The spread of antibiotic resistance genes (ARGs), particularly those carried on plasmids, poses a major risk to global health. However, the extent and frequency of ARGs transfer in microbial communities among human, animal, and environmental sectors is not well understood due to a lack of effective tracking tools. We have developed a novel fluorescent tracing tool, CRISPR-AMRtracker, to study ARG transfer. It combines CRISPR/Cas9 fluorescence tagging, fluorescence-activated cell sorting, 16S rRNA gene sequencing, and microbial community analysis. CRISPR-AMRtracker integrates a fluorescent tag immediately downstream of ARGs, enabling the tracking of ARG transfer without compromising the host cell's antibiotic susceptibility, fitness, conjugation, and transposition. Notably, our experiments demonstrate that sfGFP-tagged plasmid-borne mcr-1 can transfer across diverse bacterial species within fecal samples. This innovative approach holds the potential to illuminate the dynamics of ARG dissemination and provide valuable insights to shape effective strategies in mitigating the escalating threat of antibiotic resistance.

中文翻译：

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CRISPR-AMRtracker：一种监测粪便微生物群中抗菌素耐药性基因转移的新型工具包


抗生素耐药基因 （ARG） 的传播，尤其是质粒上携带的抗生素耐药基因 （ARG） 的传播，对全球健康构成重大风险。然而，由于缺乏有效的跟踪工具，ARGs 在人类、动物和环境部门之间的微生物群落中转移的范围和频率尚不清楚。我们开发了一种新型荧光示踪工具 CRISPR-AMRtracker 来研究 ARG 转移。它结合了 CRISPR/Cas9 荧光标记、荧光激活细胞分选、16S rRNA 基因测序和微生物群落分析。CRISPR-AMRtracker 在 ARG 的下游集成了一个荧光标签，能够在不影响宿主细胞的抗生素敏感性、适应性、偶联和转座性的情况下追踪 ARG 转移。值得注意的是，我们的实验表明，sfGFP 标记的质粒携带的 mcr-1 可以在粪便样本中的不同细菌种类之间转移。这种创新方法有可能阐明 ARG 传播的动态，并为制定有效的策略来减轻抗生素耐药性不断升级的威胁提供有价值的见解。