To gain some insight into the mechanisms that allow the reduced susceptibility of S. maltophilia
, we decided to study the presence of MDR determinants in this bacterial species. MDR efflux pumps from gram-negative bacteria are composed of three proteins located in the inner membrane, the periplasmic space, and the outer membrane. These proteins form a channel capable of extruding a broad range of substances from inside the bacterial cell through a proton motive force-dependent mechanism (36
). Synthesis of MDR determinants is usually down-regulated under standard laboratory conditions (36
), so that we decided to clone S. maltophilia
MDR determinants from a spontaneous nonrepressed MDR mutant previously obtained in our laboratory (2
). To make that, a cosmid-based library was made and expressed in the ΔacrAB E. coli
strain AA81. Functional selection for antibiotic-resistant clones allowed the isolation and further sequencing of the first MDR efflux pump determinant (smeDEF
) so far characterized for S. maltophilia
. The results of Northern and Western blot analyses, together with mass spectrometry data, presented in this work support the identity of Omp54 and SmeF. Another determinant sharing the characteristics of an efflux determinant has also been recently sequenced from S. maltophilia
; however, those authors indicate that it is not involved in antibiotic resistance (L. Zhang et al., Pseudomonas '99, abstr. 22).
SmeDEF overexpression increased the MICs of several antibiotics both for S. maltophilia
and for the heterologous host E. coli
, indicating that it is a broad-range MDR determinant. The fact that its expression in E. coli
reduces the accumulation of structurally different compounds by a mechanism that is dependent on bacterial membrane potential indicates that smeDEF
encodes all the elements needed for the synthesis of a functionally active MDR efflux pump similar to others so far described. Indeed, the gene organization of the smeDEF
operon is similar to that of operons of other efflux systems of gram-negative bacteria (37
). Highest homology was found between SmeE and components of the RND family. The membrane fusion proteins of these systems also showed high similarities, although the similarities were lower than for the members of the RND family. The lowest similarities were found between the outer membrane components of efflux pumps; interestingly, SmeF showed the highest homology to SmeC, an OMP from another efflux determinant recently described for S. maltophilia
(accession no. AF173226
). Protein sequence analysis strongly suggests that SmeD and SmeF display lipid attachment sites at the N terminus; indeed, early attempts at sequencing the N terminus of SmeF failed. Predictions of TMS regions in SmeE were also consistent with structural characteristics of the members of the RND family (37
); 12 TMS regions and two external loops situated between TMS regions 1 and 2 and TMS regions 7 and 8 were identified.
Together, these data indicate that SmeDEF is an antibiotic efflux determinant similar to others so far described for gram-negative bacteria (37
), which thus might contribute to the intrinsic susceptibility of S. maltophilia
to different drugs. A recent work has shown that this bacterial species might have several different MDR determinants, some of which are immunologically related with those previously characterized for P. aeruginosa
). It is noteworthy that expression of SmeDEF strongly increases the MIC of erythromycin. Erythromycin is commonly used for the treatment of infections by gram-positive bacteria; however, gram-negative organisms are barely susceptible to this antibiotic. It has been speculated that this reduced susceptibility may be due to a reduced permeability of cellular envelopes to erythromycin. However, some MDR determinants from gram-negative bacteria are capable of extruding this antibiotic (1
), as occurs with SmeDEF. Searching for inhibitors of those MDR determinants involved in erythromycin extrusion might allow us to increase the susceptibilities of gram-negative bacteria and thus allow us to introduce this antibiotic into the armamentarium for the treatment of gram-negative infections.
Expression of SmeDEF has been analyzed by Northern and Western blotting both for the wild-type D457 strain and for the derepressed D457R mutant. In both cases, a maximum amount of smeDEF was observed at the beginning of the exponential phase and decreased to undetectable levels after 24 h of growth. Western blot analysis demonstrated, however, that the amount of SmeF is nearly constant throughout cell cycle, with a small reduction at 24 h. These data might be explained either by the presence of another internal promoter which drives smeF expression and is not regulated by the cell cycle or because this protein is very stable and thus its amount is maintained, although smeDEF RNA levels decrease.
The regulation of the expression of smeDEF
occurs then at two independent levels. First, the system is repressed in the wild-type D457 strain, a repression that is retrieved in the MDR D457R strain. Second, the expression of the system is regulated by growth phase, a regulation that is maintained in both strains. The signals which allow expression of the usually down-regulated MDR systems are poorly understood. Some of these systems are activatable by natural signal molecules like salicylate (7
). Induction of an MDR phenotype by toxic substances such as solvents (19
) and heavy metals (18
) has also been described. Gene fusion experiments have demonstrated that the expression of acrAB
from E. coli
) and mexABOprM
from P. aeruginosa
) is increased by stress conditions and in stationary growth phase. Unlike with this growth-phase regulation, increased expression of smeDEF
is observed at early exponential phase whereas expression of the system in stationary phase is nearly null. It is for the first time that this type of regulation is observed for MDR systems. Based on the expression of other determinants (see above), it was speculated that MDR systems, might have a physiological role during stationary-phase stress (29
). However, our data do not support such a role for smeDEF
. A search of published sequences of bacterial genomes has demonstrated the presence of multiple MDR determinants in bacterial chromosomes (42
). This high redundancy probably indicates that they do not share the same physiological function. Hence, it is not strange that their expression responds in different ways to environmental and physiological signals.