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Journal of Antimicrobial Chemotherapy (2006) 57, 252–259doi:10.1093/jac/dki436Advance Access publication 22 December 2005 Characterization of tetracycline resistance mediated by the efflux Santiago Ramo´n-Garcı´a1, Carlos Martı´n1, Jose´ A. Aı´nsa1* and Edda De Rossi2 1Departamento de Microbiologı´a, Medicina Preventiva y Salud Pu´blica, Universidad de Zaragoza, 50009 Zaragoza, Spain; 2Dipartimento di Genetica e Microbiologia, Universita` degli Studi di Pavia, 27100 Pavia, Italy Received 2 August 2005; returned 8 September 2005; revised 27 October 2005; accepted 6 November 2005 Objectives: The aim of this study was to characterize the efflux pump Tap from Mycobacterium fortuitum, totest its sensitivity to well known efflux inhibitors, to study the interaction between tetracycline and thesecompounds and to test the ability of these compounds to overcome efflux pump-mediated tetracyclineresistance. For all these studies, we produced Tap protein in Mycobacterium smegmatis.
Methods: Antibiotic susceptibility tests, tetracycline uptake/efflux experiments and chequerboard synergytests.
Results: Tetracycline uptake/efflux experiments showed that Tap protein from M. fortuitum uses theelectrochemical gradient across the cytoplasmic membrane to extrude tetracycline from the cell. Thisefflux activity is inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and reserpine, consistentwith the decrease in MIC observed in antibiotic susceptibility testing in the presence of these inhibitors.
Accumulation was not inhibited in experiments in which o-vanadate and chlorpromazine (CPZ) were tested.
Inhibitor-treated cells used glycerol as a carbon source to re-establish the electrochemical gradient acrossthe membrane and to restore efflux activity. CCCP, reserpine and CPZ reduced the MIC of tetracycline in theM. smegmatis strain expressing the Tap protein, whereas o-vanadate increased the MIC. We also observedsynergy between tetracycline and CPZ or reserpine, and antagonism with o-vanadate.
Conclusions: The Tapfor efflux pump uses the electrochemical gradient to extrude tetracycline from the cell.
This efflux activity can be inhibited by several compounds. This suggests that similar compounds could beused to overcome antibiotic resistance mediated by efflux pumps.
Keywords: tetracycline efflux, antibiotic resistance, mycobacteria, efflux pump inhibitors, synergy has become an additional handicap, especially in the case ofmultidrug-resistant Mycobacterium tuberculosis strains. Mutations Non-tuberculous mycobacteria (NTMB) are environmental species in genes encoding drug targets or the enzymes required to activate found in water, soil, milk and in fish, birds and other animals.
the drug are frequently found in resistant strains. However, no NTMB include rapidly growing mycobacteria, such as Mycobac- mutation has been found in a number of drug-resistant strains.
terium fortuitum, Mycobacterium peregrinum, Mycobacterium The resistance of these strains may be accounted for by mutations abcessus and Mycobacterium chelonae. M. fortuitum is an oppor- in new, as yet unidentified loci, changes in drug permeability, or the tunistic pathogen of humans, causing abscesses and soft-tissue involvement of efflux pumps extruding the antibiotic. Efflux infections, especially at injection sites or surgical wounds, and pump-mediated antibiotic resistance has been widely reported is often associated with indwelling catheters, breast implants Bacterial membrane efflux pumps form a large, heterogeneous Prolonged antibiotic therapy is generally required for the treat- family of energy-dependent membrane proteins capable of extrud- ment of M. fortuitum infections.1 Mycobacterial infections are ing either a single antibiotic, such as tetracycline, or a wide variety generally difficult to treat because these bacteria are intrinsically of chemically and structurally unrelated substances, allowing bac- resistant to most common antibiotics and chemotherapeutic teria to adapt themselves to a hostile environment. Furthermore, as agents.2 Furthermore, the emergence of drug-resistant strains efflux pumps have been shown to be of clinical relevance in *Corresponding author. Tel: +34-976-762420; Fax: +34-976-762604; E-mail: [email protected] Ó The Author 2005. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved.
For Permissions, please e-mail: [email protected] bacterial infections,5 the characterization of such pumps (mode of action, source of energy used and substrate profile) is important for [3H]Tetracycline (0.91 Ci/mmol) was obtained from Perkin Elmer.
the design of new therapeutic strategies.
CCCP, kanamycin, reserpine, tetracycline and o-vanadate were pro- Many compounds capable of inhibiting efflux pumps have been vided by Sigma. CPZ was obtained from Fluka. CCCP and CPZ were described. Some affect the electrochemical gradient across the dissolved in DMSO; reserpine was dissolved in glacial acetic acid; and membrane used by some efflux pumps as a source of energy.
kanamycin, tetracycline and o-vanadate were dissolved in water.
These compounds include the proton uncoupler carbonyl cyanidem-chlorophenylhydrazone (CCCP), nigericin (which dissipates thetransmembrane ion gradient), the calcium channel blocker verapa- mil, and valinomycin (which dissipates the electrical potential).
The MICs of the drugs listed above for the M. smegmatis SUM36 and Other pumps using ATP as an energy source may be inhibited by AC48 strains were determined on microplates by the Resazurin the P-type ATPase inhibitor o-vanadate. Other inhibitors interact assay.17 Briefly, we inoculated 100 mL of 2-fold serial dilutions of more specifically with the efflux pump. An example of one such antibiotic with 100 mL of a 105 cells/mL suspension of mycobacteria in inhibitor is the mammalian P-glycoprotein inhibitor reserpine,6 microtitre plates. The plates were incubated for 4 days at 37C, 30 mL which also inhibits bacterial efflux pumps, such as NorA.7 of the redox indicator Resazurin (1 g/L) was added to each well and the Thus, efflux pump inhibitors can be used to make bacteria more plates were incubated for an additional day. A change in colour from susceptible to antimicrobial agents.
blue to pink indicated the growth of bacteria, and the MIC was defined The first efflux pump described for the genus Mycobacterium, as the lowest concentration of drug preventing this colour change. We the LfrA protein of Mycobacterium smegmatis, confers low-level prevented the formation of biofilms, which might affect drug resist- resistance to fluoroquinolones and other compounds.8,9 Many other ance, by using polystyrene microtitre plates from TPPÒ for antibiotic efflux pumps have since been described.10,11 Efflux pump com- ponents also seem to play a role in mycobacterial drug tolerance12 We also determined the MIC of tetracycline in the presence of CCCP and CPZ (both at a concentration of 20 mg/L), reserpine Tapfor and Taptub proteins are membrane efflux pumps of the All antibiotic susceptibility tests were carried out at least three major facilitator superfamily (MFS) isolated from M. fortuitum and M. tuberculosis, respectively.14 Note that in this study the tap geneand Tap protein from M. fortuitum will be designated tapfor andTap for, respectively. The homologous gene and protein from M. tuberculosis will be referred to as taptub and Taptub. MFS pro- For a given strain, we determined interactions between tetracycline and teins extrude substrates, using the proton gradient through the CPZ, CCCP, reserpine or o-vanadate. Using a 96-well plate, we membrane, in an antiporter mechanism.
assayed 2-fold dilutions of tetracycline against a range of 2-fold These membrane proteins confer low-level resistance and dilutions of CCCP, CPZ or reserpine by the Resazurin assay.17 have broad substrate specificity. When cloned on a plasmid, Tapfor The fractional inhibitory concentration of tetracycline (FICTET) in protein increases the resistance of M. smegmatis mc2155 to genta- the presence of CCCP, CPZ or reserpine was calculated as follows: micin, 2-N0-ethylnetilmicin, 6-N0-ethylnetilmicin, streptomycin FICTET = (MIC of tetracycline in the presence of CCCP, CPZ or and tetracycline.15 Rifampicin and ofloxacin increase expression reserpine)/(MIC of tetracycline alone). Similar FICs were calculated of the Rv1258c gene, encoding Taptub, in a clinical strain of for CCCP, CPZ and reserpine in the presence of tetracycline. The FICTET values were plotted graphically against the FIC values for In this work, we describe the characterization of tetracycline CCCP, CPZ and reserpine. A concave curve indicates synergy, whereas a convex curve indicates antagonism.19 The point closest protein uses the electrochemical gradient as a source of energy to the intersection of the axes (point zero) corresponds to the most to extrude tetracycline from the cell and is inhibited by CCCP effective combination for inhibiting bacterial growth.
and reserpine, but not by o-vanadate or chlorpromazine (CPZ).
Given the high MIC of o-vanadate (>640 mg/L), we tested a series Cells use glycerol as a carbon source to establish the electrochem- of 2-fold dilutions of tetracycline against a reference concentration of ical gradient through the membrane and to restore the activity of the o-vanadate (9 mg/L), as previously described.20 In parallel, reference pump. We also describe interactions between tetracycline and the concentrations (0.5· MIC values) were fixed for the other drugs: tet- inhibitory compounds, and the ability of these compounds to over- racycline, 1 mg/L; CCCP, 20 mg/L; reserpine, 48 mg/L; and CPZ,20 mg/L. We used these reference concentrations to calculate the FIC come efflux pump-mediated tetracycline resistance.
index (FICI) as follows: FICI = [(MIC of CCCP, CPZ or reserpine inthe presence of reference tetracycline concentration)/(MIC of CCCP,CPZ or reserpine alone)] + [(MIC of tetracycline in the presence of reference CCCP, CPZ or reserpine concentration)/(MIC of tetracyclinealone)]. Synergy was defined by FICI values £0.5, antagonism by FICI Bacterial strains and general growth conditions values >4.0, and no interaction by FICI values between >0.5 and 4.0.21 M. smegmatis mc2155 cells harbouring the cloning vector pSUM36and its derivative pAC48 containing the tapfor gene—strains SUM36 and AC48, respectively15—were grown in Middlebrook 7H9 broth(Difco) supplemented with 10% Middlebrook ADC (Difco) and Tetracycline accumulation experiments. Uptake experiments were 0.2% glycerol. Kanamycin was added to the cultures at a final con- performed as previously described.22 We inoculated 20 mL of Mid- centration of 20 mg/L to maintain the plasmids. We used Luria-Bertani dlebrook 7H9 broth with M. smegmatis SUM36 and AC48 strains, (LB) agar for colony counts. All the cultures were incubated at 37C, which were cultured to the exponential growth phase, harvested by centrifugation, washed twice with 0.1 M potassium phosphate (pH 7.0) and resuspended in assay buffer [0.1 M potassium phosphate (pH 7.0); Table 1. Antimicrobial activity of tetracycline and efflux pump inhibitors against M. smegmatis mc2155, SUM36 and AC48 We used several strategies to prevent aggregate formation and cell clumping. First, bacterial cultures were filtered through a syringe filter with 5 mm pores (Sartorius).23 Filtrates were cultured overnight at37C, then centrifuged, with the pellet washed and resuspended, as described above. Second, cells resuspended in assay buffer were allowed to stand for 15 min to allow clumps to sediment out. Aliquots(1 mL) were collected from the upper part of the cultures and used for Cells were incubated at 37C (the optimal growth temperature for M. smegmatis) for 15 min, and the uptake assay was initiated by adding [3H]tetracycline to a final concentration of 5 mM. At various time intervals, we removed 50 mL of the suspension in triplicate, dilutedit in 1 mL of ice-cold 0.1 M potassium phosphate (pH 7.0) containing TET, tetracycline; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CPZ, 0.1 M LiCl, and immediately filtered it through a filter with 0.45 mm pores (Millipore). The filter was rapidly washed twice with 5 mL of thesame buffer and dried. Radioactivity was then determined in a Beck-man LS 7000 liquid scintillation counter, using Ecolume scintillation Table 2. Antimicrobial activity of tetracycline in the presence of cocktail (ICN Biomedicals). Experiments were carried out at least three times, to ensure that the results were reproducible. Finally,after uptake experiments, serial dilutions of the remaining cells were plated on LB agar. Colonies were counted after 4 days of incuba-tion at 37C.
Inhibitor experiments. We used several compounds to assess theenergy dependence of the accumulation process (40 mg/L of CCCP, 12 mg/L of reserpine, 20 mg/L of CPZ and 9 mg/L of o-vanadate). The inhibitors were added to the cells 2.5 min after the addition of [3H]tet- racycline and the suspension was treated as described above. For o- vanadate experiments, HEPES buffer (pH 7.0) was used as the assay In another set of experiments, cell suspensions were incubated with 40 mg/L of CCCP in phosphate buffer, with gentle shaking at 37C for 60 min, to exhaust the endogenous energy reserve of the cells. Thestarved cells were then washed three times with phosphate buffer at TET, tetracycline; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CPZ, room temperature to remove CCCP and resuspended in assay buffer.
The assay was started by adding [3H]tetracycline and the cells were aThe MICs were assayed in a range of 2-fold dilutions of tetracycline in the incubated for 5 min to allow antibiotic uptake. The cells were then re- presence of non-lethal concentrations (below the MIC for M. smegmatis mc2155) energized, by adding 200 mM glycerol as an energy source, and treated of the following compounds: CCCP, 20 mg/L; reserpine, 12 mg/L; CPZ, 20 mg/L; and o-vanadate, 9 mg/L. Values in brackets correspond to the increaseor decrease in MIC with respect to the MIC of tetracycline alone.
baseline tetracycline resistance in M. smegmatis depends on inhib- Susceptibility profiles of M. smegmatis SUM36 and itor-sensitive efflux systems, such as the tet(V) determinant,22 butnot excluding other putative intrinsic non-efflux systems. Tetra- cycline MICs in the presence of these inhibitors were always higher The MICs of CCCP, reserpine, CPZ, o-vanadate and tetracycline for AC48 than for SUM36, indicating that these two efflux pump for the M. smegmatis mc2155, SUM36 and AC48 strains were blockers do not completely block the Tapfor efflux pump.
determined by the Resazurin assay.17 The MIC values (mg/L) We also determined the MIC of tetracycline for SUM36 and AC48 in the presence of CPZ. The same MIC was obtained for both The MICs of the solvents used (DMSO and glacial acetic acid) strains (0.12 mg/L; Table 2), indicating that, in the presence of this were also determined for M. smegmatis mc2155. The MICs of compound, the Tapfor pump confers no advantage. Moreover, CPZ DMSO and glacial acetic acid were 12.5–25% (v/v). In a standard decreased the MIC for SUM36 and AC48, but it is noteworthy that MIC determination, solvent concentration does not exceed 1% of this decrease was larger for AC48 (factor of 128) than for CCCP the final volume. We, therefore, conclude that the solvents had no (factor of only 16). There are two possible explanations for these appreciable effect on the growth of M. smegmatis mc2155.
results. First, it has been suggested that phenothiazines inhibit The MIC of tetracycline was determined for strains SUM36 and efflux pumps,25,26 which suggests that CPZ is a more powerful, AC48, in the presence of subinhibitory concentrations of the known or more selective efflux pump inhibitor than CCCP or reserpine.
efflux pump inhibitors CCCP and reserpine (Table 2). Generally, Second, CPZ has also been reported to bind to membranes, increas- susceptibility to tetracycline increased. CCCP and reserpine ing their permeability.27,28 CPZ may, therefore, affect the integrity decreased the MIC of tetracycline by a factor of 2–4 for the of the cell wall of M. smegmatis, allowing tetracycline to enter M. smegmatis strain SUM36 carrying the cloning vector. Thus, cells and to accumulate in large amounts, rendering Tapfor protein activity inefficient. We carried out uptake experiments to deter- For SUM36, the MIC of tetracycline increased from 2 to 32 mg/L mine whether CPZ inhibited Tapfor protein function or interacted (factor of 16), whereas for AC48, the MIC increased from 16 to with this protein in an unknown manner, by reducing tetracycline 64 mg/L (factor of 4) (Table 2). It has been suggested that the influx of tetracycline into Escherichia coli involves both phosphate bond A different effect was noted when o-vanadate was tested. Unlike hydrolysis and proton motive force.29,30 The higher MIC of tetra- the other inhibitors, o-vanadate increased tetracycline resistance.
cycline in the presence of o-vanadate may therefore be due to Figure 1. Tetracycline uptake/efflux experiments with SUM36 and AC48. (a) Tetracycline uptake of SUM36 cells (open circles) and AC48 cells (closed circles).
[3H]Tetracycline was added to the cells at time zero. (b) Tetracycline accumulation of AC48 in the absence (closed circles) and presence (open triangles) of CCCP.
CCCP was added after 2.5 min. (c) Tetracycline accumulation in de-energized AC48 cells (closed squares) and upon re-energization (open squares) following theaddition of glycerol at 5 min. (d) Tetracycline accumulation in AC48 cells (closed circles) and upon addition of reserpine after 2.5 min and glycerol after 10 min(inverted filled triangles). (e) Tetracycline accumulation in AC48 cells (closed circles) and upon addition of CPZ (inverted open triangles) after 2.5 min.
inhibition of the ATP-dependent uptake system. It has been sug- is attracted by the Donnan potential across the outer membrane, gested that tetracycline and vanadium compounds may form leading to accumulation in the periplasm, in which the metal ion– complexes, affecting the action of tetracycline.31 This provides tetracycline complex probably dissociates to release uncharged an alternative explanation for the increase in the MIC of tetracyc- tetracycline, a weakly lipophilic molecule that diffuses through the lipid bilayer regions of the cytoplasmic membrane. The elec-troneutral, lipophilic form is presumed to be the species transferred across the cytoplasmic membrane of Gram-positive bacteria. Tet-racycline uptake also depends on phosphate bond hydrolysis and We investigated the mechanism of tetracycline resistance con- proton motive force.29,30,35,36 As tetracycline is extruded primarily ferred by the Tapfor protein, by measuring tetracycline accumula- by proton motive force-driven efflux pumps, tetracycline efflux is tion in M. smegmatis SUM36 and AC48 cells. As shown in abolished in the presence of a proton motive force uncoupler, such Figure 1(a), SUM36 cells accumulated [3H]tetracycline rapidly, as CCCP, whereas tetracycline influx is affected only mildly, res- reaching steady-state levels within 2.5 min of incubation. This ulting in an increase in tetracycline accumulation.
level of accumulation is about five times higher than that in AC48cells harbouring the tapfor-containing plasmid. These results sug- o-Vanadate. Tetracycline accumulation was not increased by the gest that the Tapfor protein has tetracycline efflux activity.
ATPase inhibitor o-vanadate in SUM36 or AC48 (data not shown).
As antibiotic efflux pump systems are energy-dependent, bac- In another report, o-vanadate also failed to increase the accumu- terial cells accumulate larger amounts of antibiotic in the presence lation of substrates transported by the LmrP protein of Lactococcus of uncouplers. We investigated whether this was the case in the lactis when cloned in a plasmid.37 These results rule out an ATP- presence of the tapfor gene, by determining the effects of various dependent efflux mechanism for the Tapfor protein.
compounds. Uptake experiments were carried out in the presenceof CCCP, o-vanadate, reserpine or CPZ.
Reserpine. We further investigated the efflux pump Tapfor by car-rying out additional uptake experiments in the presence of reser- CCCP. Efflux pumps belonging to the MFS family extrude com- pine. Reserpine was added to cells 2.5 min after the addition of pounds, using proton motive force as a source of energy, in an [3H]tetracycline. Tetracycline accumulation increased by a factor antiporter mechanism.32 We investigated whether Tapfor function of 4 in AC48, suggesting that reserpine inhibits the efflux pump was an energy-dependent process, using the protonophore CCCP at (Figure 1d). Similar results were obtained in antibiotic susceptib- a concentration of 40 mg/L in uptake experiments. This concen- ility tests, in which reserpine reduced the MIC of tetracycline tration is lethal for M. smegmatis cells in antibiotic susceptibility (Table 2). The inhibitory effect of reserpine may be owing to an test conditions, but had no effect on cell viability in experiments interaction with the reserpine-binding site in the Tapfor protein, as measuring accumulation over a period of 30 min.22,33 described for the multidrug transporter Bmr from Bacillus sub- Upon CCCP addition, [3H]tetracycline accumulation increased tilis.38 We investigated whether the pump could be reactivated by a factor of 4 in AC48 (Figure 1b), consistent with antibiotic by adding glycerol 7.5 min after reserpine. Efflux activity was susceptibility test results (Table 2). In contrast, under our condi- rapidly restored in AC48 cells, which extruded the drug. The mech- tions, CCCP had no detectable effect on the level of tetracycline anism by which glycerol reactivates the pump remains unknown.
accumulation in SUM36 (data not shown). One of the possiblelimitations of our experiments is that small changes in accumula- CPZ. We also carried out accumulation experiments in the pres- tion in SUM36 (carrying single-copy efflux pump genes on the ence of CPZ. We added CPZ to cells 2.5 min after the addition of chromosome) may not have been detectable following the addition [3H]tetracycline. No difference in tetracycline accumulation of inhibitor. When efflux pump genes are present on multicopyplasmids (tapfor in the AC48 strain and the tet(V) determinantdescribed elsewhere22), the changes in accumulation are greater Table 3. FICI of efflux pump inhibitors and CPZ when combined and are therefore more readily detected.
A second experiment was carried out to determine whether energizing de-energized cells could restore tetracycline efflux in AC48 cells. De-energized SUM36 and AC48 cells accumulatedsimilar amounts of [3H]tetracycline (data not shown). However, following the addition of glycerol as an energy source, AC48 cellsrapidly extruded tetracycline (Figure 1c), whereas no effect was observed in SUM36 cells (data not shown). Thus, glycerol is used by cells to produce energy, which is used to re-establish the proton gradient across the membrane used by the Tapfor pump for tetra- cycline extrusion, probably via an antiporter mechanism.
These results are consistent with the proposed mechanism of TET, tetracycline; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CPZ, tetracycline uptake. Tetracyclines must cross one or more mem- brane systems, depending on whether the susceptible microorgan- aFICI was calculated as follows: FICI = [(MIC of compound in the presence of ism is Gram-positive or Gram-negative, to reach their targets.
TET at the reference concentration)/(MIC of compound alone)] + [(MIC of TET Tetracyclines cross the outer membrane of Gram-negative enteric in the presence of any compound at the reference concentration)/(MIC ofTet alone)]. Reference concentrations (0.5· MIC values) were fixed as follows: bacteria via the OmpF and OmpC porin channels, as positively tetracycline, 1 mg/L; CCCP, 20 mg/L; reserpine, 48 mg/L; CPZ, 20 mg/L; and o- charged cation (probably magnesium)–tetracycline coordina- vanadate, 9 mg/L. Synergy was defined as FICI values of £0.5, antagonism as tion complexes.34,35 The cationic metal ion-antibiotic complex FICI values >4.0 and no interaction as FICI values ranging between >0.5 and 4.0.
Figure 2. FIC values of reserpine (Rp), CCCP and CPZ versus FIC values of tetracycline (TET) (FICRp, CCCP or CPZ versus FICTET) for SUM36 and AC48. (a) FICTETversus FICRp for AC48. (b) FICTET versus FICCPZ for AC48. (c) FICTET versus FICCCCP for AC48. (d) FICTET versus FICRp for SUM36. (e) FICTET versus FICCPZfor SUM36. (f) FICTET versus FICCCCP for SUM36. A concave curve indicates synergy, a convex curve indicates antagonism and the closest point to the intersection ofthe axes (point zero) is the most effective combination for inhibiting bacterial growth.
was observed (Figure 1e), suggesting that CPZ had no inhibitory this putative efflux mechanism was not affected by CPZ. However, effect on the efflux activity of Tapfor. Despite suggestions that these authors also reported that CPZ and other phenothiazines may phenothiazines act as efflux pump inhibitors,25,26 CPZ did not be useful for managing antibiotic-resistant tuberculosis.11,39 block Tapfor protein activity under our conditions. These results CPZ rendered SUM36 and AC48 equally susceptible to tetra- are largely consistent with those of Viveiros et al.13 who showed cycline (0.12 mg/L) (Table 2), with both strains considerably more that reserpine decreased the MIC of isoniazid for M. tuberculosis susceptible to tetracycline than in the absence of CPZ. These res- cells with induced resistance, whereas CPZ did not, suggesting that ults, together with the lack of effect of CPZ on Tapfor efflux, suggest that CPZ may affect tetracycline resistance levels through This work was supported by the European Union research project ‘New strategies for treatment and prevention of mycobacterialdiseases’ (contract no. QLK2-2000-01761) (E. D. R. and C. M.), by the Ministerio de Ciencia y Tecnologı´a BIO-2002-01287(J. A. A.), by FAR 2004 of the University of Pavia (E. D. R.) Compounds affecting efflux pumps would be expected to react and by the Integrated Research actions Italy-Spain (E. D. R. and synergically with antibiotics with targets located within the bac- J. A. A.). S. R.-G. holds a grant from the Ministerio de Educacio´n y terial cell (i.e. tetracycline), the internal concentrations of which depend on the efficacy of efflux pumps.43 We, therefore, studiedthe interaction of tetracycline with CCCP, reserpine, o-vanadateand CPZ and the ability of these compounds to affect tetracycline resistance in the SUM36 and AC48 strains. We calculated FICIvalues. The FICI value >4 obtained for o-vanadate indicated No declarations were made by the authors of this paper.
antagonism, whereas reserpine had an FICI of 0.4 for SUM36and 0.13 for AC48, suggesting synergy. CCCP had FICI values between 0.5 and 1 for both strains, indicating no interaction withtetracycline. Finally, the FICI clearly indicated synergy between 1. Brown-Elliott BA, Wallace RJ, Jr. Clinical and taxonomic status of CPZ and tetracycline for both strains (Table 3).
pathogenic nonpigmented or late-pigmenting rapidly growing mycobac- The FIC values of reserpine, CPZ, CCCP and tetracycline were teria. Clin Microbiol Rev 2002; 15: 716–46.
calculated for SUM36 and AC48, as described in the Materials and 2. Liu J, Rosenberg EY, Nikaido H. Fluidity of the lipid domain of cell methods section. All the FIC values of each compound (reserpine, wall from Mycobacterium chelonae. Proc Natl Acad Sci USA 1995; 92: CPZ, CCCP) were then plotted against the corresponding FIC values of tetracycline (Figure 2). For reserpine (Figure 2a and d) 3. Paulsen IT. Multidrug efflux pumps and resistance: regulation and evolution. Curr Opin Microbiol 2003; 6: 446–51.
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12. Colangeli R, Helb D, Sridharan S et al. The Mycobacterium tuber- In conclusion, we describe here the sensitivity of the Tapfor culosis iniA gene is essential for activity of an efflux pump that confers drug protein to efflux pump inhibitors and the interactions of tolerance to both isoniazid and ethambutol. Mol Microbiol 2005; 55: tetracycline with these compounds. The Tapfor and Taptub proteins were initially described as increasing resistance to several com- 13. Viveiros M, Portugal I, Bettencourt R et al. Isoniazid-induced pounds.14,15 More recently, the Taptub efflux pump has been shown transient high-level resistance in Mycobacterium tuberculosis. Antimicrob to be induced by drugs in a clinical strain of M. tuberculosis.16 It Agents Chemother 2002; 46: 2804–10.
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We thank Giovanna Riccardi for her help and interest in the 16. Siddiqi N, Das R, Pathak N et al. Mycobacterium tuberculosis isolate project. We also thank Alberto Cebollada for enthusiastic help in with a distinct genomic identity overexpresses a tap-like efflux pump.
the writing of this manuscript, Maria Rosalia Pasca and Silvia Buroni for technical assistance in accumulation experiments and 17. Martin A, Camacho M, Portaels F et al. Resazurin microtiter assay Pilar G. Beltra´n for her support during the writing of this manuscript.
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