Research Article
23 August 2019

Efflux-Mediated Resistance to New Oxazolidinones and Pleuromutilin Derivatives in Escherichia coli with Class Specificities in the Resistance-Nodulation-Cell Division-Type Drug Transport Pathways

ABSTRACT

A major contribution of the resistance-nodulation-cell division (RND)-transporter AcrB to resistance to oxazolidinones and pleuromutilin derivatives in Escherichia coli was confirmed. However, we discovered significant differences in efflux inhibitor activities, specificities of the homologous pump YhiV (MdtF), and the impact of AcrB pathway mutations. Particularly, entrance channel double-mutation I38F I671T and distal binding pocket mutation F615A revealed class-specific transport routes of oxazolidinones and pleuromutilin derivatives. The findings could contribute to the understanding of the RND-type multidrug transport pathways.

INTRODUCTION

The emergence of multidrug resistance (MDR) in Gram-negative pathogens has surpassed the supply of new antibiotics by far. Resistance-nodulation-cell division (RND)-type efflux transporters, such as AcrB from Escherichia coli, play a crucial role in MDR development (1). Their function is proton-motive force (PMF) dependent, and many of them are characterized by a remarkably broad substrate compatibility (2). AcrB constitutes the major MDR efflux pump in E. coli, with constitutive expression in wild-type strains and stable overexpression due to drug exposure (3). Close homologs of AcrB are the E. coli MDR transporters AcrF and YhiV (MdtF), which supposedly play a minor role, since at least in vitro, their expression could only be triggered after AcrB inactivation (4, 5). Among substrates extruded by AcrB are “Gram-positive drugs,” including members from the relatively new class of fully synthetic oxazolidinones, such as linezolid and tedizolid (6), and the upcoming semisynthetic pleuromutilin derivatives (7). Agents from both classes target intracellular bacterial protein biosynthesis at the translational level. Only the oxazolidinones linezolid and tedizolid and the pleuromutilin retapamulin have been in use for human treatment, but new compounds are under development even against Gram-negative pathogens (710).
We evaluated the impact of RND-type transporters on the susceptibility to available oxazolidinones and pleuromutilins in E. coli using standard broth microdilution assays as described previously (11). We included wild-type acrB and efflux-deficient strains (3, 5, 12), mutants expressing acrF and yhiV, and eight AcrB substrate pathway mutants (11, 13, 14) (Table 1). The oxazolidinones tested comprised the experimental agents sutezolid (10) and radezolid (9) and the oxazolidinone-fluoroquinolone conjugate cadazolid (15). For completeness, we included previous data for linezolid (5, 13) and tedizolid (data for the latter are already available for wild-type E. coli, a ΔAcrB mutant, and the I38F I671T strain [6, 11]). Among the pleuromutilins, we tested the veterinary drugs tiamulin and valnemulin as well as retapamulin and their precursor pleuromutilin.
TABLE 1
TABLE 1 Strains and mutants used in this study
E. coli strain or mutantDescriptionSource or reference
Strains  
    ATCC 25922Reference E. coli strain (constitutive wild-type acrB expression)DSMZa, no. DSM-1103
    AG100K-12 derivative [argE3 thi-1 rpsL xyl mtl Δ(gal-uvrB) supE44], parent of 3-AG100 (constitutive wild-type acrB expression)12
    3-AG100AG100 derivative (wild-type acrB overexpressed)3
    2-DC14PSAG100 derivative, acrAB knockout mutant (wild-type acrF overexpressed)4
    DKO20/12-DC14PS derivative, acrAB acrF double-knockout (wild-type yhiV overexpressed)5
    TKOacrAB acrF yhiV triple knockout5
AcrB mutantsb  
    F610ADistal binding pocket mutant13
    F136ADistal binding pocket mutant13
    F178ADistal binding pocket mutant13
    F615ADistal binding pocket (G-loop) mutant13
    F617ADistal binding pocket (G-loop) mutant13
    F628ADistal binding pocket mutant13
    G616NDistal binding pocket (G-loop) mutant14
    I38F I671TEntrance pathway mutant11
    ΔAcrBDeletion mutant (whole acrB; resistance cassette removed)This studyc
    D408ATransmembrane mutant (impeded proton motive force generation)This studyc
a
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (https://www.dsmz.de).
b
Chromosomal mutants, derived from E. coli 3-AG100.
c
Recombination method used for site-directed mutagenesis as described previously (13), with oligonucleotides given in the supplemental material.
High efflux compatibility was confirmed for both drug classes as demonstrated by ≥64-fold increased susceptibilities due to acrB deletion. Notably, overexpression versus constitutive acrB expression caused 16- and ≥128-fold increased resistance to radezolid and cadazolid, respectively (Table 2). To exclude efflux-independent effects due to smaller amounts of integral membrane proteins in a deletion mutant, we engineered an AcrB mutant D408A (Table 1). This mutation has been shown to prevent PMF generation without compromising the incorporation of functionless AcrB proteins (16, 17). We proved similar susceptibilities of the D408A and ΔAcrB mutants and the triple-knockout (TKO) mutant additionally lacking AcrF and YhiV (Table 2). The contribution of other transporters to MDR appeared negligible as shown by previous expression studies with acrB-deficient E. coli mutants (5, 6). Among the drugs tested, only the susceptibility to linezolid was significantly increased further in the ΔAcrB mutant by the PMF-inhibitor CCCP at 10 μM (Table 2), a concentration not far from the intrinsic MIC of the mutant (20 μM), but 5 μM CCCP did not show any impact (data not shown). Notably, resistance breakpoints for linezolid and tedizolid (4 μg/ml and 0.5 μg/ml, respectively, with Staphylococcus spp. [EUCAST clinical breakpoint tables v. 6.0]) were not reached in the efflux-deficient mutants. However, susceptibility increases appeared promising with radezolid, cadazolid, and the pleuromutilins regarding repurposing options for Gram-negative application under efflux inhibition.
TABLE 2
TABLE 2 Susceptibilities of E. coli strains expressing wild-type and mutated RND-type transporters
E. coli strain or mutantMIC (μg/ml) for:a
OxazolidinonesPleuromutilins
LinezolidTedizolidSutezolidRadezolidCadazolid (FQb conjugate)TiamulinValnemulinRetapamulinPleuromutilin
Wild-type RND-transporter-expressing strains         
    ATCC 25922256>128>5121622566432256
    AG10025632512821283216128
    3-AG100 (acrB overexpressed)1024>128>512128>2565126464>256
        With 25 μg/ml PAβN128161281628424
        With 100 μg/ml NMP321282564256256643264
    2-DC14PS (ΔacrAB acrF overexpressed)256>12851232>2562566464256
    DKO20/1 (ΔacrAB ΔacrF yhiV overexpressed)83232125664161616
AcrB drug pathway mutantsc         
    I38F I671T1616322641281283232
    F178A64>12812832641281616128
    F615A512>1285126432324832
    G616N512>12851264>2562566432256
    F136A1024>128512641281281616128
    F628A512>128512321282563232256
    F617A512>12851264128128816256
    F610A25632512832328832
Efflux-deficient mutantsc         
    ΔAcrB16280.50.250.50.250.1250.5
        With 25 μg/ml PAβN80.540.50.060.1250.030.030.25
        With 100 μg/ml NMP16180.50.1250.250.1250.060.25
        With 10 μM CCCP4140.50.1250.250.1250.060.25
    D408Ad16480.50.510.50.250.5
    TKO (ΔacrAB ΔacrF ΔyhiV)8140.250.50.250.1250.060.125
a
MIC decreases >4-fold compared to the MIC detected with parental E. coli 3-AG100 (acrB overexpressed) are in boldface font; >, MIC determination limited due to drug precipitation at concentrations above the indicated values.
b
FQ, fluoroquinolone.
c
Mutants derived from parental E. coli strain 3-AG100 (TKO from DKO20/1).
d
Efflux deficiency due to a point mutation preventing the proton generation for PMF.
The efflux pump inhibitor (EPI) 1-(1-naphthylmethyl)-piperazine (NMP) (18) enhanced the activity of oxazolidinones up to 32-fold but only up to 2-fold that of the pleuromutilin derivatives. In contrast, with Phe-Arg-β-naphthylamide (PAβN), an EPI with proven outer membrane activity (19), at least 16-fold enhancement was found with all of the pleuromutilins tested (Table 2). The differing EPI activities might give an additional hint on the drug pathway specificities.
The homologous transporter AcrF can almost completely replace AcrB (1, 4, 5), also with respect to the oxazolidinones and pleuromutilins tested (Table 2). In contrast, the substrate range of YhiV is narrower. For instance, linezolid is a poor substrate (5), and this was also the case with radezolid. However, the efflux of tedizolid, sutezolid, and the pleuromutilins appeared significantly less impeded as shown by MIC decreases in the yhiV-expressing mutant that were far from those occurring due to efflux deficiency (Table 2). Surprisingly, YhiV mediated high resistance to the fluoroquinolone-oxazolidinone conjugate cadazolid, whose activity was restored when knocking out yhiV (Table 2). The reason for the distinct specificities of YhiV remains to be elucidated.
Regarding AcrB substrate pathway mutations investigated in this study, we could broadly define those revealing a single compound effect, marginal effects on resistance to drugs from both classes, class-independent overall resistance decreases, and an oxazolidinone or a pleuromutilin specificity. A unique impact occurred from mutation F617A located at the so-called G-loop (switch-loop) separating the distal from the proximal substrate binding pockets (20). It significantly increased the susceptibility only to the largest member of the pleuromutilin derivatives, valnemulin (molecular weight [MW], 564.8), whereas minor (≤4-fold) or no effects were seen with the other agents tested (Table 2). Notably, adjacent G-loop mutation G616N severely affecting macrolide resistance in E. coli (14, 20) did not cause any significant alterations with either pleuromutilins or oxazolidinones. Similar marginal effects with drugs from both classes appeared from distal binding pocket mutations F136A and F628A. In contrast, mutation F610A located across the G-loop (Fig. 1) severely increased the susceptibility to nearly all drugs from both classes. A minor impact was only found on resistances to linezolid and sutezolid, with MIC decreases of no more than 4-fold (Table 2). Sutezolid differs from linezolid only by sulfur replacing the oxygen of the morpholine ring.
FIG 1
FIG 1 AcrB binding state monomer (PDB 2HRT, chain B); visualization using PyMOL (Molecular Graphics System version 2.1.1, Schrödinger, LLC). (A) Part of the AcrB binding state monomer shown as cartoon (side view from the periplasmic cleft). PC1, PC2, PN1, and PN2, subdomains of the porter domain; Dd, docking domain; TM, transmembrane domain. (B) Enlarged part of the porter domain, with PC1 clipped. Blue sticks, phenylalanine side chains of the distal binding pocket; gray sphere, position of G616 (no side chain); red sticks, isoleucine side chains constituting a narrowing in the lower porter domain; red and black dashed-line ovals, residues critical for the efflux of oxazolidinones and pleuromutilin derivatives, respectively.
A significant oxazolidinone-specific impact appeared from double mutation I38F I671T located in a putative entrance channel of the lower porter domain (Fig. 1). It decreased the resistance to all of the tested oxazolidinones up to 64-fold, whereas the effects with pleuromutilin derivatives remained negligible (Table 2). It was shown previously that substitution of I38 and/or I671 by bulkier residues predominantly impedes the efflux of smaller drugs, whereas larger compounds, such as macrolides and rifamycins, supposedly use another entrance pathway (11). Notably, in contrast to that for the pleuromutilin derivatives (MW, >490), resistance to the smaller pleuromutilin (MW, 378.5) was significantly affected by I38F I671T (Table 2). Among the distal binding pocket mutations, the most remarkable oxazolidinone-specific impact occurred with F178A (Table 2, Fig. 1), whereas the largest pleuromutilin specificity arose from mutation F615A removing a phenyl residue from the G-loop (Fig. 1). In this mutant, the susceptibility to all pleuromutilins was significantly increased but not that to the oxazolidinones (except cadazolid) (Table 2).
Our study reveals evidence of different pathways for oxazolidinones and pleuromutilin derivatives through RND-type transporters. The knowledge of class-specific efflux determinants could elucidate structural requirements of competitive inhibitors or efflux-incompatible drugs.

ACKNOWLEDGMENTS

This work was supported in part by the Innovative Medicines Initiative (IMI) Joint Undertaking project no. 115525 ND4BB Translocation (http://www.translocation.eu, contributions from the European Union Seventh Framework Program and EFPIA companies).
We have no conflicts of interest to declare.

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REFERENCES

1.
Li XZ, Plesiat P, Nikaido H. 2015. The challenge of efflux-mediated antibiotic resistance in Gram-negative bacteria. Clin Microbiol Rev 28:337–418.
2.
Pos KM. 2009. Drug transport mechanism of the AcrB efflux pump. Biochim Biophys Acta 1794:782–793.
3.
Kern WV, Oethinger M, Jellen-Ritter AS, Levy SB. 2000. Non-target gene mutations in the development of fluoroquinolone resistance in Escherichia coli. Antimicrob Agents Chemother 44:814–820.
4.
Jellen-Ritter AS, Kern WV. 2001. Enhanced expression of the multidrug efflux pumps AcrAB and AcrEF associated with insertion element transposition in Escherichia coli mutants selected with a fluoroquinolone. Antimicrob Agents Chemother 45:1467–1472.
5.
Bohnert JA, Schuster S, Fähnrich E, Trittler R, Kern WV. 2007. Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF). J Antimicrob Chemother 59:1216–1222.
6.
Schuster S, Vavra M, Schweigger TM, Rossen JWA, Matsumura Y, Kern WV. 2017. Contribution of AcrAB-TolC to multidrug resistance in an Escherichia coli sequence type 131 isolate. Int J Antimicrob Agents 50:477–481.
7.
Paukner S, Riedl R. 2017. Pleuromutilins: potent drugs for resistant bugs-mode of action and resistance. Cold Spring Harb Perspect Med 7:a027110.
8.
Novak R, Shlaes DM. 2010. The pleuromutilin antibiotics: a new class for human use. Curr Opin Investig Drugs 11:182–191.
9.
Sutcliffe JA. 2011. Antibiotics in development targeting protein synthesis. Ann N Y Acad Sci 1241:122–152.
10.
Yip PC, Kam KM, Lam ET, Chan RC, Yew WW. 2013. In vitro activities of PNU-100480 and linezolid against drug-susceptible and drug-resistant Mycobacterium tuberculosis isolates. Int J Antimicrob Agents 42:96–97.
11.
Schuster S, Vavra M, Kern WV. 2016. Evidence of a substrate-discriminating entrance channel in the lower porter domain of the multidrug resistance efflux pump AcrB. Antimicrob Agents Chemother 60:4315–4323.
12.
George AM, Levy SB. 1983. Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline. J Bacteriol 155:531–540.
13.
Bohnert JA, Schuster S, Seeger MA, Fähnrich E, Pos KM, Kern WV. 2008. Site-directed mutagenesis reveals putative substrate binding residues in the Escherichia coli RND efflux pump AcrB. J Bacteriol 190:8225–8229.
14.
Wehmeier C, Schuster S, Fähnrich E, Kern WV, Bohnert JA. 2009. Site-directed mutagenesis reveals amino acid residues in the Escherichia coli RND efflux pump AcrB that confer macrolide resistance. Antimicrob Agents Chemother 53:329–330.
15.
Locher HH, Seiler P, Chen X, Schroeder S, Pfaff P, Enderlin M, Klenk A, Fournier E, Hubschwerlen C, Ritz D, Kelly CP, Keck W. 2014. In vitro and in vivo antibacterial evaluation of cadazolid, a new antibiotic for treatment of Clostridium difficile infections. Antimicrob Agents Chemother 58:892–900.
16.
Takatsuka Y, Nikaido H. 2006. Threonine-978 in the transmembrane segment of the multidrug efflux pump AcrB of Escherichia coli is crucial for drug transport as a probable component of the proton relay network. J Bacteriol 188:7284–7289.
17.
Wang-Kan X, Blair JMA, Chirullo B, Betts J, La Ragione RM, Ivens A, Ricci V, Opperman TJ, Piddock L. 2017. Lack of AcrB efflux function confers loss of virulence on Salmonella enterica serovar Typhimurium. mBio 8:e00968-17.
18.
Bohnert JA, Kern WV. 2005. Selected arylpiperazines are capable of reversing multidrug resistance in Escherichia coli overexpressing RND efflux pumps. Antimicrob Agents Chemother 49:849–852.
19.
Schuster S, Bohnert JA, Vavra M, Rossen JW, Kern WV. 2019. Proof of an outer membrane target of the efflux inhibitor Phe-Arg-β-naphthylamide from random mutagenesis. Molecules 24:470.
20.
Eicher T, Cha HJ, Seeger MA, Brandstätter L, El-Delik J, Bohnert JA, Kern WV, Verrey F, Grütter MG, Diederichs K, Pos KM. 2012. Transport of drugs by the multidrug transporter AcrB involves an access and a deep binding pocket that are separated by a switch-loop. Proc Natl Acad Sci U S A 109:5687–5692.

Information & Contributors

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Published In

cover image Antimicrobial Agents and Chemotherapy
Antimicrobial Agents and Chemotherapy
Volume 63Number 9September 2019
eLocator: 10.1128/aac.01041-19

History

Received: 21 May 2019
Returned for modification: 7 June 2019
Accepted: 13 June 2019
Published online: 23 August 2019

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Keywords

  1. AcrB
  2. RND-type transporter
  3. YhiV (MdtF)
  4. efflux
  5. oxazolidinones
  6. pleuromutilins

Contributors

Authors

Sabine Schuster
Division of Infectious Diseases, Department of Medicine II, University Hospital and Medical Center, Freiburg, Germany
Martina Vavra
Division of Infectious Diseases, Department of Medicine II, University Hospital and Medical Center, Freiburg, Germany
Winfried V. Kern
Division of Infectious Diseases, Department of Medicine II, University Hospital and Medical Center, Freiburg, Germany
Faculty of Medicine, Albert-Ludwigs-University, Freiburg, Germany

Notes

Address correspondence to Sabine Schuster, [email protected].

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