CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli

Abstract

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Reversal of carbapenem-resistance in Shewanella algae by CRISPR/Cas9 genome editing

“…Recently, the clustered regularly interspaced short palindromic repeat (CRISPR) system and CRISPR-associated protein 9 (Cas9) were shown to provide adaptive immunity in prokaryotic defense systems, and the CRISP/Cas9 system has since been harnessed as a genome editing tool [18]. Attempts have been made to re-sensitize resistant E. coli using this system [19], [20]. In this study, we sequenced and annotated the genome of the carbapenem-resistant strain of Shewanella algae VGH117 and performed genome editing of 3 candidate genes using CRISPR/Cas9 and recE/recT recombinase to reverse the carbapenem resistance in S. algae .…”

Genotypic antimicrobial resistance characterization of E. coli from dairy calves at high risk of respiratory disease administered enrofloxacin or tulathromycin

“…Mutation in gyr A 248, resulting in resistance to quinolone drugs has previously been reported in E. coli, and Salmonella isolates from both humans and animals 27 , 28 . One study screening 818 E. coli clinical isolates (77% resistant to nalidixic acid) from cows, chicken and pigs revealed that the most common nucleotide mutation site in gyr A was at position 248 (C → T), resulting in an amino acid change from serine to leucine at position 83 29 . Furthermore, this study used CRISPR/Cas9 to investigate the causal role of gyr A mutation in the quinolone resistance by inducing gyr A mutations in nucleotide 248 C to T and was able to successfully demonstrate the role of this mutation resulting in resistance to quinolone drugs.…”

“…Specificity for the absence of the referred resistance gene and prediction of lack of phenotypic resistance to the corresponding antimicrobial drug. tetA tetB (n = 1) % 3 % 3 % 3 % 3 % 3 % 3 % 3 % 3 % 3 % 3 % 3 % 3 % 3 % 3 AuAmEn www.nature.com/scientificreports/ isolates (77% resistant to nalidixic acid) from cows, chicken and pigs revealed that the most common nucleotide mutation site in gyrA was at position 248 (C → T), resulting in an amino acid change from serine to leucine at position 83 29 . Furthermore, this study used CRISPR/Cas9 to investigate the causal role of gyrA mutation in the quinolone resistance by inducing gyrA mutations in nucleotide 248 C to T and was able to successfully demonstrate the role of this mutation resulting in resistance to quinolone drugs.…”

<p>Reversal of <em>mcr-1</em>-Mediated Colistin Resistance in <em>Escherichia coli</em> by CRISPR-Cas9 System</p>

“…10 Based on the CRISPR-Cas9 system, reported studies have been transformed E. coli with plasmids that coded sgRNAs to specifically cleave quinolone resistance gene and Extended-spectrum β-lactamases (ESBLs) resistance gene, respectively. 11,12 Together, using the CRISPR-Cas9 system to resensitize colistin-resistant E. coli by curing mcr-1-carrying plasmid is considered as a potentially effective method to resist the dissemination of colistin resistance genes.…”