Université catholique de Louvain (UCL-Bruxelles)
Louvain Drug Research Institute > Cellular and Molecular Pharmacology
Antibiotic efflux and permeability resistance mechanisms
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Mutidrug transporters, present in both bacteria and eucaryotic cells, modulate the accumulation and disposition of antibiotics, thereby affecting their activity.  

In bacteria, we study the prevalence and expression of efflux pumps in clinical isolates. We also develop diagnostic tools for the early detection of resistance mediated by active efflux and by porin alteration.

In eucaryotic cells, we characterize the efflux transporters of antibiotics in macrophages and epithelial cells, in relation to the modulation of their intracellular pharmacokinetics and activity.

These programs are closely linked to those exploring the chemotherapy of intracellular infection, drug-membrane interactions, and the clinical evaluation of new therapeutic approaches.

 

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Collaborations

Main current research programs

General overview

Amphiphilic molecules easily cross biomembranes.  This has created for cells, probably very early on in evolution, a need to protect them-selves from inorderly invasion by these diffusible, potentially harmful molecules, and has resulted in the emergence of of a large array of efflux pumps with broad substrate specificities. 

Because antibiotics are often amphiphilic, many of them are recognized by these efflux pumps. Figure 1 shows the topology, the mechanisms of action, and the main classes of efflux pumps acting on antibiotics which have been described so far. 

Figure 1: Main classes of efflux pumps acting on antibiotics.

A. within the class of secondary active transporters (symports, antiports, uniports):
4 superfamilies (comprising at least 10 families of gene products) including the SMR (Small Multidrug Resistance), the RND (Resistance Nodulation Division), and the MFS (Major Facilitator Superfamily). 

B. within the class of primary active transporters (energized by ATP): 1 superfamily (ABC) comrpising at last 6 families of gene products  including the PgP (in the MDR1 [Multiple Drug Resistance] group) and the MRP (Multiple Resistance Protein).

from: Van Bambeke et al., 2000

Efflux in bacteria

Antibiotic efflux pumps are now recognized as significantly contributing to both innate and acquired bacterial resistance to many antibiotic because of the very broad variety of substrates they recognize. Their expression and their cooperation with other mechanisms of resistance, accounts for so far ill-explained the apparent intrinsic resistance and "poor susceptibilities" of important pathogens (e.g. Pseudomonas aeruginosa). Their overexpression may also explain therapeutic failures, and stable mutations in regulatory genes could also produce phenotypes of irreversible multidrug resistance.

We evaluate the epidemiology of efflux-mediated resistance to current antibiotics in 3 main pathogens (S. aureus, S. pneumoniae, P. aeruginosa) starting from collections of isolates obtained from clinically-confirmed cases of respiratory tract and skin and skin structures infections in Belgium. Efflux is detected by a combination of genomic and phenotypic methods. Figure 2 shows a typical example of the detection of the Mex pumps in Pseudomonas aeruginosa, and the correlation between the levels of transcription of the genes and the increase in MIC as assessed by the use of reporter antibiotics.

We also compare the transport of different antibiotics within a given pharmacological class, with the aim to delineate to molecular determinants responsible for their recognition by efflux pumps.

Figure 2:
Genomic detection of Mex pumps in Pseudomonas aeruginosa and correlation of their level of trannscription and the corresponding increase in MIC.
(from Mesaros et al.,2007)
A: mexA quantification by QC-RT-PCR in the wild-type PAO1 and in the MexAB-OprM overproducer strain SLF30. Ethidium bromide stained gels; from left to right: amplification product of cDNA of the target gene (252 bp) and decreasing amounts (in ag) of the internal competitor (127 bp).
0 : negative control; L : DNA ladder.		 B: Detection of mexC (left) and mexE (right) expression by semi-quantitative RT-PCR. The figure shows the ethidium bromide stained gels of amplified RT-PCR for PAO1 and EryR (left) and for PAO1 and PAO7H (right). The inducible target genes (mexC, 374bp; mexE, 516bp) are amplified together with the constitutive mexA gene (252bp) used as positive control. C: correlation between the level of expression of constitutive MexA and MexB and the effect of a broad spectrum efflux inhbitor on the MIC of reporter antibiotics (carbenicillin for mexA and gentamicin for mexX).

Selected references on efflux in bacteria (by reverse chronological order; for a full reference list, see our publication list)

Efflux in eucaryotic cells

Efflux pumps can modulate the accumulation of antibiotics in phagocytic cells and also play significant roles in the transepithelial transport of these drugs. 

We study the efflux of antibiotics in phagocytic cells and  try to identify, at the phenotypic and genotypic levels, the transporter(s) involved (Figures 3 A and 3B). 

An original approach in these studies has been the obtention and characterization of macrophages overexpressing efflux transporters for fluoroquinolone antibiotics (by exposure to progressively increasing concentrations, so as to obtain 'resistant cells'). We currently analyze the protein expression of these cells by molecular biology approaches and proteomics (Figure 3C).

inhibition cipro by probenecid

Figure 3A: Demonstrating ciprofloxacin efflux in J774 macrophages

Kinetics of accumulation (left) and efflux (right) of ciprofloxacin (extracellular concentration, 17 mg/liter [50 µM]) in J774 macrophages incubated in the presence or absence of 10 mM probenecid (the drugs were added simultaneously for the accumulation experiment).

This figure illustrates that the quinolone antibiotic ciprofloxacin is substrate for a probenecid-inhibitable organic anion transporter, which we tentitatively identify as a member of the MRP family (see figure 1).  This is compatible with the physicochemical properties of the drug, which contains a ionizable carboxylic acid.

From Michot et al. 2004
azithro + verapamil

Figure 3B : Demonstrating the role of P-glycoprotein in reducing the accumulation of azithromycin in J774 macrophages

Kinetics of accumulation (left) of azithromycin (extracellular concentration, 5 mg/liter) in J774 murine macrophages incubated for up to 24 h in the absence (open squares) or in the presence (closed squares) of 20 µM verapamil; kinetics of efflux of azithromycin (extracellular concentration, 20 mg/liter) from J774 murine macrophages incubated for 3 h with azithromycin (right).

In this case, the figure illustrates that azithromycin, an amphiphilic dicationic macrolide antibiotic, is transported by P-glycoprotein, since its accumulation and efflux can be modulated by the addition of an inhibitor of this pump.

from Seral et al, 2003

Figure 3C: Study of the expression of Mrp transporters in macrophages made resistant to ciprofloxacin by prolonged exposure to high concentrations of this drug (RS), as compared to wild type cells (WT) or revertant cells (obtained by cultivation of resistance cells in the absence of ciprofloxacin)

Left: Quantification of mRNA transcripts of Mrps 1 to 7 in ciprofloxacin-resistant (RS) and revertant (Rev) J774 macrophages in comparison with wild-type cells (WT). Results are expressed as the increase in expression over that in WT cells (set arbitrarily at 1 [dotted line]).

Right: Western Blot revealing Mrp2 or Mrp4 in membrane-ennriched preparation and colocalization of these transporers at the membrane of WT or RS cells.

from Marquez et al, 2009


In a broader pharmacological context, we also try

efflux from eucaryotic cells
 Figure 4:  Active transport of antibiotics in eucaryotes

Schematic representation of the main transporters potentially involved in antibiotic movement at the level of epithelial cells in the main organs (liver, bronchial tree, intestine, kidney), the blood-brain barriers, and in leucocytes (PMN are not considered here since the role of drug transporters in these cells is unclear).  
Black arrows denote transport towards extracorporeal compartments such as urine, bile, intestine and airways (i.e. transporters involved in drug elimination from the body). Grey arrows describe uptake processes from extracorporeal fluids into cells (i.e. allowing drugs to accumulate in tissues), or from cells to body fluids (i.e. causing the drug to be transported from one body fluid to another [from blood to cerebrospinal fluid, e.g.]).  
The level of expression of each transporter may differ between species (arrows with a checkerboard background indicate transporters that have been, so far, evidenced  in animals only).  The direction of transport of bidirectional transporters may differ according to the cell type.  

from: Van Bambeke et al., 2003.

Selected references on efflux in eucaryotic cells (by reverse chronological order; for a full reference list, see our publication list)

Expertise and original material

Additional information:  <tulkens@facm.ucl.ac.be>
Last significant update: December 2010