Phospholipid bilayer permeability of .BETA.-lactam antibiotics.

Abstract

Liposomes containing penicillinase or cephalosporinase were prepared from the phospholipids of Escherichia coli. After free p-lactamase was inactivated by clavulanic acid or penicillanic acid sulfone followed by separation of inactivated enzyme and inhibitor from liposomes by gel filtration, the permeability of these liposomes to ampicillin, cefazolin and cephaloridine was estimated by measuring the hydrolysis of these antibiotics by the entrapped enzymes. The permeability parameter C (minute-1 ttM lipid-1) of ampicillin, cefazolin and cephaloridine was calculated to be 2.35 x 10-4, 0.33 x 10-4 and 0.52x 10-4, respectively.The lipid bilayer permeability of these antibiotics was also measured by using the liposomes containing these antibiotics.About half of the initially entrapped ampicillin was released from the liposomes within 80 minutes, while no significant release of cefazolin and cephaloridine could be detected during the same period.These results clearly indicates that the lipid bilayer membrane is more permeable to ampicillin than cefazolin and cephaloridine, and they are consistent with the observations of SAWAI et al.1), who showed that ampicillin was a more effective antibacterial drug than cefazolin and cephaloridine against the porin-deficient mutants.The ability of i3-lactam antibiotics to pass through the bacterial outer membrane is an important factor for their efficacy as antibacterial drugs.The permeability of (3-lactam antibiotics to intact cells was first measured by SAWAI et al.2) and ZIMMERMAN & ROSSELET3j by using the enzyme activity of ;3-lactamase located in the periplasm. In the wild type strains, antibiotic permeability depends on the hydrophilicity of the molecules4,5) ; for the cephalosporins, the permeability depends also on the charges of the compounds5). NIKAIOO et al.6) and ALPHEN et al.7) showed that cephaloridine uses the porin pores as permeation route in Salmonella typhimurium and Escherichia coli. We also suggested that cephalosporins pass through the outer membrane via the porin pore even in Proteus inirabilis and Enterobacter cloacae1). However, we hypothesized in our previous paper1) that a pathway other than the porin pore might play a significant role in outer membrane permeation of penicillins, since the decrease in susceptibility of porin-deficient mutants to the penicillins was significantly smaller than with cephalosporins.In fact, significant amounts of penicillins could penetrate the porin-deficient mutants1,7). A possible pathway for penicillins is through the lipid bilayer of the outer membrane, but it is not easy to show that a penicillin can penetrate the lipid bilayer of intact cells because the outer membrane contains various materials that could interfere with the assay. Phospholipids extracted from E. coli can form closed vesicles such as liposomes without lipopolysaccharides8). The liposomes may be a useful model of the bacterial hydrophobic barrier composed of a lipid bilayer. When j3-lactamase is entrapped in the liposome and concealed from the substrat...

The Lipopolysaccharide Barrier: Correlation of Antibiotic Susceptibility with Antibiotic Permeability and Fluorescent Probe Binding Kinetics

“…The resulting multilamellar vesicle preparations were heated to 37 °C, hydrated, and converted to LUVs as described above, and separated from unincorporated β-lactamase by gel filtration on BioGel A15M at 37 °C. Permeability to cephaloridine was measured by the method of Yamaguchi et al (30) by adding 454 µM cephaloridine as well as a 0.2% starch iodide complex to the outside of LUVs containing the entrapped β-lactamase. The rate of the enzymatic reaction was followed by monitoring the decrease in absorbance at 620 nm following reaction of the hydrolyzed β-lactam with the starch iodide.…”

“…To determine bilayer area, we used an average area per molecule of 60 Å 2 for the phospholipids and an area of 160 Å 2 for LPS (6). To determine S i , we used the following formula, derived from the Michaelis-Menton equation by Sawai et al (31), and used by Yamaguchi et al (30) and Sawai et al (31) in their membrane permeability measurements of β-lactams:…”

Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux as a contributing factor to beta-lactam resistance

“…The use of ['4C]ceftriaxone allowed us to circumvent the oholipid bilayers [42].) problem of enzymatic hydrolysis.…”

Diffusion of beta-lactam antibiotics through the porin channels of Escherichia coli K-12

“…It has been claimed that penicillins tended to penetrate through the outer membrane via a nonporin pathway (24), and the relatively high rate of penetration of ampicillin through the phospholipid bilayer (30) has been correlated with this notion. Since many penicillins have very poor permeability through the porin channel due to their high hydrophobicity and their size, it is conceivable that any leakage through a nonporin pathway would become a significant part of their penetration mechanism.…”