New Topoisomerase Inhibitors: Evaluating the Potency of Gepotidacin and Zoliflodacin in Fluoroquinolone-Resistant Escherichia coli upon tolC Inactivation and Differentiating Their Efflux Pump Substrate Nature

Abstract

Inactivating tolC in multidrug-resistant Escherichia coli with differing sequence types and quinolone resistance-determining mutations reveals remarkably potentiated activity of the first-in-class topoisomerase inhibitors gepotidacin and zoliflodacin. Differences between both structurally unrelated compounds in comparison to fluoroquinolones regarding the selectivity of E. coli RND-type transporters, efflux inhibitors, and AcrB porter domain mutations were demonstrated. The findings should reinforce efforts to develop efflux bypassing drugs and provide AcrB targets with critical relevance for this purpose.

Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate

“…In order to evaluate the contribution of MdtF to total TolC-dependent efflux in MDR E. coli strains, we aimed to compare an mdtF / acrB double-knockout of the patient isolate KUN9180 (besides acrB also showing mdtF expression) with the acrB (KUN∆ acrB ) and tolC single-knockout (KUN∆ tolC ) mutants from previous studies [ 3 , 11 ]. Because selection options are limited in MDR strains, the reutilization of the kanamycin/neomycin selection cassette used for switching off acrB was an option.…”

“…The resulting double-knockout was subjected to susceptibility testing, and the results were compared with those from the KUN∆ acrB and KUN∆ tolC single-knockout mutants. Between the latter, significant differences in the susceptibilities to nadifloxacin, zoliflodacin [ 11 ], and novobiocin ( Table 1 ) had been shown, but not to several other proven AcrB substrates, including the more hydrophilic fluoroquinolones levofloxacin and moxifloxacin and the non-fluoroquinolone gyrase inhibitor gepotidacin [ 11 ], to tetracycline, chloramphenicol, linezolid, clindamycin, and rifaximin ( Table S1 ), all of which were demonstrably effluxed in isolate KUN9180 [ 3 , 11 ]. Regarding those results, only nadifloxacin, zoliflodacin, and novobiocin are potentially extruded from a TolC-dependent transporter other than AcrB.…”

“…This could be due to another TolC-dependent efflux pump. However, we did not find any significant expression of such an additional transporter in the KUN9180 isolate [ 11 ]. Residual differences in the susceptibilities of the ∆ acrB ∆ mdtF and the tolC inactivated mutants could be explained by further physiological functions of TolC [ 13 ].…”

“…In recent studies with such isolates, a significant percentage of the collection revealed mdtF expression in addition to acrB expression [ 10 , 11 ]. MdtF (formerly YhiV), exhibiting a sequence similarity of 79% compared with AcrB [ 12 ], is complexed with the membrane fusion protein MdtE (formerly YhiU) and with TolC, the ubiquitous outer membrane channel in E. coli not only working together with RND-type efflux pumps but also with transporters of other families, such as the ABC-transporter MacB and the MFS-transporter EmrB [ 13 ].…”

“…The principal capability of MdtF to efflux drugs from different classes, with the exception of the oxazolidinone linezolid, has been shown [ 9 ], but little is known about a potential role in MDR development. In the present study, we aimed to explore the MdtF functionality and its contribution to drug resistance in an MDR E. coli isolate with verified expression of this RND-type efflux pump [ 3 , 11 ].…”