Aoki Kotaro, Komori Kohji, Yamaguchi Tetsuo, Harada Sohei, Tsukada Mayumi, Murakami Hinako, Tateda Kazuhiro
Department of Microbiology and Infectious Diseases, Toho University School of Medicine, Tokyo, Japan.
Division of Collaborative Regional Infection Control, Department of Community Well-being, Toho University School of Medicine, Tokyo, Japan.
JAC Antimicrob Resist. 2025 May 7;7(3):dlaf069. doi: 10.1093/jacamr/dlaf069. eCollection 2025 Jun.
Effective infection prevention and control (IPC) interventions in hospitals require timely information to determine the potential transmission of antimicrobial-resistant (AMR) organisms. We proposed and developed a successive core-genome SNP (cgSNP)-based phylogenetic analysis workflow, 'Tracking Antimicrobial Resistant Organisms Timely' (TAROT), using the Oxford Nanopore Technologies (ONT) sequencer for MRSA, and compared the results with those obtained using the Illumina sequencer.
We have developed a TAROT workflow for successive phylogenetic analysis using ONT data. We sequenced 34 MRSA strains isolated from Toho University Omori Medical Center using MinION (ONT) and MiSeq (Illumina). Each strain's ONT data were inputted into TAROT (TAROT-ONT), and successive cgSNP-based phylogenetic analyses were conducted. Illumina data were processed with a batched cgSNP-based phylogenetic analysis. Assembly-based analysis identified AMR genes, AMR mutations and virulence genes.
MinION generated an average sequence depth of 262× for the ST8 reference genome within 3 h. TAROT-ONT successively generated 11 phylogenetic trees for 14 ST8 strains, 7 trees for 10 ST1 strains and 2 trees for 5 ST5 strains. Highly suspected transmission pairs (pairwise cgSNP< 5) were detected in trees #6 through #11 for ST8, trees #3, #5 and #7 for ST1, and tree #2 for ST5. Differences in pairwise cgSNP value between TAROT-ONT and Illumina ranged from zero to two within pairs with fewer than 20 cgSNPs using Illumina. TAROT-ONT bioinformatic analysis for each strain required 5-42 min. The identification of AMR genes, mutations and virulence genes showed high concordance between ONT and Illumina.
TAROT-ONT can facilitate effective IPC intervention for MRSA nosocomial transmissions by providing timely feedback through successive phylogenetic analyses based on cgSNPs.
医院有效的感染预防与控制（IPC）干预措施需要及时的信息来确定耐抗菌药物（AMR）微生物的潜在传播情况。我们提出并开发了一种基于连续核心基因组单核苷酸多态性（cgSNP）的系统发育分析工作流程，即“及时追踪抗菌耐药性生物体”（TAROT），使用牛津纳米孔技术（ONT）测序仪对耐甲氧西林金黄色葡萄球菌（MRSA）进行分析，并将结果与使用Illumina测序仪获得的结果进行比较。
我们开发了一种使用ONT数据进行连续系统发育分析的TAROT工作流程。我们使用MinION（ONT）和MiSeq（Illumina）对从东京都立大学大森医疗中心分离出的34株MRSA菌株进行了测序。将每个菌株的ONT数据输入到TAROT（TAROT-ONT）中，并进行基于cgSNP的连续系统发育分析。Illumina数据通过基于成批cgSNP的系统发育分析进行处理。基于组装的分析鉴定了AMR基因、AMR突变和毒力基因。
MinION在3小时内为ST8参考基因组生成了平均262倍的序列深度。TAROT-ONT为14株ST8菌株连续生成了11个系统发育树，为10株ST1菌株生成了7个系统发育树，为5株ST5菌株生成了2个系统发育树。在ST8的第6至11号树、ST1的第3、5和7号树以及ST5的第2号树中检测到高度可疑的传播配对（成对cgSNP<5）。使用Illumina时，在cgSNP少于20个的配对中，TAROT-ONT和Illumina之间的成对cgSNP值差异在0到2之间。对每个菌株进行TAROT-ONT生物信息学分析需要5至42分钟。ONT和Illumina在AMR基因、突变和毒力基因的鉴定方面显示出高度一致性。
TAROT-ONT可以通过基于cgSNP的连续系统发育分析提供及时反馈，促进对MRSA医院内传播的有效IPC干预。
