenced) were successfully sequenced. In all 12 cases, the highest percentage of bacterial reads matched with the causative organism found during routine microbiology diagnostics (E. coli). A variety of clinically relevant AMR genes were detected, such as dfrA and bla CTX-M genes, which matched the phenotypic antimicrobial susceptibility testing results. Comparison of genomic assemblies obtained through Nanopore sequencing of 10 urine samples and Illumina sequencing of the corresponding clinical isolates showed a high degree of matching, validating Nanopore sequencing as an alternative option to support diagnosis and treatment choice. We have successfully optimized the protocols to extract and sequence pathogenic bacterial DNA directly from blood and urine samples. Short-read sequencing confirmed results acquired by Nanopore sequencer, endorsing long-read sequencing in clinical settings to support timely diagnosis and treatment.