A Novel Genome-Editing Platform for Drug-Resistant Acinetobacter baumannii Reveals an AdeR-Unrelated Tigecycline Resistance Mechanism

Abstract

bInfections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC 90 of tigecycline from 25 g/ml in the parental strains to 3.1 g/ml in the ⌬adeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains. O ne of the greatest global health problems results from the limited treatment options to fight bacterial infections caused by multidrug-resistant (MDR) organisms. The group of ESKAPE organisms that is comprised of Enterobacter spp., Staphylococcus aureus/epidermidis, Klebsiella pneumonia, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterococcus faecalis/faecium is considered to cause the vast majority of, often untreatable, nosocomial infections (1). Among these ESKAPE pathogens A. baumannii is most difficult to treat due to its multiple intrinsic and acquired resistance mechanisms that resulted in the development of MDR, extensively drug resistant (XDR), or even pan-drug-resistant (PDR) phenotypes (2-5).Bacteria have evolved multiple ways to evade antibiotic-mediated cell death, such as (i) enzymatic modification/cleavage of the antibiotic (e.g., beta-lactams), (ii) modification/protection of the antibiotic target (e.g., fluoroquinolones), or (iii) reduction of the intracellular concentration by antibiotic efflux or reduced influx (e.g., tetracyclines) (6). The expression of such defense mechanisms may require an extensive metabolic investment, often leading to a reduced fitness of these resistant bacteria in the absence of the external selection pressure (7). To overcome these ecol...

Characteristics and diversity of mutations in regulatory genes of resistance-nodulation-cell division efflux pumps in association with drug-resistant clinical isolates of Acinetobacter baumannii

“…In previous investigations, coexistence of these two amino acid substitutions have been detected in both tigecycline resistant isolates, like our results, and tigecycline susceptible ones; hence, their detailed effect is in debate [3,4,6,14]. [17]. Tigecycline non-susceptibility can occur as a result of synergistic contribution of AdeIJK with AdeABC [10], while AdeABC has superior in uence [18,38].…”

Alternative Evolutionary Paths to Bacterial Antibiotic Resistance Cause Distinct Collateral Effects

“…Deletion of nalC and single nucleotide mutations in pmrB and mexZ were introduced in P. aeruginosa PA14 based on a two-step recombination method previously described ( Trebosc et al 2016 ). DNA fragments corresponding to 700-bp up- and downstream of the nalC region to be deleted (position 1,391,565–1,390,977 on PA14 genome, GenBank CP000438.1) were amplified by PCR using primers oVT464/oVT465 and oVT466/oVT467, respectively.…”

Trends, Epidemiology, and Management of Multi-Drug Resistant Gram-Negative Bacterial Infections in the Hospitalized Setting

“…AdeABC is the superfamily of efflux pumps responsible for aminoglycoside resistance. Gene targeted therapy for the gene Ade-R, which regulates AdeABC, was examined as a possible solution to tigecycline resistant A. baumannii; however, the results were not promising, indicating that Gram-negative bacteria like A. baumannii likely have multiple mechanisms of resistance to tigecycline [38]. Use is now falling out of favor while cure rates are dropping in comparison with carbapenems such as imipenem, as well as a higher mortality rate when compared to colistin [17].…”

“…Although the knock-out of Ade-R was successful, the isolates still remained largely multi-drug resistant, indicating other mechanisms of resistance likely exist. The research still showed that this kind of genome editing can be used to study and manipulate MDR and XDR bacteria [38].…”