Properties of AdeABC and AdeIJK Efflux Systems of Acinetobacter baumannii Compared with Those of the AcrAB-TolC System of Escherichia coli
01 Dec 2014-Antimicrobial Agents and Chemotherapy (American Society for Microbiology)-Vol. 58, Iss: 12, pp 7250-7257
TL;DR: Both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux.
Abstract: Acinetobacter baumannii contains RND-family efflux systems AdeABC and AdeIJK, which pump out a wide range of antimicrobial compounds, as judged from the MIC changes occurring upon deletion of the responsible genes. However, these studies may miss changes because of the high backgrounds generated by the remaining pumps and by β-lactamases, and it is unclear how the activities of these pumps compare quantitatively with those of the well-studied AcrAB-TolC system of Escherichia coli. We expressed adeABC and adeIJK of A. baumannii, as well as E. coli acrAB, in an E. coli host from which acrAB was deleted. The A. baumannii pumps were functional in E. coli, and the MIC changes that were observed largely confirmed the substrate range already reported, with important differences. Thus, the AdeABC system pumped out all β-lactams, an activity that was often missed in deletion studies. When the expression level of the pump genes was adjusted to a similar level for a comparison with AcrAB-TolC, we found that both A. baumannii efflux systems pumped out a wide range of compounds, but AdeABC was less effective than AcrAB-TolC in the extrusion of lipophilic β-lactams, novobiocin, and ethidium bromide, although it was more effective at tetracycline efflux. AdeIJK was remarkably more effective than a similar level of AcrAB-TolC in the efflux of β-lactams, novobiocin, and ethidium bromide, although it was less so in the efflux of erythromycin. These results thus allow us to compare these efflux systems on a quantitative basis, if we can assume that the heterologous systems are fully functional in the E. coli host.
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TL;DR: This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps, with particular focus on AcrAB-TolC and Mex pumps.
Abstract: The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.
1,016 citations
Washington University in St. Louis1, Dalhousie University2, University of California, Berkeley3, Universities Space Research Association4, Goddard Space Flight Center5, California Institute of Technology6, Institute for Health Metrics and Evaluation7, University of British Columbia8, University of Texas at Austin9, University of Colorado Boulder10, Earth System Research Laboratory11, Tsinghua University12, Colorado State University13, Harvard University14
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TL;DR: It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance.
Abstract: Acinetobacter baumannii is a nosocomial pathogen of increasing importance due to its multiple resistance to antibiotics and ability to survive in the hospital environment linked to its capacity to form biofilms. To fully characterize the contribution of AdeABC, AdeFGH, and AdeIJK resistance-nodulation-cell division (RND)-type efflux systems to acquired and intrinsic resistance, we constructed, from an entirely sequenced susceptible A. baumannii strain, a set of isogenic mutants overexpressing each system following introduction of a point mutation in their cognate regulator or a deletion for the pump by allelic replacement. Pairwise comparison of every derivative with the parental strain indicated that AdeABC and AdeFGH are tightly regulated and contribute to acquisition of antibiotic resistance when overproduced. AdeABC had a broad substrate range, including β-lactams, fluoroquinolones, tetracyclines-tigecycline, macrolides-lincosamides, and chloramphenicol, and conferred clinical resistance to aminoglycosides. Importantly, when combined with enzymatic resistance to carbapenems and aminoglycosides, this pump contributed in a synergistic fashion to the level of resistance of the host. In contrast, AdeIJK was expressed constitutively and was responsible for intrinsic resistance to the same major drug classes as AdeABC as well as antifolates and fusidic acid. Surprisingly, overproduction of AdeABC and AdeIJK altered bacterial membrane composition, resulting in decreased biofilm formation but not motility. Natural transformation and plasmid transfer were diminished in recipients overproducing AdeABC. It thus appears that alteration in the expression of efflux systems leads to multiple changes in the relationship between the host and its environment, in addition to antibiotic resistance. IMPORTANCE Increased expression of chromosomal genes for RND-type efflux systems plays a major role in bacterial multidrug resistance. Acinetobacter baumannii has recently emerged as an important human pathogen responsible for epidemics of hospital-acquired infections. Besides its remarkable ability to horizontally acquire resistance determinants, it has a broad intrinsic resistance due to low membrane permeability, endogenous resistance genes, and antibiotic efflux. The study of isogenic mutants from a susceptible A. baumannii clinical isolate overproducing or deleted for each of the three major RND-type pumps demonstrated their major contribution to intrinsic resistance and to the synergism between overproduction of an efflux system and acquisition of a resistance gene. We have also shown that modulation of expression of the structural genes for the efflux systems results in numerous alterations in membrane-associated cellular functions, in particular, in a decrease in biofilm formation and resistance gene acquisition.
159 citations
Cites result from "Properties of AdeABC and AdeIJK Eff..."
...This was not found in another study for minocycline-AdeABC and tetracycline-AdeIJK (22) and could have been due to the fact that the experiments were carried out in different bacterial hosts, A....
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References
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TL;DR: This review details the significant advances that have been made in understanding of this remarkable organism over the last 10 years, including current taxonomy and species identification, issues with susceptibility testing, mechanisms of antibiotic resistance, global epidemiology, clinical impact of infection, host-pathogen interactions, and infection control and therapeutic considerations.
Abstract: Acinetobacter baumannii has emerged as a highly troublesome pathogen for many institutions globally. As a consequence of its immense ability to acquire or upregulate antibiotic drug resistance determinants, it has justifiably been propelled to the forefront of scientific attention. Apart from its predilection for the seriously ill within intensive care units, A. baumannii has more recently caused a range of infectious syndromes in military personnel injured in the Iraq and Afghanistan conflicts. This review details the significant advances that have been made in our understanding of this remarkable organism over the last 10 years, including current taxonomy and species identification, issues with susceptibility testing, mechanisms of antibiotic resistance, global epidemiology, clinical impact of infection, host-pathogen interactions, and infection control and therapeutic considerations.
2,915 citations
"Properties of AdeABC and AdeIJK Eff..." refers background in this paper
...Acinetobacter species, similar to Pseudomonas aeruginosa, display high levels of multidrug resistance to a broad range of antimicrobial agents (1, 2)....
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TL;DR: Recent studies showed that multipledrug efflux pumps, many with unusually broad specificities, play a major role in the intrinsic resistance of gram-negative bacteria.
Abstract: Gram-negative bacteria tend to be more resistant to lipophilic and amphiphilic inhibitors than gram-positive bacteria. Such inhibitors include dyes, detergents, free fatty acids, antibiotics, and other chemotherapeutic agents. This property is used in the selective enrichment of gram-negative bacteria, especially of members of the family Enterobacteriaceae, for example with MacConkey agar (containing crystal violet and bile salts), EMB agar (containing dyes), and deoxycholate agar (containing sodium deoxycholate). The fact that Escherichia coli K-12 can grow in the presence of 1% sodium dodecyl sulfate has been rediscovered many times. Many lipophilic antibiotics, such as penicillin G, erythromycin, fusidic acid, and rifamycin SV, are much less active against most gram-negative bacteria. In fact, a survey of recently reported antibiotics of natural origin showed that, among those compounds that showed activity against gram-positive bacteria, more than 90% lacked activity at a useful level against E. coli (51). This intrinsic resistance of gram-negative bacteria has often been attributed entirely to the presence of the outer membrane barrier. This barrier does contribute to the resistance, as the narrow porin channels slow down the penetration of even small hydrophilic solutes, and the low fluidity of the lipopolysaccharide leaflet decreases the rate of transmembrane diffusion of lipophilic solutes (38, 40). However, the outer membrane barrier cannot be the whole explanation, even with species such as Pseudomonas aeruginosa which produces an outer membrane of exceptionally low permeability (1, 57). This is seen from the fact that equilibration across the outer membrane is achieved very rapidly, in part because the surface-to-volume ratio is very large in a small bacterial cell. Thus, the periplasmic concentrations of many antibiotics are expected to reach 50% of their external concentrations in 10 to 30 s in P. aeruginosa and in a much shorter time period in E. coli (34). Additional mechanisms are therefore needed to explain the level of intrinsic resistance. With the earlier b-lactam compounds, this second contributing factor is the hydrolysis by the periplasmic b-lactamases that are encoded by chromosomal genes in many gram-negative bacteria, and the levels of resistance can be explained quantitatively, in many cases, by the synergy between the outer membrane barrier and b-lactamase (37). However, with dyes, detergents, other classes of antibiotics, and even the b-lactams developed more recently that are not hydrolyzed easily by common b-lactamases, the second factor was completely unknown (34). Recent studies showed that multipledrug efflux pumps, many with unusually broad specificities, play a major role in the intrinsic resistance of gram-negative bacteria. Active efflux processes have been known to cause drug resistance in gram-negative bacteria, through the pioneering studies of S. Levy (19) on Tet-mediated tetracycline resistance. Each of these traditional efflux pumps excretes only one drug or one class of drugs (35). In contrast, a multidrug efflux pump can pump out a wide range of compounds, and it is often difficult to discern any common structural features among the substrates (20, 35).
1,042 citations
"Properties of AdeABC and AdeIJK Eff..." refers background in this paper
...Because increases in MICs require the synergistic interaction between the outer membrane permeability barrier and RND-type pumps (19), aminoglycoside efflux is nearly impossible to detect in E....
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TL;DR: The AcrAB system is identified as the major pump responsible for making the Mar mutants resistant to many agents, including tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin.
Abstract: Multiple-antibiotic-resistance (Mar) mutants of Escherichia coli are resistant to a wide variety of antibiotics, and increased active efflux is known to be responsible for the resistance to some drugs. The identity of the efflux system, however, has remained unknown. By constructing an isogenic set of E. coli K-12 strains, we showed that the marR1 mutation was incapable of increasing the resistance level in the absence of the AcrAB efflux system. This experiment identified the AcrAB system as the major pump responsible for making the Mar mutants resistant to many agents, including tetracycline, chloramphenicol, ampicillin, nalidixic acid, and rifampin.
782 citations
"Properties of AdeABC and AdeIJK Eff..." refers background in this paper
...AG100A ( acrAB::spc) is a acrAB derivative of AG100 [K-12 argE3 thi-1 rpsL xyl mtl (gal-uvrB) supE44] (13)....
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TL;DR: In addition, diminished expression of outer membrane proteins, mutations in topoisomerases, and up-regulation of efflux pumps play an important part in antibiotic resistance as discussed by the authors. Unfortunately, the accumulation of multiple mechanisms of resistance leads to the development of multiply resistant or even "panresistant" strains.
Abstract: Acinetobacter species and Pseudomonas aeruginosa are noted for their intrinsic resistance to antibiotics and for their ability to acquire genes encoding resistance determinants. Foremost among the mechanisms of resistance in both of these pathogens is the production of beta -lactamases and aminoglycoside-modifying enzymes. Additionally, diminished expression of outer membrane proteins, mutations in topoisomerases, and up-regulation of efflux pumps play an important part in antibiotic resistance. Unfortunately, the accumulation of multiple mechanisms of resistance leads to the development of multiply resistant or even "panresistant" strains.
681 citations
"Properties of AdeABC and AdeIJK Eff..." refers background in this paper
...Acinetobacter species, similar to Pseudomonas aeruginosa, display high levels of multidrug resistance to a broad range of antimicrobial agents (1, 2)....
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TL;DR: Three types of alpha-complementation plasmid vectors were constructed which contain a chloramphenicol- or kanamycin-resistance (CmR or KmR) gene and polylinker cloning sites within the coding region of lacZ'.
675 citations