Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis.

Abstract

Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, ofStreptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.The worldwide reemergence of tuberculosis as a major public health problem has been accompanied by an ominous increase in multidrug resistant strains (1). This increase has stimulated an intense search for new antimycobacterial agents, but at present only one additional class of drugs, the fluoroquinolones (FQ), has been added to the traditional anti-tuberculosis armamentarium (2). Introduced into clinical practice in the 1980s, the FQ were initially active against many pathogens (3), but their use has been limited by the rapid appearance of resistance in a large percentage of clinical isolates, especially in Staphylococcus aureus and Pseudomonas aeruginosa (41,42). The therapeutic use of the FQ in tuberculosis began only within the past 3-4 years, and they are generally reserved for infections resistant to other agents. However, most FQ are only moderately active against the mycobacteria, and unfortunately, FQ-resistant (FQr) clinical isolates of Mycobacterium tuberculosis have already appeared (4,5). If more can be learned about what determines the effectiveness of a particular FQ against the mycobacteria and the mechanisms by which resistance develops, new agents or strategies may be designed that can prevent or circumvent this resistance.The principal targets of the FQ are bacterial type II topoisomerases, including both the bacterial DNA gyrase, an essential type II topoisomerase that introduces supercoils into the DNA chromosome (6), and the highly homologous topoisomerase IV, which deconcatenates the chromosome after DNA replication (7,8). Mutations in a particular region of gyrA, which encodes the gyrase A subunit, have been associated with moderate-to-high level [>5x minimal inhibitory concentration (MIC)] FQ resistance in many species of bacteria, including M. tuberculosis (5), and similar mutations have been found in the homologous region of topoisomerase IV (9, 10). Mutations conferring low-level resistance have also beenThe publication costs of this article were defrayed in part by page charge p...

Noncanonical SMC protein in Mycobacterium smegmatis restricts maintenance of Mycobacterium fortuitum plasmids

“…Perhaps more importantly, M. smegmatis has proven to be a more suitable surrogate host than conventional host organisms such as E. coli, B. subtilis, or Streptomyces for investigating the molecular biology of mycobacteria. Isolation of mc 2 155 allowed for the identification of genes involved in isoniazid (68)(69)(70)(71), ethionamide (72,73), isoxyl (74,75) or thioacetazole (75), ethambutol (76)(77)(78), capreomycin (79), streptomycin (80), fuoroquinolone (81), and kanamycin (79) resistance. The ability to transform and express genes in M. smegmatis allowed the discovery of the first auxiliary secA2 secretion system (82), a novel way to study iron metabolism (83), and the analyses of unique carbohydrate antigens (84).…”

Molecular Properties of Bacterial Multidrug Transporters

“…LfrA confers resistance to hydrophilic quinolones but not to the more hydrophobic quinolones (134). Hybridization analysis showed that LfrA homologs are present in M. tuberculosis and Mycobacterium avium (244).…”

Characterization of the MSMEG_2631 Gene ( mmp ) Encoding a Multidrug and Toxic Compound Extrusion (MATE) Family Protein in Mycobacterium smegmatis and Exploration of Its Polyspecific Nature Using Biolog Phenotype MicroArray

“…LfrA, an MFS efflux pump that confers resistance to fluoroquinolones, EtBr, acridine, and quaternary ammonium compounds, was the first such pump characterized in mycobacteria (53). More recently, other efflux pumps have been demonstrated to be involved in mycobacterial drug resistance (17).…”