a Laboratoire de Bactériologie, Faculté de Médecine, Besançon, France b Department of Bacteriology and Immunology, University of Helsinki c Department of Bacteriology, National Public Health Institute, Helsinki, Finland
Sir,
Many clinically important Gram-negative bacteria possess effective outer membrane (OM) permeability barriers that markedly limit the penetration and, thus, the activities of hydrophobicantibiotics, such as erythromycin and rifampicin, and large antibiotic molecules, such asvancomycin. 1,2 This, in part, explains why these pathogens are resistant to most of the recently discoveredagents (e.g. the oxazolidinones and everninomicin) that exhibit potent activities against Gram-positive bacteria. 3 The barrier function of the OM relies principally on the compact leaflet of lipopolysaccharide (LPS) molecules that covers the cell surface and which is fairly impermeable to hydrophilic and (moderately) hydrophobic compounds. This is exemplified by the recently described Escherichia coli mutants, lpxA and lpxD, in which the lipid A moieties of the LPS have undergone profound alterations and which arealmost as susceptible to hydrophobic antibiotics as the Gram-positive reference strains. 3,4 The MICs of a large number of antibiotics for the E. coli lpxA mutant are 30- to 1000-fold lower than those for clinical isolates of E. coli. 4 Other LPS mutants, such as those in which the inner core polysaccharide moieties of the LPS aredefective (Re type), also exhibit antibiotic supersusceptibility, although to a lesser extent than thelipid A mutants.
The most reliable method of quantifying the permeability of the OM to hydrophobic compounds is that developed by Plésiat & Nikaido 5 which allows the rates of penetration of various steroid probes across the lipid portion of the bilayer to be accurately determined. The results are expressed in terms of the permeability coefficient (P) in nanometres per second (nm/s). With this approach, these investigators showed that the OM of a Re mutant of Salmonella typhimurium was up to 25- and 16-fold more permeable to uncharged steroids and to testosterone hemisuccinate (a monoanionic probe) respectively than that of the parent strain. 5 P values for the most antibiotic-hypersusceptible strains, i.e. the lipid A mutants, have not yetbeen reported. In the present study, we demonstrate that the diffusion rate of testosteronehemisuccinate through the lpxA-type OM is much higher than that through the Re-type OM.
The strains used in the study were as follows: the lipid A mutants of E. coli SM101 (lpxA) and E. coli CDH23-213 (lpxD, the gene formerly known as omsA firA and ssc), both of which are thermosensitive, i.e. SM101 grows well at 28°C, but not at 37°C, andCDH23-213 grows well at 20°C and 37°C, but not at 42°C; the corresponding isogenic parent-type strains, SM105 (lpxA+) and CDH23-210 (lpxD +), which were used as controls; and the E. coli mutant strain, D21f2 (rfa), which has a defective inner core oligosaccharide and which elaborates Re-type (heptoseless)LPS. The antibiotic susceptibilities of all of the strains have been characterized previously. 3,4
The study strains were transformed with the recombinant plasmid, pLE689, that mediates the
3-oxosteroid 1-dehydrogenase enzyme of Comamonas testosteroni.
5 The media used were LB agar and broth, both containing
10 g of tryptone (Oxoid, Basingstoke,UK), 5 g of yeast extract (Difco, Detroit, MI, USA) and 5 g
of NaCl, at pH 7.2, and supplemented with kanamycin at a concentration of 25 mg/L. The agar
plates and broth cultures were incubated at 30°C. The steroid permeability assay was
performed as described previously.
6 Briefly, cells in the exponential growth phase were
collected by centrifugation at roomtemperature and resuspended in 50 mM HEPES, pH 7.4, to an
A
650 of 3. Tubes containing 700 µL of bacterial suspension diluted three-, six-
or 30-fold in the same buffer were incubated at30°C with gentle agitation. The neutral
steroid, testosterone, or its hemisuccinate derivative was added at time 0 to give final
concentrations of 50 µM and 200 µM respectively. Following incubation for 10
min, >95% of the steroid was removed by two brief extractions with ethyl acetate. The
concentration ofthe
-dehydrogenated product was then determined by high-performance
liquid chromatography (HPLC) and the permeability coefficients were calculated according to an
equation based on Ficks' First Law of Diffusion.
6
The P values for testosterone hemisuccinate in the lipid A mutants, SM101 (lpxA) and CDH23-213 (lpxD), were approximately 9001100 nm/sapproximately five- to six-fold greater than that in the E. coli Re mutant, D21f2 (Table), approximately ten-fold greater than that previously determined in the Re mutant of S. typhimurium (data not shown) and approximately 50- to 100-fold greater than those in the control strains (Table). Of considerable note, the high P values for the lipid A mutants demonstrated here are comparable to that (approximately 1000 nm/s) previously determined for the wild-type strain, S. typhimurium SL696, 5 following exposure to deacylpolymyxin B (DAPB), a potent permeabilizer of the OM. 7 This suggests that the OM permeability barrier to a representative anionic hydrophobic probemolecule is quantitatively equally defective in both the lipid A mutants and theDAPB-permeabilized cells.
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Studies such as the one described here add to our knowledge of the properties of the permeability barriers of mutationally defective OMs. This in turn provides information that might help in designing novelantibiotics with activities against Gram-negative bacteria. In the light of increasing antibioticresistance worldwide, such agents are needed desperately.
Acknowledgments
The excellent technical assistance of Colette Godard is gratefullyacknowledged.
Notes
* Correspondence address. Haartmaninkatu 3, 00014 Helsinki, Finland. Tel: +358-9-1912-6302;
Fax: +358-9-1912-6382; E-mail: martti.vaara{at}helsinki.fi
References
1 . Nikaido, H. (1996). Outer membrane. In Escherichia coli and Salmonella typhimurium, Cellular and Molecular Biology, (Neidhardt, F. C., Ed.), pp. 2947. American Society for Microbiology, Washington, DC.
2 . Nikaido, H. & Vaara, M. (1985). Molecular basis of bacterial outer membrane permeability. Microbiological Reviews 49, 132.[ISI]
3 . Vaara, M. (1993). Antibiotic-supersusceptible mutants of Escherichia coli and Salmonella typhimurium. Antimicrobial Agents and Chemotherapy 37, 225560.[ISI][Medline]
4 . Vuorio, R. & Vaara, M. (1992). The lipid A biosynthesis mutation lpxA2 of Escherichia coli results in drastic antibiotic supersusceptibility. Antimicrobial Agents and Chemotherapy 36, 8269.[Abstract]
5 . Plésiat, P. & Nikaido, H. (1992). Outer membranes of Gram-negative bacteria arepermeable to steroid probes. Molecular Microbiology 6, 132333.[ISI][Medline]
6 . Plésiat, P., Aires, J. R., Godard, C. & Köhler, T. (1997). Use of steroids to monitoralterations in the outer membrane of Pseudomonas aeruginosa. Journal of Bacteriology 179, 700410.[Abstract]
7 . Vaara, M. (1992). Agents that increase the permeability of the outer membrane. Microbiological Reviews 56, 395411.[Abstract]