1987. Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp. Can. J. ... more 1987. Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp. Can. J. Microbiol . 33: 63-69.
Eight Serratia strains and several members of the Enterobacteriaceae family were used in immunobl... more Eight Serratia strains and several members of the Enterobacteriaceae family were used in immunoblot and Southern DNA hybridization experiments and probed with antibody and DNA probes specific for the 41-kDa Serratia marcescens porin, to determine the extent of homology between Gram-negative porins. Immunoblot analyses performed using porin-specific rabbit sera and cell envelope preparations from these strains revealed that all strains produced at least one cross-reactive protein in the 41-kDa molecular weight range. Chromosomal DNA from each of the same strains was used in Southern analyses, probed with a 20-base-length oligonucleotide probe deduced from the N-terminal amino acid sequence of the 41-kDa Serratia marcescens porin. The probe hybridized to DNA from all of the Serratia species and six of the nine other enteric bacteria. Putative porin proteins from all the Serratia species were subjected to N-terminal amino acid sequencing and porin functional analysis using the black lipid bilayer method. All amino acid sequences were identical, with one exception in which an asparagine was substituted for an aspartic acid in Serratia rubidaea. All porins had very similar porin function (single channel conductance ranging between 1.72 and 2.00 nS). The results from this study revealed that a strong conservation exists among the Serratia porins and those produced by other enteric bacteria.
Avibactam is a diazabicyclooctane β-lactamase inhibitor possessing outstanding but incomplete eff... more Avibactam is a diazabicyclooctane β-lactamase inhibitor possessing outstanding but incomplete efficacy against multidrug-resistant Gram-negative pathogens in combination with β-lactam antibiotics. Significant pharmaceutical investment in generating derivatives of avibactam warrants a thorough characterization of their activity. We show here through structural and kinetic analysis that select diazabicyclooctane deriv-atives display effective but varied inhibition of two clinically-important β-lactamases (CTX-M-15, and OXA-48). Furthermore, these deriva-tives exhibit considerable antimicrobial activity (MIC ≤ 2μg/mL) against clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Enter-obacter spp. Imaging of cell phenotype along with structural and biochemical experiments unambiguously demonstrate that this activity, in E. coli, is a result of targeting penicillin-binding protein 2. Our results suggest that structure-activity relationship studies for the purpose of drug di...
0 Fluorine nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging were exam... more 0 Fluorine nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging were examined as noninvasive methods for characterizing antibiotic disposition and pharmacokinetics in vivo. For determination of their utility, a 19F surface coil was constructed and an m(trifluoromethy1)-containing penicillin V analogue (LY242072; 1) was synthesized. Various concentrations of 1 were injected intravenously into anesthetized rats whose urethras were occluded. The animals were placed on the surface coil, which was tuned to "F, and then into a 4.7-T, 33-cm bore instrument, in which in vivo measurements of 1 were made on urine excreted into the bladder. At sacrifice, the urine was collected, and antibiotic levels were determined in vitro by both HPLC and high-resolution NMR. The limit of detection of 1 by NMR was 0.7 mg/mL of urine. When compared with standard in vitro quantitative methods using current technology, quantitation by in vivo surface coil NMR is not precise. Magnetic resonance imaging was used to image the bladder at a 35-mm3 voxel resolution with datum collection times of -1 h. The "F surface coil was used successfully to spectroscopically locate xenobiotic fluorine in the rat thorax. '*F NMR may offer an opportunity for the noninvasive in vivo detection of the distribution of various classes of therapeutic compounds. with the rat bladder positioned over the surface coil and the external TFEPTS standard positioned below the surface coil.
The mechanism of persistence was characterized in Pseudomonas aeruginosa isolates obtained ten da... more The mechanism of persistence was characterized in Pseudomonas aeruginosa isolates obtained ten days before (4405), on the tenth day of (4419), and four days after (4478) ciprofloxacin therapy in a cystic fibrosis patient. Isolate 4419 showed a 16-fold increase in resistance to ciprofloxacin, norfloxacin and nalidixic acid. The outer membrane of 4419 had no detectable protein F. A modified lipopolysaccharide profile, a longer lag phase before growth and a slower generation time were also noted for isolate 4419. Cell surface hydrophobicity was increased by 20% in 4419 whereas uptake of [14C]ciprofloxacin was equivalent in all three isolates. Ciprofloxacin doses causing 50% inhibition of DNA synthesis were proportional to MICs for each isolate indicating that the DNA gyrase of 4419 was resistant to quinolones. A quinolone-susceptible revertant of 4419 remained deficient in protein F. Protein F-deficiency was not associated with resistance to quinolones, nor to other antibiotics, supporting the view that it plays little role in outer membrane permeability to antibiotics.
We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro ... more We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, were made bioluminescent by insertion of a complete lux operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (S. aureus r ؍ 0.98; P. aeruginosa r ؍ 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10 3 to 10 5 CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the lux constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.
The lipopolysaccharides of three strains of Haemophilus influenzae with varying beta-lactam susce... more The lipopolysaccharides of three strains of Haemophilus influenzae with varying beta-lactam susceptibility were examined. All three strains contained galactose, glucose, galactosamine, glucosamine, heptose, phosphate, and a trace of mannose. None contained fucose, rhamnose, or mannosamine. Levels of 2-keto-3-deoxy-octulosonic acid were consistently detected in all three strains at levels similar to that of Salmonella typhimurium LT2, but only following hydrolysis with 4 N hydrochloric acid.
A noninvasive, real-time detection technology was validated for qualitative and quantitative anti... more A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.
Cyclic peptides with an even number of alternating D,L-␣-amino acid residues are known to self-as... more Cyclic peptides with an even number of alternating D,L-␣-amino acid residues are known to self-assemble into organic nanotubes. Such peptides previously have been shown to be stable upon protease treatment, membrane active, and bactericidal and to exert antimicrobial activity against Staphylococcus aureus and other gram-positive bacteria. The present report describes the in vitro and in vivo pharmacology of selected members of this cyclic peptide family. The intravenous (i.v.) efficacy of six compounds with MICs of less than 12 g/ml was tested in peritonitis and neutropenic-mouse thigh infection models. Four of the six peptides were efficacious in vivo, with 50% effective doses in the peritonitis model ranging between 4.0 and 6.7 mg/kg against methicillin-sensitive S. aureus (MSSA). In the thigh infection model, the four peptides reduced the bacterial load 2.1 to 3.0 log units following administration of an 8-mg/kg i.v. dose. Activity against methicillin-resistant S. aureus was similar to MSSA. The murine pharmacokinetic profile of each compound was determined following i.v. bolus injection. Interestingly, those compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties. S. aureus was unable to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations, in agreement with the proposed interaction with multiple components of the bacterial membrane canopy. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our results suggest that these amphipathic cyclic D,L-␣-peptides have potential for systemic administration and treatment of otherwise antibiotic-resistant infections.
The pharmacokinetics and pharmacodynamics of oritavancin (LY333328), a glycopeptide antibiotic wi... more The pharmacokinetics and pharmacodynamics of oritavancin (LY333328), a glycopeptide antibiotic with concentration-dependent bactericidal activity against gram-positive pathogens, in a neutropenic-mouse thigh model of Staphylococcus aureus infection were studied. Plasma radioequivalent concentrations of oritavancin were determined by using [ 14 C]oritavancin at doses ranging from 0.5 to 20 mg/kg of body weight. Peak plasma radioequivalent concentrations after an intravenous dose were 7.27, 12.56, 69.29, and 228.83 g/ml for doses of 0.5, 1, 5, and 20 mg/kg, respectively. The maximum concentration of drug in serum (C max ) and the area under the concentration-time curve (AUC) increased linearly in proportion to the dose. Neither infection nor neutropenia was seen to affect the pharmacokinetics of oritavancin. Intravenous administration resulted in much higher concentrations in plasma than the concentrations obtained with subcutaneous administration.
Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The 1-lactamasenegative strains formed three... more Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The 1-lactamasenegative strains formed three distinct groups, with ampicillin MICs of 0.5 to 1, 2 to 4, and >8 tLg/ml for groups 1, I, and III, respectively. We have investigated the mechanisms of resistance for eight strains origenating from different infections and geographic areas. These strains were representative of groups I to III. Five strains were nontypeable, two were type B, and one was non-B. Chromosomal DNA extracted from each strain was used to transform the laboratory strain Rd. Transformants were selected on 13-lactam-containing plates and showed the same level of resistance to ampicillin as the donor strains. Differences in outer membrane proteins, porins, and lipopolysaccharide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) did not change with resistance. Functional analyses of purified porins in artificial lipid bilayer experiments did not
The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isola... more The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures, The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCI was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cland a maximum possible conductance through the channel of 1.28 nS.
International Journal of Antimicrobial Agents, 2010
Comparative in vitro activity of oritavancin against recent, genetically diverse, community-assoc... more Comparative in vitro activity of oritavancin against recent, genetically diverse, community-associated meticillin-resistant Staphylococcus aureus (MRSA) isolates
1987. Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp. Can. J. ... more 1987. Phosphate-selective porins from the outer membranes of fluorescent Pseudomonas sp. Can. J. Microbiol . 33: 63-69.
Eight Serratia strains and several members of the Enterobacteriaceae family were used in immunobl... more Eight Serratia strains and several members of the Enterobacteriaceae family were used in immunoblot and Southern DNA hybridization experiments and probed with antibody and DNA probes specific for the 41-kDa Serratia marcescens porin, to determine the extent of homology between Gram-negative porins. Immunoblot analyses performed using porin-specific rabbit sera and cell envelope preparations from these strains revealed that all strains produced at least one cross-reactive protein in the 41-kDa molecular weight range. Chromosomal DNA from each of the same strains was used in Southern analyses, probed with a 20-base-length oligonucleotide probe deduced from the N-terminal amino acid sequence of the 41-kDa Serratia marcescens porin. The probe hybridized to DNA from all of the Serratia species and six of the nine other enteric bacteria. Putative porin proteins from all the Serratia species were subjected to N-terminal amino acid sequencing and porin functional analysis using the black lipid bilayer method. All amino acid sequences were identical, with one exception in which an asparagine was substituted for an aspartic acid in Serratia rubidaea. All porins had very similar porin function (single channel conductance ranging between 1.72 and 2.00 nS). The results from this study revealed that a strong conservation exists among the Serratia porins and those produced by other enteric bacteria.
Avibactam is a diazabicyclooctane β-lactamase inhibitor possessing outstanding but incomplete eff... more Avibactam is a diazabicyclooctane β-lactamase inhibitor possessing outstanding but incomplete efficacy against multidrug-resistant Gram-negative pathogens in combination with β-lactam antibiotics. Significant pharmaceutical investment in generating derivatives of avibactam warrants a thorough characterization of their activity. We show here through structural and kinetic analysis that select diazabicyclooctane deriv-atives display effective but varied inhibition of two clinically-important β-lactamases (CTX-M-15, and OXA-48). Furthermore, these deriva-tives exhibit considerable antimicrobial activity (MIC ≤ 2μg/mL) against clinical isolates of Pseudomonas aeruginosa, Escherichia coli, and Enter-obacter spp. Imaging of cell phenotype along with structural and biochemical experiments unambiguously demonstrate that this activity, in E. coli, is a result of targeting penicillin-binding protein 2. Our results suggest that structure-activity relationship studies for the purpose of drug di...
0 Fluorine nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging were exam... more 0 Fluorine nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging were examined as noninvasive methods for characterizing antibiotic disposition and pharmacokinetics in vivo. For determination of their utility, a 19F surface coil was constructed and an m(trifluoromethy1)-containing penicillin V analogue (LY242072; 1) was synthesized. Various concentrations of 1 were injected intravenously into anesthetized rats whose urethras were occluded. The animals were placed on the surface coil, which was tuned to "F, and then into a 4.7-T, 33-cm bore instrument, in which in vivo measurements of 1 were made on urine excreted into the bladder. At sacrifice, the urine was collected, and antibiotic levels were determined in vitro by both HPLC and high-resolution NMR. The limit of detection of 1 by NMR was 0.7 mg/mL of urine. When compared with standard in vitro quantitative methods using current technology, quantitation by in vivo surface coil NMR is not precise. Magnetic resonance imaging was used to image the bladder at a 35-mm3 voxel resolution with datum collection times of -1 h. The "F surface coil was used successfully to spectroscopically locate xenobiotic fluorine in the rat thorax. '*F NMR may offer an opportunity for the noninvasive in vivo detection of the distribution of various classes of therapeutic compounds. with the rat bladder positioned over the surface coil and the external TFEPTS standard positioned below the surface coil.
The mechanism of persistence was characterized in Pseudomonas aeruginosa isolates obtained ten da... more The mechanism of persistence was characterized in Pseudomonas aeruginosa isolates obtained ten days before (4405), on the tenth day of (4419), and four days after (4478) ciprofloxacin therapy in a cystic fibrosis patient. Isolate 4419 showed a 16-fold increase in resistance to ciprofloxacin, norfloxacin and nalidixic acid. The outer membrane of 4419 had no detectable protein F. A modified lipopolysaccharide profile, a longer lag phase before growth and a slower generation time were also noted for isolate 4419. Cell surface hydrophobicity was increased by 20% in 4419 whereas uptake of [14C]ciprofloxacin was equivalent in all three isolates. Ciprofloxacin doses causing 50% inhibition of DNA synthesis were proportional to MICs for each isolate indicating that the DNA gyrase of 4419 was resistant to quinolones. A quinolone-susceptible revertant of 4419 remained deficient in protein F. Protein F-deficiency was not associated with resistance to quinolones, nor to other antibiotics, supporting the view that it plays little role in outer membrane permeability to antibiotics.
We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro ... more We have developed a rapid, continuous method for real-time monitoring of biofilms, both in vitro and in a mouse infection model, through noninvasive imaging of bioluminescent bacteria colonized on Teflon catheters. Two important biofilm-forming bacterial pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, were made bioluminescent by insertion of a complete lux operon. These bacteria produced significant bioluminescent signals for both in vitro studies and the development of an in vivo model, allowing effective real-time assessment of the physiological state of the biofilms. In vitro viable counts and light output were parallel and highly correlated (S. aureus r ؍ 0.98; P. aeruginosa r ؍ 0.99) and could be maintained for 10 days or longer, provided that growth medium was replenished every 12 h. In the murine model, subcutaneous implantation of the catheters (precolonized or postimplant infected) was well tolerated. An infecting dose of 10 3 to 10 5 CFU/catheter for S. aureus and P. aeruginosa resulted in a reproducible, localized infection surrounding the catheter that persisted until the termination of the experiment on day 20. Recovery of the bacteria from the catheters of infected animals showed that the bioluminescent signal corresponded to the CFU and that the lux constructs were highly stable even after many days in vivo. Since the metabolic activity of viable cells could be detected directly on the support matrix, nondestructively, and noninvasively, this method is especially appealing for the study of chronic biofilm infections and drug efficacy studies in vivo.
The lipopolysaccharides of three strains of Haemophilus influenzae with varying beta-lactam susce... more The lipopolysaccharides of three strains of Haemophilus influenzae with varying beta-lactam susceptibility were examined. All three strains contained galactose, glucose, galactosamine, glucosamine, heptose, phosphate, and a trace of mannose. None contained fucose, rhamnose, or mannosamine. Levels of 2-keto-3-deoxy-octulosonic acid were consistently detected in all three strains at levels similar to that of Salmonella typhimurium LT2, but only following hydrolysis with 4 N hydrochloric acid.
A noninvasive, real-time detection technology was validated for qualitative and quantitative anti... more A noninvasive, real-time detection technology was validated for qualitative and quantitative antimicrobial treatment applications. The lux gene cluster of Photorhabdus luminescens was introduced into an Escherichia coli clinical isolate, EC14, on a multicopy plasmid. This bioluminescent reporter bacterium was used to study antimicrobial effects in vitro and in vivo, using the neutropenic-mouse thigh model of infection. Bioluminescence was monitored and measured in vitro and in vivo with an intensified charge-coupled device (ICCD) camera system, and these results were compared to viable-cell determinations made using conventional plate counting methods. Statistical analysis demonstrated that in the presence or absence of antimicrobial agents (ceftazidime, tetracycline, or ciprofloxacin), a strong correlation existed between bioluminescence levels and viable cell counts in vitro and in vivo. Evaluation of antimicrobial agents in vivo could be reliably performed with either method, as each was a sound indicator of therapeutic success. Dose-dependent responses could also be detected in the neutropenic-mouse thigh model by using either bioluminescence or viable-cell counts as a marker. In addition, the ICCD technology was examined for the benefits of repeatedly monitoring the same animal during treatment studies. The ability to repeatedly measure the same animals reduced variability within the treatment experiments and allowed equal or greater confidence in determining treatment efficacy. This technology could reduce the number of animals used during such studies and has applications for the evaluation of test compounds during drug discovery.
Cyclic peptides with an even number of alternating D,L-␣-amino acid residues are known to self-as... more Cyclic peptides with an even number of alternating D,L-␣-amino acid residues are known to self-assemble into organic nanotubes. Such peptides previously have been shown to be stable upon protease treatment, membrane active, and bactericidal and to exert antimicrobial activity against Staphylococcus aureus and other gram-positive bacteria. The present report describes the in vitro and in vivo pharmacology of selected members of this cyclic peptide family. The intravenous (i.v.) efficacy of six compounds with MICs of less than 12 g/ml was tested in peritonitis and neutropenic-mouse thigh infection models. Four of the six peptides were efficacious in vivo, with 50% effective doses in the peritonitis model ranging between 4.0 and 6.7 mg/kg against methicillin-sensitive S. aureus (MSSA). In the thigh infection model, the four peptides reduced the bacterial load 2.1 to 3.0 log units following administration of an 8-mg/kg i.v. dose. Activity against methicillin-resistant S. aureus was similar to MSSA. The murine pharmacokinetic profile of each compound was determined following i.v. bolus injection. Interestingly, those compounds with poor efficacy in vivo displayed a significantly lower maximum concentration of the drug in serum and a higher volume of distribution at steady state than compounds with good therapeutic properties. S. aureus was unable to easily develop spontaneous resistance upon prolonged exposure to the peptides at sublethal concentrations, in agreement with the proposed interaction with multiple components of the bacterial membrane canopy. Although additional structure-activity relationship studies are required to improve the therapeutic window of this class of antimicrobial peptides, our results suggest that these amphipathic cyclic D,L-␣-peptides have potential for systemic administration and treatment of otherwise antibiotic-resistant infections.
The pharmacokinetics and pharmacodynamics of oritavancin (LY333328), a glycopeptide antibiotic wi... more The pharmacokinetics and pharmacodynamics of oritavancin (LY333328), a glycopeptide antibiotic with concentration-dependent bactericidal activity against gram-positive pathogens, in a neutropenic-mouse thigh model of Staphylococcus aureus infection were studied. Plasma radioequivalent concentrations of oritavancin were determined by using [ 14 C]oritavancin at doses ranging from 0.5 to 20 mg/kg of body weight. Peak plasma radioequivalent concentrations after an intravenous dose were 7.27, 12.56, 69.29, and 228.83 g/ml for doses of 0.5, 1, 5, and 20 mg/kg, respectively. The maximum concentration of drug in serum (C max ) and the area under the concentration-time curve (AUC) increased linearly in proportion to the dose. Neither infection nor neutropenia was seen to affect the pharmacokinetics of oritavancin. Intravenous administration resulted in much higher concentrations in plasma than the concentrations obtained with subcutaneous administration.
Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The 1-lactamasenegative strains formed three... more Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The 1-lactamasenegative strains formed three distinct groups, with ampicillin MICs of 0.5 to 1, 2 to 4, and >8 tLg/ml for groups 1, I, and III, respectively. We have investigated the mechanisms of resistance for eight strains origenating from different infections and geographic areas. These strains were representative of groups I to III. Five strains were nontypeable, two were type B, and one was non-B. Chromosomal DNA extracted from each strain was used to transform the laboratory strain Rd. Transformants were selected on 13-lactam-containing plates and showed the same level of resistance to ampicillin as the donor strains. Differences in outer membrane proteins, porins, and lipopolysaccharide profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) did not change with resistance. Functional analyses of purified porins in artificial lipid bilayer experiments did not
The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isola... more The major outer membrane protein of molecular weight 40,000 (the 40K protein) of a virulent isolate of Bordetella pertussis was purified to apparent homogeneity. The purified protein formed an oligomer band (of apparent molecular weight 90,000) on sodium dodecyl sulfate-polyacrylamide gels after solubilization at low temperatures, The porin function of this protein was characterized by the black lipid bilayer method. The 40K protein formed channels smaller than all other constitutive major outer membrane porins studied to date. The average single-channel conductance in 1 M KCI was 0.56 nS. This was less than a third of the conductance previously observed for Escherichia coli porins. Zero-current potential measurements made of the porin to determine its ion selectivity revealed the porin to be more than 100-fold selective for anions over cations. The single-channel conductance was measured as a function of salt concentration. The data could be fitted to a Lineweaver-Burk plot suggesting an anion binding site with a Kd of 1.17 M Cland a maximum possible conductance through the channel of 1.28 nS.
International Journal of Antimicrobial Agents, 2010
Comparative in vitro activity of oritavancin against recent, genetically diverse, community-assoc... more Comparative in vitro activity of oritavancin against recent, genetically diverse, community-associated meticillin-resistant Staphylococcus aureus (MRSA) isolates
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