Data_Sheet_2_Exploring the Potential of CRISPR-Cas9 Under Challenging Conditions: Facing High-Copy Plasmids and Counteracting Beta-Lactam Resistance in Clinical Strains of Enterobacteriaceae.PDF

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 blaTEM–1 AMR gene located on a high-copy plasmid (i.e., 100–300 copies/cell) and by directly tackling blaTEM–1-positive clinical isolates. Upon CRISPR-Cas9 insertion into a model strain of E. coli harboring blaTEM–1 on the plasmid pSB1A2, the plasmid number and, accordingly, the blaTEM–1 gene expression decreased but did not become extinct in a subpopulation of CRISPR-Cas9 treated bacteria. Sequence alterations in blaTEM–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.

抗生素耐药性（antimicrobial resistance, AMR）危机迫切需要采取对策以遏制质粒携带耐药基因的传播。其中尤为值得关注的是肠杆菌科（Enterobacteriaceae）的条件致病菌。一种创新性策略便是CRISPR-Cas9系统，该系统近期已被用于定向清除大肠杆菌（Escherichia coli）特定菌株中的质粒。本研究在此基础上，于极具挑战性的条件下进一步拓展了该系统的应用：靶向位于高拷贝质粒（即每个细胞100~300个拷贝）上的blaTEM–1耐药基因，并直接针对blaTEM–1阳性临床分离株开展实验。将CRISPR-Cas9导入携带质粒pSB1A2上blaTEM–1的大肠杆菌模式菌株后，质粒拷贝数及相应的blaTEM–1基因表达水平均出现下降，但经CRISPR-Cas9处理的细菌亚群中并未完全清除质粒。研究中观察到blaTEM–1基因发生序列变异，这可能导致其编码产物功能失活。尽管质粒仍得以保留，但最终实现了对抗生素敏感表型的完全逆转。在一株大肠杆菌临床分离株中，实现了质粒的完全清除，并同时使其对五种β-内酰胺类抗生素重新敏感。通过用经CRISPR-Cas9处理的大肠杆菌感染大蜡螟（Galleria mellonella）幼虫并使其获救，证实了抗生素的可重复使用性；而未经过修饰的临床分离株感染则无法达到这一效果。对于霍氏肠杆菌（Enterobacter hormaechei）的一株临床分离株，同样可提升其药物敏感性；在变栖克雷伯菌（Klebsiella variicola）中效果稍弱，二者均携带可影响β-内酰胺类抗生素耐药性的额外耐药基因。本研究数据表明，即便面对高拷贝质粒，靶向耐药基因的策略仍颇具应用前景。对于临床分离株而言，同时干扰介导多重重叠耐药性的多个基因，或许是实现表型逆转成功的关键。