BACKGROUND Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR). RESULTS The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network-based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, β-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes. CONCLUSION Overall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.

中文翻译：

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使用天然 DNA 和 RNA 纳米孔测序评估广泛耐药肺炎克雷伯菌的基因组和耐药组。


背景肺炎克雷伯氏菌经常具有多重耐药性，并且当前的诊断方法难以快速识别合适的抗生素来治疗这些细菌感染。 MinION 设备可以实时对天然 DNA 和 RNA 进行测序，从而提供了比较 DNA 和 RNA 在预测抗生素敏感性方面的效用的机会。然而，细菌直接 RNA 测序和碱基识别的有效性此前尚未得到研究。本研究探讨了 4 种广泛耐药 (XDR) 肺炎克雷伯菌临床分离株的基因组和转录组；然而，进一步的抗菌药物敏感性测试发现 3 个分离株具有全耐药性 (PDR)。结果 大多数获得性抗性（≥75%）存在于质粒上，包括几个大质粒（≥100 kb）。 DNA测序在测序2小时内检测到大多数耐药基因（≥70%）。基于神经网络的直接 RNA 碱基识别实现了高达 86% 的同一率，尽管 ≤23% 的读数可以对齐。直接 RNA 测序（孔易位速度慢 6 倍）能够识别（10 小时内）≥35% 的耐药基因，包括与氨基糖苷类、β-内酰胺类、甲氧苄啶和磺胺类以及喹诺酮类、利福平、某些分离株中含有磷霉素和苯酚。直接 RNA 测序还鉴定出含有多达 3 个抗性基因的操纵子的存在。多粘菌素抗性分离株显示 phoPQ（≥2 倍）和 pmrHFIJKLM 操纵子（≥8 倍）的转录增强。与 11 个抗性基因的定量实时 PCR 相比，直接 RNA 测序估计的表达水平显示出很强的相关性（Pearson：0.86）。结论 总的来说，MinION 测序快速检测到了 XDR/PDR K。 肺炎链球菌耐药组和直接RNA测序可以准确估计这些基因的表达水平。