A balancing act: efflux/influx in mycobacterial drug resistance.

Since the discovery of the tubercle bacillus by Robert Koch in 1882 (110), a greater understanding of the dynamics and survival mechanisms of this pathogen has led to more questions than answers. Despite stringent control strategies and many advances in our knowledge of the epidemiology of tuberculosis (TB) and the biology of the causative agent Mycobacterium tuberculosis, TB still remains one of the most common and deadly infectious diseases worldwide. The emergences of multidrug-resistant TB (MDR-TB) (with resistance to at least the first-line drugs isoniazid [INH] and rifampin [rifampicin] [RIF]) (39) and extensively drug-resistant TB (XDR-TB) (with additional resistance to a fluoroquinolone [FQ] and any one of the injectable drugs kanamycin [KAN], amikacin [AMI], and capreomycin [CAP]) (43, 67) are a major concern in the control of the global TB epidemic. Drug resistance is not acquired through horizontal gene transfer in M. tuberculosis, since this pathogen does not contain plasmids and the transfer of genomic DNA has not been demonstrated (125). Thus, resistance to anti-TB drugs develops by spontaneous mutation and the resulting resistant mutants are selected by subsequent treatment with anti-TB drugs to which the mutants are resistant. Resistance to various first-line anti-TB drugs, such as INH, RIF, pyrazinamide (PZA), ethambutol (EMB), and classes of second-line drugs (FQs, aminoglycosides, thionamides, peptides, and cycloserines) is attributed to specific mutations in target genes or regulatory domains (10, 11, 28, 69, 107–109) (Table 1). It is thus believed that a specific gene alteration (mutation, insertion, or deletion) will alter the structure of the target protein, thereby influencing the degree of susceptibility to the drug (116). For example, the katG gene codes for both catalase and peroxidase enzyme activity, which is essential for the conversion of INH to its active form. Mutations in the katG gene lead to a decrease in catalase activity, thereby resulting in less INH being activated and M. tuberculosis being resistant to high levels of INH (40). This relationship was confirmed by Ramaswamy et al., who showed that INH-resistant isolates with MICs of 256 g/ml INH all had low or no catalase activity levels (89). In contrast, mutations in the regulatory or structural regions of the inhA gene result in low-level resistance to INH in M. tuberculosis (41, 89). Interestingly, mutations within the promoter and the coding region of inhA were found to also confer ethionamide resistance (7, 69). This demonstrates that mutations in the same genes or regulatory domain can result in different drug resistance phenotypes. However, resistance in a proportion of clinical M. tuberculosis isolates cannot be explained by classical gene mutations such as those described above. For example, approximately 20 to 30% of clinical INH-resistant M. tuberculosis isolates do not have mutations in any of the known genes (Table 1) associated with INH resistance (88, 89). Similarly, approximately 5% of clinical RIF-resistant M. tuberculosis isolates do not harbor mutations in the RIF resistance-determining region of the rpoB gene (112). Therefore, it is evident that other, moreundefined mechanisms could play a role in drug resistance. Additional mechanisms that contribute to drug resistance in mycobacteria exist. These mechanisms include the production of drug-modifying and -inactivating enzymes, low cell wall permeability, and efflux-related mechanisms (1, 9, 12, 88, 120, 121). Mycobacteria produce enzymes that degrade or modify certain antibiotics, leading to their inactivation (61, 111). For example, Mycobacterium smegmatis is naturally resistant to RIF, although no mutations have been identified in the rpoB gene (87). This suggests that an alternative mechanism or mechanisms play a role in conferring resistance to RIF. In 1995, it was reported that M. smegmatis DSM43756 inactivates RIF by ribosylation, whereby a ribose ring is covalently linked to the RIF molecule (17, 46). Gene disruption experiments provided evidence that RIF inactivation via ribosylation was the principal contributor of RIF resistance in M. smegmatis (87). However, only limited data exist for the production of degrading and drug-modifying enzymes in M. tuberculosis. It has previously been reported that bacterial resistance to aminoglycosides can be attributed to enzymatic inactivation of aminoglycosides by phosphotransferases, nucleotidyltransferases, and acetyltransferases (18). Acetyltransferase AAC (2 )-Ic and the phosphotransferase encoded by the Rv3225c gene have been shown to display aminoglycoside-modifying activity (25) that resulted in resistance to aminoglycosides in mycobacteria. Individual mechanisms may not be sufficient to confer clinical resistance but may interact with other resistance mechanisms to cause high-level resistance. This accumulation * Corresponding author. Mailing address: DST/NRF Centre of Excellence in Biomedical Tuberculosis Research/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health Sciences, Stellenbosch University, P.O. Box 19063, Tygerberg 7505, South Africa. Phone: 27-21-9389251. Fax: 27-21-9389476. E-mail: tv @sun.ac.za. Published ahead of print on 18 May 2009.