Innate Aminoglycoside Resistance of Achromobacter xylosoxidans Is Due to AxyXY-OprZ, an RND-Type Multidrug Efflux Pump

ABSTRACTAchromobacter xylosoxidansis an innately multidrug-resistant pathogen which is emerging in cystic fibrosis (CF) patients. We characterized a new resistance-nodulation-cell division (RND)-type multidrug efflux pump, AxyXY-OprZ. This system is responsible for the intrinsic high-level resistance ofA. xylosoxidansto aminoglycosides (tobramycin, amikacin, and gentamicin). Furthermore, it can extrude cefepime, carbapenems, some fluoroquinolones, tetracyclines, and erythromycin. Some of the AxyXY-OprZ substrates are major components widely used to treat pulmonary infections in CF patients.

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First Description of an RND-Type Multidrug Efflux Pump in Achromobacter xylosoxidans, AxyABM

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00341-11 ◽

2011 ◽

Vol 55 (10) ◽

pp. 4912-4914 ◽

Cited By ~ 42

Author(s):

Julien Bador ◽

Lucie Amoureux ◽

Jean-Marie Duez ◽

Anthony Drabowicz ◽

Eliane Siebor ◽

...

Keyword(s):

Cystic Fibrosis ◽

Multidrug Resistance ◽

Cell Division ◽

Efflux Pump ◽

Achromobacter Xylosoxidans ◽

Clinical Strain ◽

Multidrug Efflux ◽

Emerging Pathogen ◽

Multidrug Efflux Pump ◽

Content Type

ABSTRACTAchromobacter xylosoxidansis an emerging pathogen in cystic fibrosis patients. The multidrug resistance of these bacteria remains poorly understood. We have characterized in a clinical strain the first resistance-nodulation-cell division (RND)-type multidrug efflux pump in this species: AxyABM. The inactivation of the transporter componentaxyBgene led to decreased MICs of cephalosporins (except cefepime), aztreonam, nalidixic acid, fluoroquinolones, and chloramphenicol.

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Characterization of Posttranslationally Modified Multidrug Efflux Pumps Reveals an Unexpected Link between Glycosylation and Antimicrobial Resistance

mBio ◽

10.1128/mbio.02604-20 ◽

2020 ◽

Vol 11 (6) ◽

Author(s):

Sherif Abouelhadid ◽

John Raynes ◽

Tam Bui ◽

Jon Cuccui ◽

Brendan W. Wren

Keyword(s):

Antimicrobial Resistance ◽

Efflux Pump ◽

Multidrug Resistant ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Gram Negative ◽

Content Type ◽

Multidrug Efflux Pumps ◽

Pump Activity

ABSTRACT The substantial rise in multidrug-resistant bacterial infections is a current global imperative. Cumulative efforts to characterize antimicrobial resistance in bacteria has demonstrated the spread of six families of multidrug efflux pumps, of which resistance-nodulation-cell division (RND) is the major mechanism of multidrug resistance in Gram-negative bacteria. RND is composed of a tripartite protein assembly and confers resistance to a range of unrelated compounds. In the major enteric pathogen Campylobacter jejuni, the three protein components of RND are posttranslationally modified with N-linked glycans. The direct role of N-linked glycans in C. jejuni and other bacteria has long been elusive. Here, we present the first detailed account of the role of N-linked glycans and the link between N-glycosylation and antimicrobial resistance in C. jejuni. We demonstrate the multifunctional role of N-linked glycans in enhancing protein thermostability, stabilizing protein complexes and the promotion of protein-protein interaction, thus mediating antimicrobial resistance via enhancing multidrug efflux pump activity. This affirms that glycosylation is critical for multidrug efflux pump assembly. We present a generalized strategy that could be used to investigate general glycosylation system in Campylobacter genus and a potential target to develop antimicrobials against multidrug-resistant pathogens. IMPORTANCE Nearly all bacterial species have at least a single glycosylation system, but the direct effects of these posttranslational protein modifications are unresolved. Glycoproteome-wide analysis of several bacterial pathogens has revealed general glycan modifications of virulence factors and protein assemblies. Using Campylobacter jejuni as a model organism, we have studied the role of general N-linked glycans in the multidrug efflux pump commonly found in Gram-negative bacteria. We show, for the first time, the direct link between N-linked glycans and multidrug efflux pump activity. At the protein level, we demonstrate that N-linked glycans play a role in enhancing protein thermostability and mediating the assembly of the multidrug efflux pump to promote antimicrobial resistance, highlighting the importance of this posttranslational modification in bacterial physiology. Similar roles for glycans are expected to be found in other Gram-negative pathogens that possess general protein glycosylation systems.

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Motility, Biofilm Formation and Antimicrobial Efflux of Sessile and Planktonic Cells of Achromobacter xylosoxidans

Pathogens ◽

10.3390/pathogens8010014 ◽

2019 ◽

Vol 8 (1) ◽

pp. 14 ◽

Cited By ~ 7

Author(s):

Signe Nielsen ◽

Line Penstoft ◽

Niels Nørskov-Lauritsen

Keyword(s):

Biofilm Formation ◽

Efflux Pump ◽

Multidrug Resistant ◽

Achromobacter Xylosoxidans ◽

Flagellar Motility ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Biofilm Growth ◽

Membrane Fusion Protein

Achromobacter xylosoxidans is an innately multidrug-resistant bacterium capable of forming biofilms in the respiratory tract of cystic fibrosis (CF) patients. During the transition from the planktonic stage to biofilm growth, bacteria undergo a transcriptionally regulated differentiation. An isolate of A. xylosoxidans cultured from the sputum of a CF patient was separated into sessile and planktonic stages in vitro, and the transcriptomes were compared. The selected genes of interest were subsequently inactivated, and flagellar motility was found to be decisive for biofilm formation in vitro. The spectrum of a new resistance-nodulation-cell division (RND)-type multidrug efflux pump (AxyEF-OprN) was characterized by inactivation of the membrane fusion protein. AxyEF-OprN is capable of extruding some fluoroquinolones (levofloxacin and ciprofloxacin), tetracyclines (doxycycline and tigecycline) and carpabenems (ertapenem and imipenem), which are classes of antimicrobials that are widely used for treatment of CF pulmonary infections.

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ArmA Methyltransferase in a Monophasic Salmonella enterica Isolate from Food

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00308-11 ◽

2011 ◽

Vol 55 (11) ◽

pp. 5262-5266 ◽

Cited By ~ 17

Author(s):

Sophie A. Granier ◽

Laura Hidalgo ◽

Alvaro San Millan ◽

Jose Antonio Escudero ◽

Belen Gutierrez ◽

...

Keyword(s):

16S Rrna ◽

Salmonella Enterica ◽

Multidrug Resistant ◽

Broad Host Range ◽

En Bloc ◽

Content Type ◽

Route Of Transmission ◽

Amoxicillin Clavulanate ◽

High Level ◽

Level Resistance

ABSTRACTThe 16S rRNA methyltransferase ArmA is a worldwide emerging determinant that confers high-level resistance to most clinically relevant aminoglycosides. We report here the identification and characterization of a multidrug-resistantSalmonella entericasubspecies I.4,12:i:− isolate recovered from chicken meat sampled in a supermarket on February 2009 in La Reunion, a French island in the Indian Ocean. Susceptibility testing showed an unusually high-level resistance to gentamicin, as well as to ampicillin, expanded-spectrum cephalosporins and amoxicillin-clavulanate. Molecular analysis of the 16S rRNA methyltransferases revealed presence of thearmAgene, together withblaTEM-1,blaCMY-2, andblaCTX-M-3. All of these genes could be transferreden blocthrough conjugation intoEscherichia coliat a frequency of 10−5CFU/donor. Replicon typing and S1 pulsed-field gel electrophoresis revealed that thearmAgene was borne on an ∼150-kb broad-host-range IncP plasmid, pB1010. To elucidate howarmAhad integrated in pB1010, a PCR mapping strategy was developed for Tn1548, the genetic platform forarmA.The gene was embedded in a Tn1548-like structure, albeit with a deletion of the macrolide resistance genes, and an IS26was inserted within themelgene. To our knowledge, this is the first report of ArmA methyltransferase in food, showing a novel route of transmission for this resistance determinant. Further surveillance in food-borne bacteria will be crucial to determine the role of food in the spread of 16S rRNA methyltransferase genes worldwide.

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Integrative and Conjugative Element-Mediated Azithromycin Resistance in Multidrug-Resistant Salmonella enterica Serovar Albany

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02634-20 ◽

2021 ◽

Vol 65 (5) ◽

Author(s):

Yu-Ping Hong ◽

Ying-Tsong Chen ◽

You-Wun Wang ◽

Bo-Han Chen ◽

Ru-Hsiou Teng ◽

...

Keyword(s):

Vibrio Cholerae ◽

Salmonella Enterica ◽

Multidrug Resistant ◽

Content Type ◽

Human Salmonellosis ◽

High Level ◽

Azithromycin Resistance ◽

Level Resistance ◽

Integrative And Conjugative Element

ABSTRACT We identified an erm42-carrying integrative and conjugative element, ICE_erm42, in 26.4% of multidrug-resistant Salmonella enterica serovar Albany isolates recovered from cases of human salmonellosis between 2014 and 2019 in Taiwan. ICE_erm42-carrying strains displayed high-level resistance to azithromycin, and the element could move into the phylogenetically distant species Vibrio cholerae via conjugation.

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Emergence of Two AcrB Substitutions Conferring Multidrug Resistance to Salmonella spp.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01589-20 ◽

2021 ◽

Vol 65 (5) ◽

Author(s):

Ling Yang ◽

Haiyang Shi ◽

Lijuan Zhang ◽

Xiaoling Lin ◽

Yinan Wei ◽

...

Keyword(s):

Efflux Pump ◽

Acid Sites ◽

Site Directed Mutagenesis ◽

Fluorescence Labeling ◽

Directed Mutagenesis ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Salmonella Spp ◽

Multiple Substrates ◽

High Level

ABSTRACT AcrAB-TolC is a major tripartite multidrug efflux pump conferring resistance to a wide variety of compounds in Gram-negative pathogens. Many AcrB mutants have been constructed through site-directed mutagenesis to probe the mechanism of AcrB function in antibiotic resistance. However, much less is known about the actual drug resistance-related mutants that naturally occur in clinically isolated pathogens. Here, we report two novel AcrB substitutions, M78I and P319L, in clinically isolated Salmonella strains with high-level ciprofloxacin resistance. Plasmids expressing the detected acrB mutations were constructed and introduced into SL1344 ΔacrB. Antimicrobial susceptibility assays showed that AcrB M78I, AcrB P319L, and AcrB M78I/319L all conferred reduced susceptibilities to multiple substrates, including fluoroquinolones, erythromycin, tetracyclines, bile salts, and dyes. Site-directed mutagenesis and MIC results revealed that the increased hydrophobicity of M78I was one of the reasons the AcrB M78I mutant had lower susceptibility to fluoroquinolones. Fluorescence labeling experiments suggested that the AcrB M78I substitution enhanced the binding of substrates to certain amino acid sites in the efflux pathway (e.g., sites Q89, E673, and F617) and weakened the binding to other amino acids (e.g., S134 and N274). Structural modeling disclosed that the increased flexibility of Leu was favorable for the functional rotation of AcrB compared to the original Pro residue. AcrA 319L makes the functional rotation of AcrB more flexible; this enables substrate efflux more efficiently. In order to understand the mechanism of AcrAB-TolC drug efflux well, the interaction between AcrA and AcrB in the role of the substrate efflux of AcrAB-TolC should be further investigated.

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A Broad-Specificity Multidrug Efflux Pump Requiring a Pair of Homologous SMR-Type Proteins

Journal of Bacteriology ◽

10.1128/jb.182.8.2311-2313.2000 ◽

2000 ◽

Vol 182 (8) ◽

pp. 2311-2313 ◽

Cited By ~ 63

Author(s):

Donald L. Jack ◽

Michael L. Storms ◽

Jason H. Tchieu ◽

Ian T. Paulsen ◽

Milton H. Saier

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Multidrug Resistant ◽

Drug Efflux ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Resistant Phenotype ◽

Small Multidrug Resistance ◽

Drug Efflux Pumps ◽

Broad Specificity

ABSTRACT The Bacillus subtilis genome encodes seven homologues of the small multidrug resistance (SMR) family of drug efflux pumps. Six of these homologues are paired in three distinct operons, and coexpression in Escherichia coli of one such operon,ykkCD, but not expression of either ykkC orykkD alone, gives rise to a broad specificity, multidrug-resistant phenotype including resistance to cationic, anionic, and neutral drugs.

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Cryo-EM Structures of a Gonococcal Multidrug Efflux Pump Illuminate a Mechanism of Drug Recognition and Resistance

mBio ◽

10.1128/mbio.00996-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 5

Author(s):

Meinan Lyu ◽

Mitchell A. Moseng ◽

Jennifer L. Reimche ◽

Concerta L. Holley ◽

Vijaya Dhulipala ◽

...

Keyword(s):

Electron Microscopy ◽

Drug Resistance ◽

Neisseria Gonorrhoeae ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Transmitted Infection ◽

Content Type ◽

Cryo Electron Microscopy

ABSTRACT Neisseria gonorrhoeae is an obligate human pathogen and causative agent of the sexually transmitted infection (STI) gonorrhea. The most predominant and clinically important multidrug efflux system in N. gonorrhoeae is the multiple transferrable resistance (Mtr) pump, which mediates resistance to a number of different classes of structurally diverse antimicrobial agents, including clinically used antibiotics (e.g., β-lactams and macrolides), dyes, detergents and host-derived antimicrobials (e.g., cationic antimicrobial peptides and bile salts). Recently, it has been found that gonococci bearing mosaic-like sequences within the mtrD gene can result in amino acid changes that increase the MtrD multidrug efflux pump activity, probably by influencing antimicrobial recognition and/or extrusion to elevate the level of antibiotic resistance. Here, we report drug-bound solution structures of the MtrD multidrug efflux pump carrying a mosaic-like sequence using single-particle cryo-electron microscopy, with the antibiotics bound deeply inside the periplasmic domain of the pump. Through this structural approach coupled with genetic studies, we identify critical amino acids that are important for drug resistance and propose a mechanism for proton translocation. IMPORTANCE Neisseria gonorrhoeae has become a highly antimicrobial-resistant Gram-negative pathogen. Multidrug efflux is a major mechanism that N. gonorrhoeae uses to counteract the action of multiple classes of antibiotics. It appears that gonococci bearing mosaic-like sequences within the gene mtrD, encoding the most predominant and clinically important transporter of any gonococcal multidrug efflux pump, significantly elevate drug resistance and enhance transport function. Here, we report cryo-electron microscopy (EM) structures of N. gonorrhoeae MtrD carrying a mosaic-like sequence that allow us to understand the mechanism of drug recognition. Our work will ultimately inform structure-guided drug design for inhibiting these critical multidrug efflux pumps.

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Multidrug Adaptive Resistance of Pseudomonas aeruginosa Swarming Cells

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01999-19 ◽

2019 ◽

Vol 64 (3) ◽

Cited By ~ 2

Author(s):

Shannon R. Coleman ◽

Travis Blimkie ◽

Reza Falsafi ◽

Robert E. W. Hancock

Keyword(s):

Pseudomonas Aeruginosa ◽

Antibiotic Resistance ◽

Efflux Pump ◽

Iron Acquisition ◽

Polymyxin B ◽

Rna Seq ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

Content Type ◽

Adaptive Resistance

ABSTRACT Swarming surface motility is a complex adaptation leading to multidrug antibiotic resistance and virulence factor production in Pseudomonas aeruginosa. Here, we expanded previous studies to demonstrate that under swarming conditions, P. aeruginosa PA14 is more resistant to multiple antibiotics, including aminoglycosides, β-lactams, chloramphenicol, ciprofloxacin, tetracycline, trimethoprim, and macrolides, than swimming cells, but is not more resistant to polymyxin B. We investigated the mechanism(s) of swarming-mediated antibiotic resistance by examining the transcriptomes of swarming cells and swarming cells treated with tobramycin by transcriptomics (RNA-Seq) and reverse transcriptase quantitative PCR (qRT-PCR). RNA-Seq of swarming cells (versus swimming) revealed 1,581 dysregulated genes, including 104 transcriptional regulators, two-component systems, and sigma factors, numerous upregulated virulence and iron acquisition factors, and downregulated ribosomal genes. Strain PA14 mutants in resistome genes that were dysregulated under swarming conditions were tested for their ability to swarm in the presence of tobramycin. In total, 41 mutants in genes dysregulated under swarming conditions were shown to be more resistant to tobramycin under swarming conditions, indicating that swarming-mediated tobramycin resistance was multideterminant. Focusing on two genes downregulated under swarming conditions, both prtN and wbpW mutants were more resistant to tobramycin, while the prtN mutant was additionally resistant to trimethoprim under swarming conditions; complementation of these mutants restored susceptibility. RNA-Seq of swarming cells treated with subinhibitory concentrations of tobramycin revealed the upregulation of the multidrug efflux pump MexXY and downregulation of virulence factors.

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Genetic Characterization of Highly Fluoroquinolone-Resistant Clinical Escherichia coli Strains from China: Role ofacrR Mutations

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.45.5.1515-1521.2001 ◽

2001 ◽

Vol 45 (5) ◽

pp. 1515-1521 ◽

Cited By ~ 181

Author(s):

Hui Wang ◽

Joann L. Dzink-Fox ◽

Minjun Chen ◽

Stuart B. Levy

Keyword(s):

Escherichia Coli ◽

Blot Analysis ◽

Genetic Basis ◽

Efflux Pump ◽

Pcr Amplification ◽

Fluoroquinolone Resistance ◽

Multidrug Efflux ◽

Multidrug Efflux Pump ◽

E Coli ◽

High Level

ABSTRACT The genetic basis for fluoroquinolone resistance was examined in 30 high-level fluoroquinolone-resistant Escherichia coliclinical isolates from Beijing, China. Each strain also demonstrated resistance to a variety of other antibiotics. PCR sequence analysis of the quinolone resistance-determining region of the topoisomerase genes (gyrA/B, parC) revealed three to five mutations known to be associated with fluoroquinolone resistance. Western blot analysis failed to demonstrate overexpression of MarA, and Northern blot analysis did not detect overexpression of soxS RNA in any of the clinical strains. The AcrA protein of the AcrAB multidrug efflux pump was overexpressed in 19 of 30 strains of E. colitested, and all 19 strains were tolerant to organic solvents. PCR amplification of the complete acrR (regulator/repressor) gene of eight isolates revealed amino acid changes in four isolates, a 9-bp deletion in another, and a 22-bp duplication in a sixth strain. Complementation with a plasmid-borne wild-type acrR gene reduced the level of AcrA in the mutants and partially restored antibiotic susceptibility 1.5- to 6-fold. This study shows that mutations in acrR are an additional genetic basis for fluoroquinolone resistance.

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