European Society of Mycobacteriology - Instituto Nacional de Saúde ...

PP-112
METHODS FOR ASSESSMENT OF ETHIDIUM BROMIDE
TRANSPORT ACROSS Mycobacterium smegmatis CELL WALL
Jorge Ramos 1* , Liliana Rodrigues 1,2 , Isabel Couto 1,3 , Leonard Amaral 1,2 and Miguel Viveiros 1
1 - Unit of Mycobacteriology, Instituto de Higiene e Medicina Tropical, Universidade Nova de Lisboa (IHMT/UNL), Lisboa, Portugal
2 - UPMM, IHMT/UNL, Lisboa, Portugal
3 - Centro de Recursos Microbiológicos (CREM), Faculdade de Ciências e Tecnologia, UNL, Caparica, Portugal
*Correspondig author: Jorge Ramos, Unit of Mycobacteriology, Instituto Higiene e Medicina Tropical, Universidade Nova
de Lisboa (IHMT/UNL), Rua da Junqueira 96, 1349-008 Lisboa, Portugal.
Tel: +351 21 3652600; Fax: +351 21 3632105; E-mail: jramos@ihmt.unl.pt
Active efflux systems and reduced cell wall permeability are considered to be the main causes of mycobacterial intrinsic
resistance to many antimicrobials. Although several mycobacterial efflux pumps have already been described, their role in
drug resistance is not yet fully understood. Recent studies showed that both LfrA and MspA, the main efflux pump and
porin in M. smegmatis, respectively, are involved in reduced susceptibility to several antimicrobials.
We have compared the M. smegmatis wild-type strain mc 2 155 with LfrA and MspA M. smegmatis deleted mutants, for
their ability to extrude ethidium bromide (EtBr), a known efflux pump substrate, under different energy conditions
and in the presence or absence of efflux pumps inhibitors (EPIs), by (i) a 96 well microplate screening assay with the
mycobacterial cells grown in Middlebrook 7H9 with 10% of OADC in presence of increasing concentrations of EtBr
and different concentrations of the EPIs and the plates examined with a UV transilluminator and photographed after
24 hours of incubation; and (ii) a semi-automated fluorimetric method that detects efflux on a real time basis during a
period of 30 minutes. The EPIs employed were chlorpromazine, thioridazine, carbonyl cyanide m-chlorophenylhydrazone
and verapamil.
The efflux activity detected for each strain by these two methods was then correlated with resistance to several antibiotics
(ATBs), by the determination of their minimal inhibitory concentrations in the presence or absence of the EPIs.
The ATBs tested were streptomycin, isoniazid (INH), rifampicin (RIF), ethambutol (ETB), amikacin, ciprofloxacin (CIP)
and clarithromycin (CLR).
In the absence of the major porin of M. smegmatis, MspA, it was observed that accumulation of EtBr decreased and the
cells became more resistant to several ATBs. On the other hand, the mutant for the major efflux pump LfrA showed
increased accumulation of EtBr. This strain also presented increased susceptibility to EtBr, INH, RIF, ETB, CIP and CLR.
These results show that MspA is an important channel for entrance of quaternary ammonium compounds and ATBs and
that the pump LfrA is involved in low-level resistance to several ATBs and quaternary ammonium compounds in M. smegmatis.
184 ESM 2009