title: Effect of efflux pump inhibitors on drug susceptibility of ofloxacin resistant Mycobacterium tuberculosis isolates.
abstract: OBJECTIVE
  In drug resistant, especially multi-drug resistant (MDR) tuberculosis, fluoroquinolones (FQs) are used as second line drugs. However, the incidence of FQ-resistant Mycobacterium tuberculosis is rapidly increasing which may be due to extensive use of FQs in the treatment of various other diseases. The most important known mechanism i.e., gyrA mutation in FQ resistance is not observed in a significant proportion of FQ resistant M. tuberculosis isolates suggesting that the resistance may be because of other mechanisms such as an active drug efflux pump. In this study we evaluated the role of the efflux pumps in quinolone resistance by using various inhibitors such as carbonyl cyanide m-chlorophenyl hydrazone (CCCP), 2,4-dinitrophenol (DNP) and verapamil, in clinical isolates of M. tuberculosis.
  
  
  METHODS
  A total of 55 M. tuberculosis clinical isolates [45 ofloxacin (OFL) resistant and 10 ofloxacin sensitive] were tested by Resazurin microtitre assay (REMA) to observe the changes in ofloxacin minimum inhibitory concentration (MIC) levels in presence of efflux inhibitors as compared to control (without efflux inhibitor).
  
  
  RESULTS
  The MIC levels of OFL showed 2-8 folds reduction in presence of CCCP (16/45; 35.5%), verapamil (24/45; 53.3%) and DNP (21/45; 46.6%) while in case of isolates identified as OFL sensitive these did not show any effect on ofloxacin MICs. In 11 of 45 (24.5%) isolates change in MIC levels was observed with all the three inhibitors. Overall 30 (66.6%) isolates had reduction in OFL MIC after treatment with these inhibitors. A total of eight isolates were sequenced for gyrA gene, of which, seven (87.5%) showed known mutations. Of the eight sequenced isolates, seven (87.5%) showed 2 to 8 fold change in MIC in presence of efflux inhibitors.
  
  
  CONCLUSIONS
  Our findings suggest the involvement of active efflux pumps of both Major Facilitator Super Family (MFS) family (inhibited by CCCP and DNP) and ATP Binding Cassette (ABC) transporters (inhibited by verapamil) in the development of OFL resistance in M. tuberculosis isolates. Epidemiological significance of these findings needs to be determined in prospective studies with appropriate number of samples/isolates.
authors.affiliation: Department of Microbiology & Molecular Biology, National JALMA Institute for Leprosy & Other Mycobacterial Diseases (ICMR), Agra, India.
authors.forename: Mradula
authors.initials: M
authors.lastname: Singh
authors.forename: G P S
authors.initials: GP
authors.lastname: Jadaun
authors.lastname: Ramdas
authors.forename: K
authors.initials: K
authors.lastname: Srivastava
authors.forename: Vipin
authors.initials: V
authors.lastname: Chauhan
authors.forename: Ritu
authors.initials: R
authors.lastname: Mishra
authors.forename: Kavita
authors.initials: K
authors.lastname: Gupta
authors.forename: Surya
authors.initials: S
authors.lastname: Nair
authors.forename: D S
authors.initials: DS
authors.lastname: Chauhan
authors.forename: V D
authors.initials: VD
authors.lastname: Sharma
authors.forename: K
authors.initials: K
authors.lastname: Venkatesan
authors.forename: V M
authors.initials: VM
authors.lastname: Katoch
mesh_terms: D019297:2,4-Dinitrophenol / Q000494:pharmacology; D018528:ATP-Binding Cassette Transporters / Q000037:antagonists & inhibitors*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D001483:Base Sequence; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D019295:Computational Biology; D027081:DNA Gyrase / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D024881:Drug Resistance, Bacterial / Q000187:drug effects* / Q000235:genetics; D008826:Microbial Sensitivity Tests; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D015242:Ofloxacin / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D014700:Verapamil / Q000494:pharmacology
chemical_list: D018528:ATP-Binding Cassette Transporters; D001426:Bacterial Proteins; D017931:DNA Primers; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone; D015242:Ofloxacin; D014700:Verapamil; D027081:DNA Gyrase; D019297:2,4-Dinitrophenol