Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

Molecular basis of bacterial outer membrane permeability.

Some species of bacteria have low-permeability membrane barriers and are thereby "intrinsically" resistant to many antibiotics; they are selected out in the multitude of antibiotics present in the hospital environment and thus cause many hospital-acquired infections. Some strains of originally antibiotic-susceptible species may also acquire resistance through decreases in the permeability of membrane barriers. Another mechanism for preventing access of drugs to targets is the membrane-associated energy-driven efflux, which plays a major role in drug resistance, especially in combination with the permeation barrier. Recent results indicate the existence of bacterial efflux systems of extremely broad substrate specificity, in many ways reminiscent of the multidrug resistance pump of mammalian cells. One such system seems to play a major role in the intrinsic resistance of Pseudomonas aeruginosa, a common opportunistic pathogen. As the pharmaceutical industry succeeds in producing agents that can overcome specific mechanisms of bacterial resistance, less specific resistance mechanisms such as permeability barriers and multidrug active efflux may become increasingly significant in the clinical setting.

https://science.sciencemag.org/content/sci/264/5157/382.full.pdf

Prevention of drug access to bacterial targets: permeability barriers and active efflux

Gram-negative bacteria tend to be more resistant to lipophilic and amphiphilic inhibitors than gram-positive bacteria. Such inhibitors include dyes, detergents, free fatty acids, antibiotics, and other chemotherapeutic agents. This property is used in the selective enrichment of gram-negative bacteria, especially of members of the family Enterobacteriaceae, for example with MacConkey agar (containing crystal violet and bile salts), EMB agar (containing dyes), and deoxycholate agar (containing sodium deoxycholate). The fact that Escherichia coli K-12 can grow in the presence of 1% sodium dodecyl sulfate has been rediscovered many times. Many lipophilic antibiotics, such as penicillin G, erythromycin, fusidic acid, and rifamycin SV, are much less active against most gram-negative bacteria. In fact, a survey of recently reported antibiotics of natural origin showed that, among those compounds that showed activity against gram-positive bacteria, more than 90% lacked activity at a useful level against E. coli (51). This intrinsic resistance of gram-negative bacteria has often been attributed entirely to the presence of the outer membrane barrier. This barrier does contribute to the resistance, as the narrow porin channels slow down the penetration of even small hydrophilic solutes, and the low fluidity of the lipopolysaccharide leaflet decreases the rate of transmembrane diffusion of lipophilic solutes (38, 40). However, the outer membrane barrier cannot be the whole explanation, even with species such as Pseudomonas aeruginosa which produces an outer membrane of exceptionally low permeability (1, 57). This is seen from the fact that equilibration across the outer membrane is achieved very rapidly, in part because the surface-to-volume ratio is very large in a small bacterial cell. Thus, the periplasmic concentrations of many antibiotics are expected to reach 50% of their external concentrations in 10 to 30 s in P. aeruginosa and in a much shorter time period in E. coli (34). Additional mechanisms are therefore needed to explain the level of intrinsic resistance. With the earlier b-lactam compounds, this second contributing factor is the hydrolysis by the periplasmic b-lactamases that are encoded by chromosomal genes in many gram-negative bacteria, and the levels of resistance can be explained quantitatively, in many cases, by the synergy between the outer membrane barrier and b-lactamase (37). However, with dyes, detergents, other classes of antibiotics, and even the b-lactams developed more recently that are not hydrolyzed easily by common b-lactamases, the second factor was completely unknown (34). Recent studies showed that multipledrug efflux pumps, many with unusually broad specificities, play a major role in the intrinsic resistance of gram-negative bacteria. Active efflux processes have been known to cause drug resistance in gram-negative bacteria, through the pioneering studies of S. Levy (19) on Tet-mediated tetracycline resistance. Each of these traditional efflux pumps excretes only one drug or one class of drugs (35). In contrast, a multidrug efflux pump can pump out a wide range of compounds, and it is often difficult to discern any common structural features among the substrates (20, 35).

Multidrug efflux pumps of gram-negative bacteria.

The global emergence of multidrug-resistant Gram-negative bacteria is a growing threat to antibiotic therapy. The chromosomally encoded drug efflux mechanisms that are ubiquitous in these bacteria greatly contribute to antibiotic resistance and present a major challenge for antibiotic development. Multidrug pumps, particularly those represented by the clinically relevant AcrAB-TolC and Mex pumps of the resistance-nodulation-division (RND) superfamily, not only mediate intrinsic and acquired multidrug resistance (MDR) but also are involved in other functions, including the bacterial stress response and pathogenicity. Additionally, efflux pumps interact synergistically with other resistance mechanisms (e.g., with the outer membrane permeability barrier) to increase resistance levels. Since the discovery of RND pumps in the early 1990s, remarkable scientific and technological advances have allowed for an in-depth understanding of the structural and biochemical basis, substrate profiles, molecular regulation, and inhibition of MDR pumps. However, the development of clinically useful efflux pump inhibitors and/or new antibiotics that can bypass pump effects continues to be a challenge. Plasmid-borne efflux pump genes (including those for RND pumps) have increasingly been identified. This article highlights the recent progress obtained for organisms of clinical significance, together with methodological considerations for the characterization of MDR pumps.

/pdf/the-challenge-of-efflux-mediated-antibiotic-resistance-in-3le37l35cs.pdf

The Challenge of Efflux-Mediated Antibiotic Resistance in Gram-Negative Bacteria

Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.

F Yoshimura

Papers

Permeability of Pseudomonas aeruginosa outer membrane to hydrophilic solutes.