Antimicrobial resistance is a significant global threat, posing major public health risks and economic costs to healthcare systems. Bacterial cultures are typically used to diagnose healthcare-acquired infections (HAI); however, culture-dependent methods provide limited presence/absence information and are not applicable to all pathogens. Next generation sequencing (NGS) has the capacity to detect a wide variety of pathogens, virulence elements, and antimicrobial resistance (AMR) signatures in healthcare settings without the need for culturing, but few research studies have explored how NGS could be used to detect viable human pathogen transmission events under different HAI-relevant scenarios. The objective of this project was to assess the capability of NGS-based methods to detect the direct and indirect transmission of high priority healthcare-related pathogens. DNA was extracted and sequenced from a previously published study exploring pathogen transfer with simulated skin containing background microorganisms, which allowed for complementary culture and metagenomic analysis comparisons. RNA was also isolated from an additional set of samples to evaluate metatranscriptomic analysis methods at different concentrations. Using various analysis methods and custom reference databases, both pathogenic and non-pathogenic members of the microbial community were identified at the species level. Virulence and AMR genes known to reside within the community were also routinely identified. Ultimately, pathogen abundance within the overall microbial community played the largest role in successful taxonomic classification and gene identification. These results illustrate the utility of metagenomic analysis in clinical settings or for epidemiological studies, but also highlight the limits associated with the detection and characterization of pathogens at low abundance in a microbial community.

中文翻译：

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使用元基因组和元转录组测序方法检测模拟皮肤传播事件中的ESKAPE病原体和难辨梭状芽胞杆菌

抗菌素耐药性是一个重大的全球性威胁，给医疗保健系统带来了重大的公共健康风险和经济成本。细菌培养通常用于诊断医疗保健获得性感染（HAI）；但是，依赖于培养物的方法只能提供有限的存在/不存在信息，并且不适用于所有病原体。下一代测序（NGS）无需检测即可在医疗机构中检测多种病原体，毒力元素和抗菌素耐药性（AMR）特征，但很少有研究探索如何使用NGS来检测可行的不同HAI相关场景下的人类病原体传播事件。该项目的目的是评估基于NGS的方法检测高优先级医疗相关病原体直接和间接传播的能力。从先前发表的研究中提取DNA并进行测序，该研究探索了含有背景微生物的模拟皮肤中病原体的转移，从而可以进行互补培养和宏基因组分析比较。还从另外一组样品中分离出RNA，以评估不同浓度的转录组分析方法。使用各种分析方法和定制参考数据库，在物种级别上确定了微生物群落的致病性和非致病性成员。还常规鉴定了已知存在于社区内的毒力和AMR基因。最终，整个微生物群落中病原体的丰度在成功的分类学分类和基因鉴定中发挥了最大的作用。这些结果说明了宏基因组学分析在临床环境或流行病学研究中的效用，但同时也突出了与微生物群落中低丰度病原体的检测和表征相关的限制。