title: RNA expression analysis of efflux pump genes in clinical isolates of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis in South Korea.
abstract: Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis, is an important communicable disease. Various mechanisms of resistance to antituberculosis drugs have been reported; these are principally mutations in target genes. However, not all M. tuberculosis resistance can be explained by mutations in such genes. Other resistance mechanisms associated with drug transport, such as efflux pumps, have also been reported. In this study, we investigated the expression levels of three putative efflux pumps and mutations in target genes associated with injectable agents and fluoroquinolones with clinical MDR and XDR-TB isolates. Thirty clinical isolates of M. tuberculosis that had been phenotypically characterized were obtained from the Korean Institute of Tuberculosis. Of these, 14 were MDR-TB isolates resistant to at least one injectable aminoglycoside (amikacin; AMK, kanamycin; KAN, and/or capreomycin; CPM) and 16 were XDR-TB isolates. M. tuberculosis H37Rv (ATCC 27249) was used as a reference strain. Five putative genes (Rv1258c, Rv2686c, Rv2687c, Rv2688c and pstB) were selected for analysis in this study. Sequencing was performed to detect mutations in rrs and eis genes. qRT-PCR was performed to investigate expression levels of five efflux pump genes. Of the 30 isolates, 25 strains had mutations in rrs associated with resistance to KAN, CPM and AMK and two strains had eis mutations, as well as mutations in rrs. pstB (Rv0933) exhibited increased expression and Rv2687c and Rv2688c exhibited decreased expression compared to the reference strain. Increased expression of pstB in clinical drug-resistant tuberculosis isolates may contribute to drug resistance in M. tuberculosis. In our case, overexpression of Rv1258c may have been associated with resistance to kanamycin. No correlation was evident between Rv2686c, Rv2687c or Rv2688c expression and fluoroquinolone resistance. To explore the details of efflux pump drug-resistance mechanisms, further studies on efflux pump inhibitors, transcriptional regulators, such as whiB7, and additional efflux pump genes are needed.
authors.affiliation: Department of Laboratory Medicine, Kyung Hee University Hospital, Seoul, Republic of Korea.
authors.forename: Tae Sang
authors.initials: TS
authors.lastname: Oh
authors.affiliation: Department of Laboratory Medicine, Kyung Hee University School of Medicine, Seoul, Republic of Korea.
authors.forename: Young Jin
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Kyung Hee University School of Medicine, Seoul, Republic of Korea.
authors.forename: Hee Yoon
authors.initials: HY
authors.lastname: Kang
authors.affiliation: Department of Laboratory Medicine, Hanyang University Guri Hospital, Guri, Republic of Korea.
authors.forename: Chang-Ki
authors.initials: CK
authors.lastname: Kim
authors.affiliation: Department of Laboratory Medicine, Kyung Hee University School of Medicine, Seoul, Republic of Korea.
authors.forename: Sun Young
authors.initials: SY
authors.lastname: Cho
authors.affiliation: Department of Laboratory Medicine, Kyung Hee University School of Medicine, Seoul, Republic of Korea. Electronic address: leehejo@gmail.com.
authors.forename: Hee Joo
authors.initials: HJ
authors.lastname: Lee
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000235:genetics*; D000583:Amikacin / Q000494:pharmacology; D000995:Antitubercular Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D002207:Capreomycin / Q000494:pharmacology; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics*; D054908:Extensively Drug-Resistant Tuberculosis / Q000382:microbiology; D015870:Gene Expression; D005798:Genes, Bacterial; D006801:Humans; D007612:Kanamycin / Q000494:pharmacology; D008826:Microbial Sensitivity Tests; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014397:Tuberculosis, Pulmonary / Q000188:drug therapy / Q000382:microbiology
chemical_list: D018528:ATP-Binding Cassette Transporters; D000995:Antitubercular Agents; D001426:Bacterial Proteins; D002207:Capreomycin; D007612:Kanamycin; D000583:Amikacin; D000251:Adenosine Triphosphatases; C120060:phosphate import ATP-binding protein, Bacteria
keywords: Aminoglycosides; Efflux pump; Fluoroquinolones; MDR-TB; Tuberculosis; XDR-TB