Characterization of tetracycline resistance mediated by the efflux pump Tap from Mycobacterium fortuitum.
OBJECTIVE
The aim of this study was to characterize the efflux pump Tap from Mycobacterium fortuitum, to test its sensitivity to well known efflux inhibitors, to study the interaction between tetracycline and these compounds and to test the ability of these compounds to overcome efflux pump-mediated tetracycline resistance. For all these studies, we produced Tap protein in Mycobacterium smegmatis.


METHODS
Antibiotic susceptibility tests, tetracycline uptake/efflux experiments and checkerboard synergy tests.


RESULTS
Tetracycline uptake/efflux experiments showed that Tap protein from M. fortuitum uses the electrochemical gradient across the cytoplasmic membrane to extrude tetracycline from the cell. This efflux activity is inhibited by carbonyl cyanide m-chlorophenylhydrazone (CCCP) and reserpine, consistent with 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 across the membrane and to restore efflux activity. CCCP, reserpine and CPZ reduced the MIC of tetracycline in the M. smegmatis strain expressing the Tap protein, whereas o-vanadate increased the MIC. We also observed synergy 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 be used to overcome antibiotic resistance mediated by efflux pumps.
Ramón-García
Santiago
S
Departamento de Microbiología, Medicina Preventiva y Salud Pública, Universidad de Zaragoza, 50009 Zaragoza, Spain.
Martín
Carlos
C
Aínsa
José A
JA
De Rossi
Edda
E
D000900:Anti-Bacterial Agents / Q000378:metabolism / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D002746:Chlorpromazine / Q000494:pharmacology; D004357:Drug Synergism; D004563:Electrochemistry; D004734:Energy Metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008826:Microbial Sensitivity Tests; D019907:Mycobacterium fortuitum / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D012110:Reserpine / Q000494:pharmacology; D012997:Solvents; D013752:Tetracycline / Q000378:metabolism; D013753:Tetracycline Resistance / Q000235:genetics*; D014475:Uncoupling Agents / Q000494:pharmacology; D014638:Vanadates / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D012997:Solvents; C115861:Tap protein, Mycobacterium; D014475:Uncoupling Agents; D014638:Vanadates; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D012110:Reserpine; D013752:Tetracycline; D002746:Chlorpromazine