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

Thaysa Leite Tagliaferri,Simone Gonçalves Dos Santos,Tiago Antônio De Oliveira Mendes,Natália Rocha Guimarães,Liza Figueiredo Felicori Vilela,Hans-Peter Horz,Marcella De Paula Martins Pereira

doi:10.3389/fmicb.2020.00578

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 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.

Highlights

Antimicrobial resistant microorganisms have become a public health concern due to their impact on human morbidity and mortality in recent decades
The blaTEM−1 gene located on the small high-copy plasmid pSB1A2 was introduced into the E. coli strain BL21, which was otherwise devoid of antimicrobial resistance (AMR)
For the design of a proper sgRNA, the conserved region most closely located at the 5 -end of the blaTEM−1 gene was selected (Supplementary Figure S1A) in order to maximize the likelihood of an early stop codon generated during an eventual bacterial DNA repair mechanism (Chayot et al, 2010)

Summary

Introduction

Antimicrobial resistant microorganisms have become a public health concern due to their impact on human morbidity and mortality in recent decades. Multiple copies of a resistance gene increase the probability of mutational adaptation, thereby rapidly generating allele variations (Hall and Harrison, 2016). The repeated exposure to beta-lactams and high levels of beta-lactam resistance have been shown to be associated with an increase of plasmid copy number, reaching values higher than 100 copies/cell (San Millan et al, 2015, 2016). The increase of plasmid copy numbers can lead to maximum levels of AMR (San Millan et al, 2015, 2016; Hall and Harrison, 2016; Schechter et al, 2018)

Methods

Results

Conclusion