Role of the Outer Membrane of Gram-Negative Bacteria in Antimicrobial Resistance

Nikaido, Hiroshi

doi:10.1007/978-3-642-74095-4_1

Cited by 22 publications

(15 citation statements)

References 137 publications

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“…For a gram-positive bacterium, these properties would not be expected to interfere with DIG-AMP binding to the freely accessible PBPs. However, conventional P-lactams largely permeate the outer membrane of E. coli through the narrow water-filled porin channels (17). It is thus more plausible that DIG-AMP would gain access to the E. coli PBPs predominantly by simple diffusion across the outer membrane, as observed for other steroid derivatives comparable to digoxigenin (19).…”

Section: Discussionmentioning

confidence: 99%

Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence

Weigel

Belisle

Radolf

et al. 1994

Antimicrob Agents Chemother

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This paper describes a highly sensitive new method for the identification of penicillin-binding proteins (PBPs) that is based on the use of an ampicillin-digoxigenin conjugate (DIG-AMP conjugate) which is detected by immunoblotting and chemiluminescence. The sensitivity of chemiluminescence permitted X-ray film exposure times to be decreased to minutes, as opposed to the days or weeks which are requisite when conventionally radiolabeled P-lactams are used. Coupling of ampicillin to digoxigenin yielded a product containing digoxigenin (detected by chemiluminescence) which also was inhibitory for Staphylococcus aureus and Escherichia coli. Unconjugated digoxigenin at concentrations of up to 100 ,ug/ml was not inhibitory for either organism. For S. aureus the MICs of DIG-AMP (0.7 ,ug of conjugated ampicillin per ml) and of free ampicillin (0.5 ,ug/ml) were comparable, indicating that ampicillin retained its bioactivity when coupled to digoxigenin. However, for E. coli the MICs of DIG-AMP (70 ,ug of conjugated ampicillin per ml) and of free ampicillin (8 ,ug/ml) Penicillin-binding proteins (PBPs) are enzymes involved in the terminal biosynthetic reactions of the peptidoglycan layer of the bacterial cell wall. These enzymes, some of which are the lethal targets for penicillins and other P-lactam antimicrobial agents, are involved in various aspects of cell growth, cell division, and the maintenance of cell shape (6,26,27,29).Previous studies to examine the interactions between PBPs and 1-lactam drugs have relied primarily on the use of radiolabeled compounds (10,25,28,29). However, there are major disadvantages associated with the use of radiolabeled 3-lactams for this purpose: (i) they typically are expensive, and some forms are unstable, (ii) they require extended periods for autoradiography or fluorography, and (iii) there are substantial hazards associated with their handling as well as increasing concerns regarding disposal and storage of radioactive waste. To circumvent these problems, we developed a method to conjugate digoxigenin to the free amino group of ampicillin. Following incubation of either cell envelopes or viable bacteria with the conjugate, PBPs were detected by immunoblotting and chemiluminescence. This new procedure should have broad applicability as a rapid and highly sensitive nonradioactive alternative for investigating the interactions of ,-lactams and their targets in bacterial cells. MATERIALS AND METHODSChemical reagents. Benzylpenicillin, sodium ampicillin, Nlauroylsarcosine (Sarkosyl), and dimethylformamide were ob-* Corresponding author. Mailing address:

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Section: Discussionmentioning

confidence: 99%

Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence

Weigel

Belisle

Radolf

et al. 1994

Antimicrob Agents Chemother

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“…Most nutrients used by gram-negative bacteria move across the outer membrane through the nonspecific channels of porin (16), and a similar mechanism of diffusion is thought to be used by most antibiotics that are effective against these bacteria, including most ,-lactams (13). Some nutrients, however, are too large for efficient diffusion through the porin channel, and these compounds, including iron-siderophore complexes, are often taken up by specific pathways present in the outer membrane.…”

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confidence: 99%

Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups

1990

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Recently, j8-lactam agents containing iron-chelating moieties, such as E0702, which contains catechol, and pirazmonam and U-78,608, which contain 3-hydroxypyridone, have been developed. By determining the susceptibility to these agents of Escherichia coli mutants lacking various iron-repressible outer membrane proteins, we showed that two of these proteins with hitherto unknown functions, Fiu and Cir, were apparently involved in the transport of monomeric catechol and its analogs. These results confirm the conclusion of Curtis and co-workers, which was obtained by using a different set of catechol-containing antibiotics (N. A. C. Curtis, R. L. Eisenstadt, S. J. East, R. J. Cornford, L. A. Walker, and A. J. White, Antimicrob. Agents Chemother. 32:1879Chemother. 32: -1886Chemother. 32: , 1988. E0702 was shown to enhance the uptake of radioactive ferric iron into wild-type cells but not into cir fiu double mutants. By combining the influx of E0702 with its hydrolysis by a periplasmic P-lactamase, we showed that the wild-type cells transported unliganded E0702 at a rate comparable to or even higher than the rate of transport of the E0702-Fe3+ complex. We postulate that the main function of Cir and Fiu may be to recapture the hydrolytic products of enterobactin, such as 2,3-dihydroxybenzoic acid and 2,3-dihydroxybenzoylserine.Most nutrients used by gram-negative bacteria move across the outer membrane through the nonspecific channels of porin (16), and a similar mechanism of diffusion is thought to be used by most antibiotics that are effective against these bacteria, including most ,-lactams (13). Some nutrients, however, are too large for efficient diffusion through the porin channel, and these compounds, including iron-siderophore complexes, are often taken up by specific pathways present in the outer membrane. Thus, Escherichia coli, for example, is known to have at least seven outer membrane proteins whose production is regulated by the availability of ferric iron in the environment; four of them, the products of the fepA, fecA, fluA, and AhuE genes, have been identified as the receptors for ferric enterobactin, ferric citrate, fermchrome, and coprogen, respectively (12,22). Recently, several P-lactam compounds with attached iron-chelating moieties have been developed (9,18,24). Moreover, evidence suggestive of the utilization of a siderophore receptor for outer membrane penetration was obtained for at least one of these compounds, E0702, because E. coli became resistant to this compound if it had tonB mutations, which abolish the transport of all known siderophores as well as vitamin B12 across the outer membrane (26). However, the identities of the outer membrane receptors used by these 1-lactams were unknown, and this study was undertaken to identify these receptors. In the course of the present study, we learned that another laboratory studied the outer membrane permeation of a group of catechol-containing cephalosporins by using a similar approach (3). Our results, however, have been obtained by using an...

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“…In gram-negative bacteria, the outer membrane barrier very often slows the influx of antibiotic molecules and constitutes one of the major factors that determine the degree of antibiotic resistance of the organism (4). Thus, the precise determination of outer membrane permeability is important in the quantitative analysis of the efficacy of antibiotics, especially those that are inactivated within bacterial cells, for example 1-lactams (5).…”

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confidence: 99%

Contribution of the cell-surface-associated enzyme in the Zimmermann-Rosselet assay of outer membrane permeability of beta-lactam antibiotics

Liu

Nikaido

1991

Antimicrob Agents Chemother

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Analysis of j8-lactam hydrolysis by intact cells of several strains of enteric bacteria suggests that a significant fraction of P-lactamase molecules is present at a location directly accessible for the substrates in the medium.We propose a method to correct for the error introduced by these "surface-exposed" enzymes in the Zimmermann-Rosselet assay of outer membrane permeability.In gram-negative bacteria, the outer membrane barrier very often slows the influx of antibiotic molecules and constitutes one of the major factors that determine the degree of antibiotic resistance of the organism (4). Thus, the precise determination of outer membrane permeability is important in the quantitative analysis of the efficacy of antibiotics, especially those that are inactivated within bacterial cells, for example 1-lactams (5). Among the several methods available for the measurement of outer membrane permeability, the one proposed by Zimmermann and Rosselet (8) appears to be most rigorously correct. In this method, hydrolysis of 1-lactam antibiotics by intact cells of gramnegative bacteria is analyzed as the consequence of two processes: the first, diffusion across the outer membrane following Fick's first law of diffusion, and the second, hydrolysis by periplasmic P-lactamase molecules following 8).In the actual application of this method, some interference is caused by ,-lactamase molecules that have leaked into the medium. This is corrected by measuring P-lactam hydrolysis catalyzed by the centrifugal supernatant of the cell suspension (6). However, if there are cells with damaged, "leaky" outer membranes but that still contain periplasmic ,-lactamase molecules or if there are enzyme molecules that are exposed on the cell surface but remain associated with the cell, the contribution of these enzymes will be difficult to correct for, as was pointed out earlier (3). Indeed, Hewinson et al. (1) reported that the Zimmermann assay of outer membrane permeability of a Pseudomonas aeruginosa strain produced different permeability coefficients depending on the concentration of the substrate, cephalosporin C, used, and it was pointed out by R. E. W. Hancock that these results may be due to the presence of surface-associated 3-lactamase (cited in reference 2). The potential problems arising from the presence of cell-surface-associated enzyme have not, however, been quantitatively studied. We propose here a method for estimating this contribution and show that it may introduce significant errors in the determination of outer membrane permeability.If cell-surface-exposed enzyme hydrolyzes the P-lactam * Corresponding author.antibiotic with a Vmax value of V., then the total rate of hydrolysis of the drug by a suspension of intact cells, vcells, is as follows: Vcells = Vs X Col(Km+ CO) + P x A x (Co-Cp) (1) where CO, Cp, P, and A represent the external concentration of the drug, its periplasmic concentration, the coefficient of outer membrane permeability to that drug, and the area of the outer membrane per unit of cell mass, respecti...

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Role of the Outer Membrane of Gram-Negative Bacteria in Antimicrobial Resistance

Cited by 22 publications

References 137 publications

Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence

Digoxigenin-ampicillin conjugate for detection of penicillin-binding proteins by chemiluminescence

Cir and Fiu proteins in the outer membrane of Escherichia coli catalyze transport of monomeric catechols: study with beta-lactam antibiotics containing catechol and analogous groups

Contribution of the cell-surface-associated enzyme in the Zimmermann-Rosselet assay of outer membrane permeability of beta-lactam antibiotics

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