Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles

Zwama, Martijn; Nishino, Kunihiko

doi:10.1128/aac.02392-21

Cited by 5 publications

(4 citation statements)

References 36 publications

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“…To our best knowledge, the genetic context of the three Salmonella serovars containing erm (42) reported in this study (on IncQ plasmids) is novel. Mutation in the RND-type transporter gene ( acrB _R717L/Q), which has been reported mostly in S. Typhi and S. Paratyphi A, has been confirmed recently to confer azithromycin resistance in E. coli by cloning ( 34 , 35 ). The first Arg717 substitution was predicted to have emerged around 2010 with travel-related R717Q mutant found in the United Kingdom ( 36 ).…”

Section: Discussionmentioning

confidence: 92%

Genomic analysis of azithromycin-resistant Salmonella from food animals at slaughter and processing, and retail meats, 2011–2021, United States

Ge,

Mukherjee,

et al. 2024

Microbiol Spectr

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Azithromycin, a 15-membered ring macrolide, is among the recommended antimicrobials for treating invasive salmonellosis in humans. It is not approved for use in veterinary medicine. We analyzed the U.S. National Antimicrobial Resistance Monitoring System (NARMS) culture collections (~40,700) between 2011 and 2021 from food animals at slaughter and processing and retail meats, and identified 31 azithromycin-resistant Salmonella spp. with the first occurrence in 2015. These isolates belonged to 12 Salmonella serovars and possessed one or more macrolide resistance determinants: erm (42), mef (C), mph (A), mph (E), mph (G), and msr (E) or a point mutation ( acrB _R717L), of which mph (A) was dominant (61.3%). Compared with azithromycin-susceptible controls, these determinants accounted for up to 256-fold MIC increases against azithromycin with MIC 50 and MIC 90 increased by 32- and 8-fold, respectively. We report the first detection of an mph (G)- mef (C)- mph (E)- msr (E)-containing Salmonella Agona isolate with very high-level azithromycin resistance (1,024 µg/mL) and the first detection of acrB _R717L accounting for azithromycin resistance in nontyphoidal Salmonella serovars in the United States. Plasmids of diverse replicon types were identified, with 86.2% carrying multidrug resistance including azithromycin and ceftriaxone, or decreased susceptibility to ciprofloxacin. This report also highlights an emerging mph (A)-containing (on an IncR plasmid) Salmonella Newport clone of cattle/beef origin with high-level azithromycin resistance (128 µg/mL) and decreased susceptibility to ciprofloxacin (0.25 µg/mL). Further work is needed to better understand the drivers of emerging azithromycin resistance in nontyphoidal Salmonella associated with food animal sources. IMPORTANCE Macrolides of different ring sizes are critically important antimicrobials for human medicine and veterinary medicine, though the widely used 15-membered ring azithromycin in humans is not approved for use in veterinary medicine. We document here the emergence of azithromycin-resistant Salmonella among the NARMS culture collections between 2011 and 2021 in food animals and retail meats, some with co-resistance to ceftriaxone or decreased susceptibility to ciprofloxacin. We also provide insights into the underlying genetic mechanisms and genomic contexts, including the first report of a novel combination of azithromycin resistance determinants and the characterization of multidrug-resistant plasmids. Further, we highlight the emergence of a multidrug-resistant Salmonella Newport clone in food animals (mainly cattle) with both azithromycin resistance and decreased susceptibility to ciprofloxacin. These findings contribute to a better understating of azithromycin resistance mechanisms in Salmonella and warrant further investigations on the drivers behind the emergence of resistant clones.

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Section: Discussionmentioning

confidence: 92%

Genomic analysis of azithromycin-resistant Salmonella from food animals at slaughter and processing, and retail meats, 2011–2021, United States

Ge,

Mukherjee,

et al. 2024

Microbiol Spectr

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“…Subsequent to our first description of R717L 51 , a recent study by Zwama and Nishino 60 has provided evidence which indicated steric hindrance and electrostatic effects to be the cause of a change in the relative accessibility of the PBP. This supports the work we report here, and we now significantly expand the scope of that study by providing a quantitative assessment of drug binding and the specific molecular environment within the binding pockets of the pump to further understand the molecular mechanisms of this mutation.…”

Section: Discussionmentioning

confidence: 93%

Functionally distinct mutations within AcrB underpin antibiotic resistance in different lifestyles

Trampari,

Prischi,

Vargiu

et al. 2023

npj Antimicrob Resist

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Antibiotic resistance is a pressing healthcare challenge and is mediated by various mechanisms, including the active export of drugs via multidrug efflux systems, which prevent drug accumulation within the cell. Here, we studied how Salmonella evolved resistance to two key antibiotics, cefotaxime and azithromycin, when grown planktonically or as a biofilm. Resistance to both drugs emerged in both conditions and was associated with different substitutions within the efflux-associated transporter, AcrB. Azithromycin exposure selected for an R717L substitution, while cefotaxime for Q176K. Additional mutations in ramR or envZ accumulated concurrently with the R717L or Q176K substitutions respectively, resulting in clinical resistance to the selective antibiotics and cross-resistance to other drugs. Structural, genetic, and phenotypic analysis showed the two AcrB substitutions confer their benefits in profoundly different ways. R717L reduces steric barriers associated with transit through the substrate channel 2 of AcrB. Q176K increases binding energy for cefotaxime, improving recognition in the distal binding pocket, resulting in increased efflux efficiency. Finally, we show the R717 substitution is present in isolates recovered around the world.

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“…4 ). All of the residues selected for measurements, except D141 and N282, have been shown to contribute to substrate selectivity ( 11 , 56 – 58 ). Largest average changes during conformational transition were found between DBP residues F178 and F628 (80%), F617 and the PBP residue R717 (43%), and the putative entrance channel residues I38 and I671 (36%) ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Comparative reassessment of AcrB efflux inhibitors reveals differential impact of specific pump mutations on the activity of potent compounds

Schuster,

Vavra,

Wirth

et al. 2024

Microbiol Spectr

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Multidrug resistance poses global challenges, particularly with regard to Gram-negative bacterial infections. In view of the lack of new antibiotics, drug enhancers, such as efflux pump inhibitors (EPIs), have increasingly come into focus. A number of chemically diverse agents have been reported to inhibit AcrB, the main multidrug transporter in Escherichia coli , and homologs in other Gram-negative bacteria. However, due to the often varying methodologies used for their characterization, results remain difficult to compare. In this study, using a defined selection of antibiotics known to be efflux substrates, we reevaluated 38 published compounds for their in vitro EPI activity. When examined in an E. coli strain with stable wild-type AcrB overexpression, we found 17 compounds showing at least fourfold enhancing potency with more than 2 out of 10 test drugs (belonging to eight antibiotic classes). Pyranopyridines (MBX series) were confirmed as the most potent inhibitors among agents reported so far. A new and surprising finding was that their activity, unlike that of the pyridylpiperazine EPI BDM88855, was highly susceptible to the AcrB double-mutation G141D_N282Y, which had previously been shown to diminish drug enhancing of 1-(1-naphthylmethyl)piperazine in a predominantly substrate-specific manner. Conversely, transmembrane region mutation V411A, while eliminating the drug potentiating of the BDM compound, did not decrease the activity of the MBX EPIs. Besides comparative reassessment of the potency of reported EPIs, the study demonstrated the usefulness of mutagenesis approaches providing tools for an initial discrimination of EPIs regarding their mode of function. IMPORTANCE Infections with difficult-to-treat multidrug-resistant bacteria pose an urgent global threat in view of the stagnating development of new antimicrobial substances. Efflux pumps in Gram-negative pathogens are known to substantially contribute to multidrug resistance making them promising targets for chemotherapeutic interventions to restore the efficacy of conventional antibiotics. In the present study, the in vitro activity of previously reported efflux pump inhibitors was reassessed using standardized conditions. Relevant drug sensitizing activity could be proven for almost half of the tested compounds. Further characterization of potent inhibitors was achieved by investigating the impact of specific efflux pump mutations. A double-mutation previously known to decrease the activity of the arylpiperazine 1-(1-naphthylmethyl)piperazine also impaired that of the highly efficient pyranopyridine efflux pump inhibitors. Our findings provide direct comparability of reported efflux pump inhibitors and contribute to the elucidation of their mode of action.

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Proximal Binding Pocket Arg717 Substitutions in Escherichia coli AcrB Cause Clinically Relevant Divergencies in Resistance Profiles

Cited by 5 publications

References 36 publications

Genomic analysis of azithromycin-resistant Salmonella from food animals at slaughter and processing, and retail meats, 2011–2021, United States

Genomic analysis of azithromycin-resistant Salmonella from food animals at slaughter and processing, and retail meats, 2011–2021, United States

Functionally distinct mutations within AcrB underpin antibiotic resistance in different lifestyles

Comparative reassessment of AcrB efflux inhibitors reveals differential impact of specific pump mutations on the activity of potent compounds

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