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

Abstract

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.

Swine liquid manure: a hotspot of mobile genetic elements and antibiotic resistance genes

“…Although ARGs may be present e.g. on some high-copy plasmids 62,63 , it is not affect our absolute quantitative results and conclusion. Nevertheless, the fact that if ARGs are present on high-copy plasmids may overestimate their relative abundance in swine waste, which is a possible limitation of this work.…”

CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria

“…Although more data are needed, these studies suggest that instead of using CRISPR antimicrobials as replacements for conventional antibiotics, they may be more effectively deployed in combination therapies. For example, in a 2-step experiment, E. coli-infected G. mellonella larvae were first treated with CRISPR/Cas9 targeting bla TEM-1 , conferring beta-lactam resistance, then treated with ceftriaxone, resulting in the survival of 70% of dual-treated larvae compared to 30% of larvae treated with antibiotics alone [32]. In another example, CRISPR antimicrobials could be used for decolonization of antibiotic-resistant bacteria, rather than infection treatment.…”

“…coli strains harboring either high or low copy number plasmids. Complete eradication of the low copy number plasmid but not of the high copy number plasmid was observed [ 32 ]. Importantly, this study highlights challenges posed by targeting high-copy plasmids and the need to optimize the system to completely clear antibiotic resistance genes encoded by these types of plasmids.…”

CRISPR-Cas. A Revolution in the Treatment and Study of ESKAPE Infections: Pre-Clinical Studies

“…In the same year, Tagliaferri and colleagues targeted the plasmid-borne blaTEM-1 gene in the E. hormaechei 4962 clinical isolate [98]. This plasmid, coding for the Cas9 protein and a specific sgRNA targeting the blaTEM-1 gene, was succesfully electroporated in the E. coli 189A clinical isolate, as seen by qPCR and phenotypic testing, with a re-sensitization to ampicillin, cefazolin, cefuroxime, ceftriaxone and cefotaxime.…”