Department of Engineering Science, University of Oxford, Parks Road, OX1 3PJ, Oxford, United Kingdom.
Key Lab of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China.
Sci Rep. 2017 Nov 30;7(1):16648. doi: 10.1038/s41598-017-16898-x.
The emergence and widespread distribution of antimicrobial resistant (AMR) bacteria has led to an increasing concern with respect to potential environmental and public health risks. Culture-independent and rapid identification of AMR bacteria in-situ in complex environments is important in understanding the role of viable but non-culturable and antibiotic persistent bacteria and in revealing potential pathogens without waiting for colony formation. In this study, a culture-independent and non-destructive phenotyping approach, so called Raman Deuterium Stable Isotope Probing (Raman-DIP), was developed to identify AMR bacteria in the River Thames. It is demonstrated that Raman-DIP was able to accurately identify resistant and susceptible bacteria within 24 hours. The work shows that, in the River Thames, the majority of the bacteria (76 ± 2%) were metabolically active, whilst AMR bacteria to carbenicillin, kanamycin and both two antibiotics were 35 ± 5%, 28 ± 3%, 25 ± 1% of the total bacterial population respectively. Raman activated cell ejection (RACE) was applied to isolate single AMR bacteria for the first time, linking AMR phenotype (reistance to antibiotics) and genotype (DNA sequence). The sequences of the RACE sorted cells indicate that they were potential human pathogens Aeromonas sp., Stenotrophomonas sp. and an unculturable bacterium. This work demonstrates Raman-DIP and RACE are effective culture-independent approach for rapid identification of AMR bacteria at the single cell level in their natural conditions.
抗微生物药物耐药（AMR）细菌的出现和广泛分布引起了人们对潜在环境和公共卫生风险的日益关注。在复杂环境中，原位、非培养依赖性和快速鉴定 AMR 细菌对于理解活而不可培养和抗生素持久细菌的作用以及揭示潜在病原体而无需等待菌落形成非常重要。在这项研究中，开发了一种非培养依赖性和非破坏性的表型鉴定方法，即所谓的拉曼氘稳定同位素探测（Raman-DIP），用于鉴定泰晤士河中的 AMR 细菌。结果表明，Raman-DIP 能够在 24 小时内准确鉴定耐药和敏感细菌。研究表明，在泰晤士河中，大多数细菌（76±2%）具有代谢活性，而耐羧苄西林、卡那霉素和两种抗生素的 AMR 细菌分别占总细菌种群的 35±5%、28±3%和 25±1%。首次应用拉曼激活细胞弹射（RACE）分离单个 AMR 细菌，将 AMR 表型（对抗生素的抗性）与基因型（DNA 序列）联系起来。RACE 分选细胞的序列表明它们是潜在的人类病原体气单胞菌、寡养单胞菌和一种不可培养的细菌。这项工作证明，Raman-DIP 和 RACE 是在其自然条件下快速鉴定单个细胞水平上 AMR 细菌的有效非培养方法。
