Abstract :
[en] Background
The resistance of Gram negative bacteria toward β-lactam antibiotics is caused by the interplay between four independent factors: i) the alteration of the sensitivity of the target enzymes, the penicillin-binding proteins, ii) the properties and concentration of the periplasmic β-lactamases, iii) the permeability of the outer membrane, iv) the efficiency of the active efflux system.
On this basis, Zimmermann and Rosselet [1] proposed a model yelding a quantitative prediction of the MICs for gram-negative bacteria which was successfully applied to Escherichia coli and Enterobacter cloacae.
This model seems to be less suitable in Pseudomonas aeruginosa due to its low outer membrane permeability which is mostly influenced by both a remarkable reduction of functional porins expression and an over-expression of efflux systems [2]. This decreased permeability causes difficulties in obtaining permeability coefficient direct measures. Moreover, the few published coefficients for P. aeruginosa are highly variable.
For this purpose, BlaR-CTD, the C-terminal domain of a highly sensitive penicillin binding protein from Bacillus licheniformis, expressed in the periplasmic space, has been used in order to directly determinate of the concentrations of different β-lactams in this cell compartment and, consequently to obtain reliable measures of the permeability coefficient [3].
Methods
P. aeruginosa PAO1 cells were incubated with different β-lactams, whose penetration into the periplasm is rapidly followed by the formation of a stable complex with BlaR-CTD. This latter was quantified in cells lysate by densitometric analysis, countermarking the free BlaR-CTD with a fluorescent β-lactam. The excess of the antibiotics will be hydrolysed by the addition of a class B β-lactamase.
We used the same protocol for P. aeruginosa TNP004 [4], a PAO1 strain with a selective deletion of OprD porin, in order to study the influence of this single mutation for the antibiotic permeability.
Results
By the approach described above we determined the permeability coefficients of the external membrane of P. aeruginosa for different antibiotics belonging to the penicillin, cephalosporin and carbapenem sub-families.
The comparison with the porin mutant strain showed similar coefficients for all the antibiotic tested except, as expected, for Imipenem
Conclusion
This work allowed a preliminary characterization of antibiotic permeability in P. aeruginosa which was poorly studied until now.
Furthermore, we could apply this method to correlate the permeability with the role of porin deletions and/or efflux pumps overexpression in antibiotic resistant strains of clinical relevance.
References
1 Zimmermann, W. and A. Rosselet. 1977. Function of the outer membrane of Escherichia coli as a permeability barrier to beta-lactam antibiotics. Antimicrob. Agents Chemother. 12:368–372.
2 Livermore D. M., and K. W. M. Davy. 1991. Invalidity for Pseudomonas aeruginosa of an accepted model of bacterial permeability to β-lactam antibiotics. Antimicrob. Agents Chemother. 35:916-921.
3 Lakaye B., Dubus A., Joris B., and J.M. Frère. 2002. Method for estimation of low outer membrane permeability to β-lactam antibiotics. Antimicrob. Agents Chemother. 46:2901-2907.
4 Yoneyama H., Yamano Y and T. Nakae. 1995 Role of porins in the antibiotic susceptibility of Pseudomonas aeruginosa: construction of mutants with deletions in the multiple porin genes. Biochem Biophys Res Commun. 213:88-95.
