A method for measuring the outer membrane-permeability of .BETA.-lactam antibiotics in gram-negative bacteria.

Sawai, Tetsuo; Matsuba, Kiyotaka; Yamagishi, Saburo

doi:10.7164/antibiotics.30.1134

Cited by 36 publications

(32 citation statements)

References 2 publications

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“…Of course the treatment decreased the f1-lactamase activity of these strains, but we could not clearly explain the difference in the susceptibility to CER among these strains by the decrease in enzyme activity alone. Recently Sawai et al (23,24), and Zimmerman and Rosselet (32) devised a method for measuring the membrane permeability of Gram-negative bacteria for f1-lactam antibiotics. The measurement was made on the basis of the difference in the rate of hydrolysis of a substrate between whole cells (Vi) and the sonicates of the corresponding cells (V d ) , and of the Michaelis' constant (K m ) (24).…”

Section: Discussionmentioning

confidence: 99%

“…Recently Sawai et al (23,24), and Zimmerman and Rosselet (32) devised a method for measuring the membrane permeability of Gram-negative bacteria for f1-lactam antibiotics. The measurement was made on the basis of the difference in the rate of hydrolysis of a substrate between whole cells (Vi) and the sonicates of the corresponding cells (V d ) , and of the Michaelis' constant (K m ) (24). Using this method, we compared the permeability for CER of strains with and without R plasmids.…”

Section: Discussionmentioning

confidence: 99%

“…The penetrability of the f3-lactam antibiotics through the outer membrane was estimated from the ratio of the substrate concentration in the reaction medium (Sl I'M) to that in the periplasm (S2 I'M), by the method of Sawai (24). S2 was calculated from the following formula which is derived from the Michaelis-Menten equation.…”

Section: Methodsmentioning

confidence: 99%

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A Possible Role of R Plasmids in Bacterial Permeability for β‐Lactam Antibiotics

Ikeuchi

Osada

1981

Microbiology and Immunology

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Four strains of clinical isolates of Serratia marcescens (13039, 13090, 13093, 14093) harboring R plasmids were highly resistant to ampicillin (ABPC) and cephaloridine (CER). With elimination of R plasmids from these strains by acriflavine treatment, ABPC-resistance levels of these strains were markedly reduced. Reduction of CER-resistance levels was also demonstrated in strains 13039 and 13093, but not in strains 13090 and 14093. The permeability of the former strains for CER was also decreased, but not in the latter strains. At the same time, fllactamase activity of these strains also almost completely disappeared when the R plasmids were eliminated. By broth matings with these strains, the recipient strains of S. marcescens 13031 (rif), Escherichia coli K-12 (rif), and E. coli 15046 (rif) all acquired a high permeability barrier against CER with inheritance of the R plasmids from strains 13039 and 13093, but not from strains 13090 and 14093. The transconjugant of strain 13031 that inherited R plasmid 13093 was resistant not only to CER but also to cefazolin, cephalothin, and cephalexin. Its permeabiiity to these antibiotics was significantly lower than that of the original strain. This fact suggests the possibility that the R plasmid from strain 13093 may be involved not only in production of p-lactamases, but also in regulation of bacterial permeability for cephalosporins.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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A Possible Role of R Plasmids in Bacterial Permeability for β‐Lactam Antibiotics

Ikeuchi

Osada

1981

Microbiology and Immunology

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“…One important aspect of the mechanism of action requires investigation of the outer membrane permeability of imipenem in Gram-negative bacteria. Previous work on the estimation of permeability of ~-lactam antibiotics through the bacterial outer membrane, known to be a barrier against penetration to the target proteins, was reported by SAWAI et al 8), and ZIMMERMANN and ROSSELET9). The procedure used was based on a kinetic treatment of the hydrolysis of 9-lactam antibiotic by the 18-lactamase located in the periplasm of intact cells, however, this method is inapplicable to B-lactamase-stable B-lactams such as imipenem.…”

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Outer membrane permeability of imipenem in comparison with other .BETA.-lactam antibiotics.

1986

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“…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).…”

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confidence: 99%

Phospholipid bilayer permeability of .BETA.-lactam antibiotics.

1982

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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...

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A method for measuring the outer membrane-permeability of .BETA.-lactam antibiotics in gram-negative bacteria.

Cited by 36 publications

References 2 publications

A Possible Role of R Plasmids in Bacterial Permeability for β‐Lactam Antibiotics

A Possible Role of R Plasmids in Bacterial Permeability for β‐Lactam Antibiotics

Outer membrane permeability of imipenem in comparison with other .BETA.-lactam antibiotics.

Phospholipid bilayer permeability of .BETA.-lactam antibiotics.

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