Structures and Transport Mechanisms of RND Efflux Pumps

ABSTRACT The RND-type efflux pumps are responsible for the multidrug resistance phenotype observed in many clinically relevant species. Also, RND pumps have been implicated in physiological processes, with roles in the virulence mechanisms of several pathogenic bacteria. We have previously shown that the BepC outer membrane factor of Brucella suis is involved in the efflux of diverse drugs, probably as part of a tripartite complex with an inner membrane translocase. In the present work, we characterize two membrane fusion protein-RND translocases of B. suis encoded by the bepDE and bepFG loci. MIC assays showed that the B. suis ΔbepE mutant was more sensitive to deoxycholate (DOC), ethidium bromide, and crystal violet. Furthermore, multicopy bepDE increased resistance to DOC and crystal violet and also to other drugs, including ampicillin, norfloxacin, ciprofloxacin, tetracycline, and doxycycline. In contrast to the ΔbepE mutant, the resistance profile of B. suis remained unaltered when the other RND gene (bepG) was deleted. However, the ΔbepE ΔbepG double mutant showed a more severe phenotype than the ΔbepE mutant, indicating that BepFG also contributes to drug resistance. An open reading frame (bepR) coding for a putative regulatory protein of the TetR family was found upstream of the bepDE locus. BepR strongly repressed the activity of the bepDE promoter, but DOC released the repression mediated by BepR. A clear induction of the bepFG promoter activity was observed only in the BepDE-defective mutant, indicating a regulatory interplay between the two RND efflux pumps. Although only the BepFG-defective mutant showed a moderate attenuation in model cells, the activities of both bepDE and bepFG promoters were induced in the intracellular environment of HeLa cells. Our results show that B. suis harbors two functional RND efflux pumps that may contribute to virulence.

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The ins and outs of RND efflux pumps in Escherichia coli

Frontiers in Microbiology ◽

10.3389/fmicb.2015.00587 ◽

2015 ◽

Vol 6 ◽

Cited By ~ 110

Author(s):

JoÃ£o Anes ◽

Matthew P. McCusker ◽

SÃ©amus Fanning ◽

Marta Martins

Keyword(s):

Escherichia Coli ◽

Efflux Pumps ◽

Rnd Efflux Pumps

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Differential Roles of RND Efflux Pumps in Antimicrobial Drug Resistance of Sessile and Planktonic Burkholderia cenocepacia Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03800-14 ◽

2014 ◽

Vol 58 (12) ◽

pp. 7424-7429 ◽

Cited By ~ 30

Author(s):

Silvia Buroni ◽

Nele Matthijs ◽

Francesca Spadaro ◽

Heleen Van Acker ◽

Viola C. Scoffone ◽

...

Keyword(s):

Efflux Pumps ◽

Burkholderia Cenocepacia ◽

Intrinsic Resistance ◽

Antimicrobial Drugs ◽

Content Type ◽

Antimicrobial Drug Resistance ◽

Rnd Efflux Pumps ◽

Ciprofloxacin Resistance ◽

High Level ◽

Tract Infections

ABSTRACTBurkholderia cenocepaciais notorious for causing respiratory tract infections in people with cystic fibrosis. Infections with this organism are particularly difficult to treat due to its high level of intrinsic resistance to most antibiotics. Multidrug resistance inB. cenocepaciacan be ascribed to different mechanisms, including the activity of efflux pumps and biofilm formation. In the present study, the effects of deletion of the 16 operons encoding resistance-nodulation-cell division (RND)-type efflux pumps inB. cenocepaciastrain J2315 were investigated by determining the MICs of various antibiotics and by investigating the antibiofilm effect of these antibiotics. Finally, the expression levels of selected RND genes in treated and untreated cultures were investigated using reverse transcriptase quantitative PCR (RT-qPCR). Our data indicate that the RND-3 and RND-4 efflux pumps are important for resistance to various antimicrobial drugs (including tobramycin and ciprofloxacin) in planktonicB. cenocepaciaJ2315 populations, while the RND-3, RND-8, and RND-9 efflux systems protect biofilm-grown cells against tobramycin. The RND-8 and RND-9 efflux pumps are not involved in ciprofloxacin resistance. Results from the RT-qPCR experiments on the wild-type strainB. cenocepaciaJ2315 suggest that there is little regulation at the level of mRNA expression for these efflux pumps under the conditions tested.

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Characterization of a Novel Pyranopyridine Inhibitor of the AcrAB Efflux Pump of Escherichia coli

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01866-13 ◽

2013 ◽

Vol 58 (2) ◽

pp. 722-733 ◽

Cited By ~ 91

Author(s):

Timothy J. Opperman ◽

Steven M. Kwasny ◽

Hong-Suk Kim ◽

Son T. Nguyen ◽

Chad Houseweart ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Efflux Pumps ◽

Gram Negative ◽

Content Type ◽

E Coli ◽

Resistance Nodulation Division ◽

Rnd Efflux Pumps ◽

Acrab Efflux Pump

ABSTRACTMembers of the resistance-nodulation-division (RND) family of efflux pumps, such as AcrAB-TolC ofEscherichia coli, play major roles in multidrug resistance (MDR) in Gram-negative bacteria. A strategy for combating MDR is to develop efflux pump inhibitors (EPIs) for use in combination with an antibacterial agent. Here, we describe MBX2319, a novel pyranopyridine EPI with potent activity against RND efflux pumps of theEnterobacteriaceae. MBX2319 decreased the MICs of ciprofloxacin (CIP), levofloxacin, and piperacillin versusE. coliAB1157 by 2-, 4-, and 8-fold, respectively, but did not exhibit antibacterial activity alone and was not active against AcrAB-TolC-deficient strains. MBX2319 (3.13 μM) in combination with 0.016 μg/ml CIP (minimally bactericidal) decreased the viability (CFU/ml) ofE. coliAB1157 by 10,000-fold after 4 h of exposure, in comparison with 0.016 μg/ml CIP alone. In contrast, phenyl-arginine-β-naphthylamide (PAβN), a known EPI, did not increase the bactericidal activity of 0.016 μg/ml CIP at concentrations as high as 100 μM. MBX2319 increased intracellular accumulation of the fluorescent dye Hoechst 33342 in wild-type but not AcrAB-TolC-deficient strains and did not perturb the transmembrane proton gradient. MBX2319 was broadly active againstEnterobacteriaceaespecies andPseudomonas aeruginosa. MBX2319 is a potent EPI with possible utility as an adjunctive therapeutic agent for the treatment of infections caused by Gram-negative pathogens.

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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

10.21203/rs.3.rs-46743/v4 ◽

2020 ◽

Author(s):

Bahare Salehi ◽

Zohreh Ghalavand ◽

Abbas Yadegar ◽

Gita Eslami

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Regulatory Genes ◽

Clinical Isolates ◽

Drug Resistant ◽

Broth Microdilution Method ◽

Rnd Efflux Pumps ◽

Multiple Antibiotics

Abstract Background: This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii. Methods: Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected. Conclusions: Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms.

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Multidrug Resistance Efflux Pumps in Acinetobacter spp.: An Update

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00514-21 ◽

2021 ◽

Author(s):

Vanessa Kornelsen ◽

Ayush Kumar

Keyword(s):

Multidrug Resistance ◽

Antimicrobial Resistance ◽

Bacterial Species ◽

Clinical Importance ◽

Efflux Pumps ◽

Major Facilitator Superfamily ◽

Small Multidrug Resistance ◽

Resistance Nodulation Division ◽

Rnd Efflux Pumps ◽

Major Facilitator

Acinetobacter spp. have become of increased clinical importance as studies have shown the antimicrobial resistant potential of these species. Efflux pumps can lead to reduced susceptibility to a variety of antibiotics and are present in large number across Acinetobacter spp. There are six families of efflux pumps that have been shown to be of clinical relevance: the Major Facilitator Superfamily (MFS), Small Multidrug Resistance (SMR) family, ATP-binding cassette (ABC) family, Multidrug and Toxic Compound Extrusion (MATE) family, Proteobacterial Antimicrobial Compound Efflux (PACE) family and Resistance-Nodulation-Division (RND) family. A lot of work has been done on understanding and characterizing the roles that these efflux pumps play in relation to antimicrobial resistance and the physiology of these bacteria. RND efflux pumps, with their expansive substrate profiles, are a major component of Acinetobacter spp. antimicrobial resistance. New discoveries over the last decade have shed a lot of light on to the complex regulation of these efflux pumps leading to greater understanding and potential of slowing the reduced susceptibility seen by these bacterial species.

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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

10.21203/rs.3.rs-46743/v3 ◽

2020 ◽

Author(s):

Bahare Salehi ◽

Zohreh Ghalavand ◽

Abbas Yadegar ◽

Gita Eslami

Keyword(s):

Cell Division ◽

Acinetobacter Baumannii ◽

Efflux Pump ◽

Efflux Pumps ◽

Regulatory Genes ◽

Clinical Isolates ◽

Drug Resistant ◽

Broth Microdilution Method ◽

Rnd Efflux Pumps ◽

Multiple Antibiotics

Abstract Background: This study aimed to characterize the regulation and expression of three putative resistance-nodulation-cell division (RND)-type efflux systems and their contribution to multidrug efflux in clinical isolates of Acinetobacter baumannii.Methods: Antimicrobial susceptibility testing (AST) of 95 A. baumannii isolates was determined by Kirby-Bauer disk diffusion for 18 antibiotics and minimum inhibitory concentration (MIC) of colistin was determined by broth microdilution method. Moreover, MIC of five classes of antibiotics was assessed using E-test strips in the presence and absence of phenylalanine-arginine beta-naphthylamide (PAβN). Regulatory genes of RND efflux pumps (AdeRS, AdeL, AdeN and BaeSR) were subjected to sequencing. The relative expression of adeB. adeG and adeJ genes was determined by quantitative real-time PCR (RT-PCR).Results: Overall, majority of isolates (93%) were extensively drug-resistant (XDR). In the phenotypic assay, efflux pump activity was observed in 40% of isolates against multiple antibiotics mainly tigecycline, but not to imipenem. Several amino acid substitutions were detected in the regulatory genes; except in AdeN. Of note, G186V in AdeS were found to be associated with overexpression of their relative efflux pumps. No insertion sequences (ISs) were detected.Conclusions: Our findings outline the role of RND efflux pumps in resistance of A. baumannii against multiple antibiotics particularly tigecycline, and point out importance of a variety of single mutations in the corresponding regulatory systems. Even though it has been concluded that multidrug resistance occurs as a result of a complex sets of different resistant mechanisms.

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