Two Distinct Major Facilitator Superfamily Drug Efflux Pumps Mediate Chloramphenicol Resistance in Streptomyces coelicolor

Abstract

Chloramphenicol, florfenicol, and thiamphenicol are used as antibacterial drugs in clinical and veterinary medicine. Two efflux pumps of the major facilitator superfamily encoded by the cmlR1 and cmlR2 genes mediate resistance to these antibiotics in Streptomyces coelicolor, a close relative of Mycobacterium tuberculosis. The transcription of both genes was observed by reverse transcription-PCR. Disruption of cmlR1 decreased the chloramphenicol MIC 1.6-fold, while disruption of cmlR2 lowered the MIC 16-fold. The chloramphenicol MIC of wild-type S. coelicolor decreased fourfold and eightfold in the presence of reserpine and Phe-Arg-␤-naphthylamide, respectively. These compounds are known to potentiate the activity of some antibacterial drugs via efflux pump inhibition. While reserpine is known to potentiate drug activity against gram-positive bacteria, this is the first time that Phe-Arg-␤-naphthylamide has been shown to potentiate drug activity against a gram-positive bacterium.Membrane-bound efflux pumps commonly underlie drug resistance in pathogenic bacteria (21,26,33). These pumps function by actively expelling drugs from the cytosol of bacterial cells, reducing the effective intracellular drug concentration. There are five major families of efflux pumps in bacteria: the ATP binding cassette family, the major facilitator superfamily (MFS), the multidrug and toxic compound extrusion family, the resistance nodulation division family, and the small multidrug resistance family (33). Some efflux pumps exhibit high specificity for certain antimicrobial agents, while others act upon drugs from unrelated structural classes and thereby confer multidrug resistance (33). In both gram-positive and gramnegative bacteria, genes encoding efflux pumps are found on the chromosome and on plasmids (33). Multidrug resistance in human pathogens is often correlated with the overexpression of efflux pump genes (18,34).Efflux is a nondestructive mechanism of antibiotic resistance commonly observed in antibiotic-producing bacteria that provides self-resistance without compromising the biological activity of the biosynthesized antibiotics (9). Given that antibioticproducing organisms are a reservoir of resistance genes (23), they are a likely source of the drug efflux pump genes found in pathogenic bacteria. Streptomyces bacteria produce two-thirds of the clinically used antibiotics and often have an efflux pump gene associated with self-resistance (1, 17, 31). In some cases, these gram-positive, soil-dwelling bacteria have efflux pumps that confer resistance to multiple antibiotics. For instance, the ptr gene in Streptomyces pristinaespiralis confers resistance to the pristinamycins and rifampin (rifampicin) (3). Likewise, an ATP binding cassette family transporter gene in Streptomyces rochei F20 confers resistance to macrolides (i.e., oleandomycin, erythromycin, and spiramycin), tetracycline, and doxorubicin (11). Although most Streptomyces bacteria are not pathogenic, they are of clinical interest because they are close relatives ...

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