Target-Based Resistance in Pseudomonas aeruginosa and Escherichia coli to NBTI 5463, a Novel Bacterial Type II Topoisomerase Inhibitor

Abstract

), a novel bacterial type II topoisomerase inhibitor, NBTI 5463, with activity against Gram-negative pathogens was described. First-step resistance mutations in Pseudomonas aeruginosa arose exclusively in the nfxB gene, a regulator of the MexCD-OprJ efflux pump system. The present report describes further resistance studies with NBTI 5463 in both Pseudomonas aeruginosa and Escherichia coli. Second-step mutations in P. aeruginosa arose at aspartate 82 of the gyrase A subunit and led to 4-to 8-fold increases in the MIC over those seen in the parental strain with a first-step nfxB efflux mutation. A third-step mutant showed additional GyrA changes, with no changes in topoisomerase IV. Despite repeated efforts, resistance mutations could not be selected in E. coli. Genetic introduction of the Asp82 mutations observed in P. aeruginosa did not significantly increase the NBTI MIC in E. coli. However, with the aspartate 82 mutation present, it was possible to select second-step mutations in topoisomerase IV that did lead to MIC increases of 16-and 128-fold. As with the gyrase aspartate 82 mutation, the mutations in topoisomerase IV did not by themselves raise the NBTI MIC in E. coli. Only the presence of mutations in both targets of E. coli led to an increase in NBTI MIC values. This represents a demonstration of the value of balanced dual-target activity in mitigating resistance development.

Insights into the Mechanism of Inhibition of Novel Bacterial Topoisomerase Inhibitors from Characterization of Resistant Mutants of Staphylococcus aureus

“…Whereas most mutants showed similar decreases in susceptibility across the NBTI compounds, the D83G mutant was the most variable. The elevation in the MICs of AZ6142 and AZ0217 against this mutant was quite modest (2-to 4-fold), yet there was a significant impact (128-fold) on MCHEM18, a level similar to that observed with other NBTI molecules in the literature (20,22,31). The modest 2-fold increase in the MIC of AZ6142 likely explains the inability to obtain this mutant despite multiple selection experiments with AZ6142.…”

“…All of the mutants contained nonsynonymous substitutions in the DNA gyrase subunits; 12 of them were in GyrA, and two mu- tants carried a GyrB substitution. The D83G and M121K substitutions, which have been seen previously with other NBTIs (22,24,31), were observed with all three compounds in this study, with the exception of D83G mutants with AZ6142, despite several independent experiments and characterization of multiple variants. The mutants were tested for cross-resistance against all three NBTI compounds, as well as two fluoroquinolones, ciprofloxacin and levofloxacin (Table 4).…”

“…The most significant and consistent MIC increases were observed with the GyrA M121K substitution, ranging from 64-to 128-fold for all of the NBTI compounds. This change, like D83G, is also located in the binding pocket and has been shown to affect all other NBTI molecules studied thus far in the literature (20,22,31), reflecting its scaffold-specific attributes. Additionally, there was no cross-resistance observed with either ciprofloxacin or levofloxacin, as previously reported (Table 4).…”

Rapid Evolution of Reduced Susceptibility against a Balanced Dual-Targeting Antibiotic through Stepping-Stone Mutations

“…Currently, designing multitarget antibiotics is a major focus of the pharmaceutical industry, but until now, relatively few drugs have been demonstrated to achieve a balanced inhibition of multiple microbial targets (8)(9)(10). Due to the shortage of in-depth resistance studies, our knowledge on the tempo and mode of resistance development against multitarget antibiotics remains limited (11)(12)(13).…”

Antibiotic usage promotes the evolution of resistance against gepotidacin, a novel multi-targeting drug

“…Such exemplary antibiotics include gepotidacin 9 , NBTI 5463 10 , Trius' C3 and C4 11 . However, due to the shortage of indepth resistance studies, our knowledge on the tempo and mode of resistance development against multi-target antibiotics remains limited (but see [12][13][14] ).…”