Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae
Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae
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DOI：
10.3389/fmicb.2020.00578
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发表时间：
2020-04-30
影响因子：
5.2
通讯作者：
de Oliveira Mendes, Tiago Antonio
中科院分区：
文献类型：
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作者：
Tagliaferri, Thaysa Leite;Guimaraes, Natalia Rocha;de Oliveira Mendes, Tiago Antonio
抗生素耐药性（AMR）危机迫切需要采取对策，以减少质粒携带的耐药基因的传播。特别值得关注的是肠杆菌科的机会致病菌。一种创新方法是CRISPR-Cas9系统，该系统最近已用于大肠杆菌的确定菌株中的质粒固化。在这里，我们在具有挑战性的条件下进一步利用该系统：通过靶向位于高拷贝质粒上的bla（TEM-）（1）AMR基因（即，100-300拷贝/细胞）和直接处理bla（TEM-）（1）阳性临床分离株。在CRISPR-Cas9插入E.在质粒pSB 1A 2上携带bla（TEM-）（1）的大肠杆菌中，质粒数量和相应的bla（TEM-）（1）基因表达减少，但在CRISPR-Cas9处理的细菌亚群中没有灭绝。观察到bla（TEM-）（1）中的序列改变，可能导致基因产物功能障碍。因此，尽管质粒维持，但实现了对抗生素敏感表型的完全逆转。在E.大肠杆菌，质粒清除和同时再致敏五β-内酰胺是可能的。抗生素的可重复使用性可以通过拯救感染CRISPR-Cas9处理的E.大肠杆菌，而不是感染未经修改的临床分离。在临床分离的霍氏肠杆菌中，药物敏感性水平也可能增加，在较小程度上在变异克雷伯氏菌中，这两种菌都含有影响β-内酰胺类的其他耐药基因。数据显示，即使面对高拷贝质粒，靶向耐药基因也是令人鼓舞的。在临床分离株中，同时干扰多个介导重叠耐药的基因可能是成功逆转表型的线索。
The antimicrobial resistance (AMR) crisis urgently requires countermeasures for reducing the dissemination of plasmid-borne resistance genes. Of particular concern are opportunistic pathogens of Enterobacteriaceae. One innovative approach is the CRISPR-Cas9 system which has recently been used for plasmid curing in defined strains of Escherichia coli. Here we exploited this system further under challenging conditions: by targeting the bla(TEM-)(1) AMR gene located on a high-copy plasmid (i.e., 100-300 copies/cell) and by directly tackling bla(TEM-)(1)-positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of E. coli harboring bla(TEM-)(1) on the plasmid pSB1A2, the plasmid number and, accordingly, the bla(TEM-)(1) gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in bla(TEM-)(1) were observed, likely resulting in a dysfunction of the gene product. As a consequence, a full reversal to an antibiotic sensitive phenotype was achieved, despite plasmid maintenance. In a clinical isolate of E. coli, plasmid clearance and simultaneous re-sensitization to five beta-lactams was possible. Reusability of antibiotics could be confirmed by rescuing larvae of Galleria mellonella infected with CRISPR-Cas9-treated E. coli, as opposed to infection with the unmodified clinical isolate. The drug sensitivity levels could also be increased in a clinical isolate of Enterobacter hormaechei and to a lesser extent in Klebsiella variicola, both of which harbored additional resistance genes affecting beta-lactams. The data show that targeting drug resistance genes is encouraging even when facing high-copy plasmids. In clinical isolates, the simultaneous interference with multiple genes mediating overlapping drug resistance might be the clue for successful phenotype reversal.