Efficacy of oritavancin in a murine model of Bacillus anthracis spore inhalation anthrax.

The inhaled form of Bacillus anthracis infection may be fatal to humans. The current standard of care for inhalational anthrax postexposure prophylaxis is ciprofloxacin therapy twice daily for 60 days. The potent in vitro activity of oritavancin, a semisynthetic lipoglycopeptide, against B. anthracis (MIC against Ames strain, 0.015 microg/ml) prompted us to test its efficacy in a mouse aerosol-anthrax model. In postexposure prophylaxis dose-ranging studies, a single intravenous (i.v.) dose of oritavancin of 5, 15, or 50 mg/kg 24 h after a challenge with 50 to 75 times the median lethal dose of Ames strain spores provided 40, 70, and 100% proportional survival, respectively, at 30 days postchallenge. Untreated animals died within 4 days of challenge, whereas 90% of control animals receiving ciprofloxacin at 30 mg/kg intraperitoneally twice daily for 14 days starting 24 h after challenge survived. Oritavancin demonstrated significant activity post symptom development; a single i.v. dose of 50 mg/kg administered 42 h after challenge provided 56% proportional survival at 30 days. In a preexposure prophylaxis study, a single i.v. oritavancin dose of 50 mg/kg administered 1, 7, 14, or 28 days before lethal challenge protected 90, 100, 100, and 20% of mice at 30 days; mice treated with ciprofloxacin 24 h or 24 and 12 h before challenge all died within 5 days. Efficacy in pre- and postexposure models of inhalation anthrax, together with a demonstrated low propensity to engender resistance, promotes further study of oritavancin pharmacokinetics and efficacy in nonhuman primate models.

Direct continuous method for monitoring biofilm infection in a mouse model.

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.

Validation of a noninvasive, real-time imaging technology using bioluminescent Escherichia coli in the neutropenic mouse thigh model of infection.

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.

Discovery of MC-02,331, a new cephalosporin exhibiting potent activity against methicillin-resistant Staphylococcus aureus.

A systematic approach toward building activity against methicillin-resistant staphylococci into the cephalosporin class of beta-lactam antibiotics is described. Initial work focused on finding the optimal linkage between the cephem nucleus and a biphenyl pharmacophore, which established that a thio linkage afforded potent activity in vitro. Efforts to optimize this activity by altering substitution on the pharmacophore afforded iodophenylthio analog MC-02,002, which although highly potent against MRSA, was also highly bound to serum proteins. Further work to decrease serum protein binding showed that replacement of the iodo substituent by the positively-charged isothiouronium group afforded potent activity and reduced serum binding, but insufficient aqueous solubility. Solubility was enhanced by incorporation of a second positively-charged group into the 7-acyl substituent. Such derivatives (MC-02,171 and MC-02,306) lacked sufficient stability to staphylococcal beta-lactamase enzymes. The second positive charge was incorporated into the cephem 3-substituent in order to utilize the beta-lactamase-stable aminothiazolyl(oximino)acetyl class of 7-substituents. These efforts culminated with the discovery of bis(isothiouroniummethyl)phenylthio analog MC-02,331, whose profile is acceptable with respect to potency against MRSA, serum binding, aqueous solubility, and beta-lactamase stability.

Characterization of a rabbit model of staphylococcal osteomyelitis.

We previously described a rabbit osteomyelitis model that involved the direct introduction of Staphylococcus aureus into devascularized bone. To further evaluate the model, we performed experiments aimed at correlating the microbiological, radiographic, and histologic parameters involved in the development of experimental osteomyelitis. Using the strain UAMS-1, we achieved an infection rate of 75% with an inoculum as small as 2 x 10(3) colony-forming units. However, development of significant radiographic and histologic signs of disease required an inoculum of at least 2 x 10(4) colony-forming units. Radiographic signs were minimal 1 week after infection and progressed steadily to a maximum 3 weeks after infection. In contrast, histologic signs of disease were observed within 1 week and remained essentially unchanged throughout the 4-week evaluation period. Unlike the results obtained with UAMS-1, rabbits infected with the heavily encapsulated Staphylococcus aureus strain Smith diffuse exhibited little evidence of disease even when infected with 2 x 10(6) colony-forming units. The reduced virulence of strain Smith diffuse was surprising given its greatly enhanced virulence (relative to UAMS-1) in a murine peritonitis model of staphylococcal disease. These results suggest that UAMS-1 expresses virulence factors that are important in the pathogenesis of osteomyelitis and that some or all of these virulence factors are either absent or are not expressed in strain Smith diffuse. Most importantly, the results suggest that our model may be appropriate for the identification and characterization of these virulence factors.

Localization of penicillin-binding proteins to the splitting system of Staphylococcus aureus septa by using a mercury-penicillin V derivative.

Precise localization of penicillin-binding protein (PBP)-antibiotic complexes in a methicillin-sensitive Staphylococcus aureus strain (BB255), its isogenic heterogeneous methicillin-resistant transductant (BB270), and a homogeneous methicillin-resistant strain (Col) was investigated by high-resolution electron microscopy. A mercury-penicillin V (Hg-pen V) derivative was used as a heavy metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells during growth. The most striking feature of thin sections was the presence of an abnormally large (17 to 24 nm in width) splitting system within the thick cross walls or septa of Hg-pen V-treated bacteria of all strains. Untreated control cells possessed a thin, condensed splitting system, 7 to 9 nm in width. A thick splitting system was also distinguishable in unstained thin sections, thereby confirming that the electron contrast of this structure was not attributed to binding of bulky heavy metal stains usually used for electron microscopy. Biochemical analyses demonstrated that Hg-pen V bound to isolated plasma membranes as well as sodium dodecyl sulfate-treated cell walls and that two or more PBPs in each strain bound to this antibiotic. In contrast, the splitting system in penicillin V-treated bacteria was rarely visible after 30 min in the presence of antibiotic. These findings suggest that while most PBPs were associated with the plasma membrane, a proportion of PBPs were located within the fabric of the cell wall, in particular, in the splitting system. Inhibition of one or more high-M(r) PBPs by beta-lactam antibiotics modified the splitting system and cross-wall structure, therefore supporting a role for these PBPs in the synthesis and architectural design of these structures in S. aureus.

Nucleotide sequence of the region between crr and cysM in Salmonella typhimurium: five novel ORFs including one encoding a putative transcriptional regulator of the phosphotransferase system.

A 4471 bp region between crr and cysM on the Salmonella typhimurium chromosome (49.5 min) has been sequenced. Five ORFs were found within this region, one of which is likely to be the putative regulatory gene, ptsJ, that corresponds in map position to a gene which when mutated allows expression of a cryptic Enzyme I of the phosphotransferase system. The deduced amino acid sequence of the encoded protein is similar to those of several open reading frames (ORFs) including ORFT2 of Rhodobacter spheroides with which it is 28% identical throughout most of its length (comparison score of 21 S.D.). PtsJ exhibits a putative, N-terminal, helix-turn-helix, DNA binding domain that is similar in sequence to those in members of the GntR family of transcriptional regulators. Analyses of the sequences of the ORFs encoded within this region are presented.

Characterization and cilofungin inhibition of solubilized Aspergillus fumigatus (1,3)-beta-D-glucan synthase.

(1,3)-beta-D-Glucan synthase, a major cell wall synthesis enzyme, is the target of antifungal drugs of the lipopeptide class. Aspergillus fumigatus (1,3)-beta-D-glucan synthase was prepared and its activity was measured by incorporation of [14C]glucose from UDP-[U-14C]glucose into an insoluble polymer in the presence of alpha-amylase. Solubilization of the (1,3)-beta-D-glucan synthase was attempted with several detergents, and the maximum percent solubilization was obtained with a polyoxyethylene ether detergent, W-1. Up to 70% of enzyme activity and 50% of total protein were recovered when 1-mg/ml membrane preparations were extracted with 0.045% W-1 at 4 degrees C overnight. Confirmation of the presence of a (1,3)-beta-D-glucose polymer synthesized by this glucan synthase was done by three methods. The first was enzymatic end product degradation by alpha-amylase (no degradation) and beta-glucanase (85 to 95% degradation). The second was gas chromatography-mass spectroscopy analysis of the partially methylated alditol acetate derivatives prepared from total carbohydrate polymers present in the sample. This method identified the presence of (1,3)- and (1,2)-glucosidic linkages. The third was high-performance anion exchange chromatography of radioactive oligosaccharides. This method allowed differentiation of the newly synthesized, radioactive polymers from the contaminating carbohydrates already present in the preparation. The results showed that the polymer synthesized comprised oligosaccharides consistent with beta-(1,3)-linked sugars. Maximal inhibition of the (1,3)-beta-D-glucan synthase by the lipopeptide antifungal agent cilofungin was 80%. Dose-response experiments with this inhibitor showed that the solubilized enzyme was maximally inhibited at a cilofungin concentration of 1.25 microgram/ml and showed <5% inhibition at 0.02 microgram/ml. The apparent K(m) (K(m app)) for the solubilized glucan synthase was 400 +/- 80 microM, and the apparent K(i) (K(i app)) for cilofungin was 0.19 +/- 0.03 microM. Inhibition of A.fumigatus (1,3)-beta-D-glucan synthase with cilofungin was noncompetitive, as it was for the Candida albicans (1,3)-beta-D-glucan synthase.

Comparative analyses of Serratia spp. outer membrane porin proteins.

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.

Correlation of cilofungin in vivo efficacy with its activity against Aspergillus fumigatus (1,3)-beta-D-glucan synthase.

(1,3)-beta-D-Glucan synthase is a cell wall synthesis enzyme that is the target of cilofungin, an antifungal agent of the lipopeptide class. Cilofungin's glucan synthase inhibitory activity, MIC, and effective dose 50% in a systemic infection mouse model tend to correlate for Candida albicans. This correlation is not seen in Aspergillus fumigatus. MICs for cilofungin against A. fumigatus were consistently > 125 micrograms/ml while the effective dose 50% in a systemic aspergillosis model was determined to be 20.6 mg/kg. To begin to understand this discrepancy, we examined the A. fumigatus glucan synthase. This cell wall enzyme was prepared and its activity was measured by [14C]-glucose incorporation from UDP-[U-14C]glucose into an acid insoluble polymer formed in the presence of alpha-amylase. Enzyme activity in crude membrane preparations was measured in the presence of several antifungal agents. Enzyme inhibition results showed that 1 microgram/ml of papulacandin B, echinochandin B, aculeacin A and cilofungin all inhibited A. fumigatus glucan synthase activity (40-71%) while 1 microgram/ml of amphotericin B, fluconazole, ketoconazole and nikkomycin did not affect enzyme activity. A correlation was therefore established between the inhibitory effect of cilofungin on the A. fumigatus glucan synthase and the effective dose 50% obtained in a systemic aspergillosis mouse model.

Epidemiological typing of Moraxella catarrhalis by using DNA probes.

Small-fragment restriction enzyme analysis and DNA-DNA hybridization were used to compare 60 strains of Moraxella catarrhalis isolated from various geographic locations. Restriction enzyme analysis with HaeIII resulted in 46 different patterns, 7 of which were shared by more than one isolate. Hybridizations with two DNA probes resulted in 18 different patterns, 11 of which were shared by more than one isolate. Strains with the same restriction enzyme pattern always had the same hybridization pattern. However, of the 50 strains that shared the 11 hybridization patterns, 39 could be further differentiated by restriction enzyme analysis. We found that hybridization is a method that is specific for the epidemiological typing of M. catarrhalis, but because of limited sensitivity, combination with small-fragment restriction enzyme analysis may be necessary to better determine the relatedness of strains.

Comparison of the in vivo and in vitro nuclear magnetic resonance detection of trifluoromethyl penicillin V in rats.

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-(trifluoromethyl)-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 19F, 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 approximately 1 h. The 19F surface coil was used successfully to spectroscopically locate xenobiotic fluorine in the rat thorax. 19F NMR may offer an opportunity for the noninvasive in vivo detection of the distribution of various classes of therapeutic compounds.

Modes of action and inhibitory activities of new siderophore-beta-lactam conjugates that use specific iron uptake pathways for entry into bacteria.

We describe here the mechanism of inhibition of two new siderophore-beta-lactam conjugates against Escherichia coli X580. One conjugate is a spermidine-based catechol siderophore-carbacephalosporin (JAM-2-263), and the other is an N5-acetyl-N5-hydroxy-L-ornithine tripeptide hydroxamate siderophore-carbacephalosporin (EKD-3-88). In an agar diffusion test, both conjugates produced large inhibitory zones against strain X580. Resistant strains (i.e., JAMR and EKDR) could be isolated after exposure of X580 to the conjugates JAM-2-263 and EKD-3-88, respectively. No cross-resistance was observed in these individual isolates. JAMR and EKDR were studied further to elucidate the mechanism of inhibition of each conjugated drug. The affinities of JAM-2-263 and EKD-3-88 for penicillin-binding proteins (PBPs) of isolated inner membranes were determined by a competition assay with 125I-penicillin V. JAM-2-263 targeted primarily PBPs 1A/B and 5/6, while EKD-3-88 targeted PBPs 1A/B and 3. Strains X580, JAMR, and EKDR showed similar PBP affinities for the conjugates. However, marked changes were observed in the iron-regulated outer membrane proteins of resistant isolates grown on agar plates depleted of iron. EKDR lost the expression of FhuA (78 kDa) and its sensitivity to phages T1 and T5, whereas JAMR lost the expression of Cir (74 kDa) and its sensitivity to colicin Ia. These results revealed the requirement of FhuA and Cir for the inhibitory activities of EKD-3-88 and JAM-2-263, respectively. In an antibiotic diffusion assay, ferrichrome (1 microM) strongly antagonized the activities of both conjugates against X580 and JAMR, including the residual activity of JAM-2-263 against JAMR. However, the susceptibility of strain EKDR lacking the ferrichrome receptor (FhuA-) to the two conjugates remained the same in the presence of ferrichrome. The antagonistic effect of ferrichrome on the activity of JAM-2-263 may also indicate a role for FhuA in the activity of this beta-lactam conjugate. A FhuA- Cir- double mutant confirmed this hypothesis, since it showed a higher level of resistance to JAM-2-263. To reproduce iron-restricted in vivo growth conditions, we grew X580 and EKDR cells in diffusion chambers implanted in the peritoneal cavities of rats. Strain EKDR showed impaired growth in such a cultivation system. This is the first report of beta-lactam drug transport into E. coli cells that involves the FhuA outer membrane protein.

Molecular basis of the non-beta-lactamase-mediated resistance to beta-lactam antibiotics in strains of Haemophilus influenzae isolated in Canada.

A study recently conducted across Canada showed that 64 of 2,503 clinical isolates of Haemophilus influenzae were resistant to beta-lactams without production of a beta-lactamase (L. D. Tremblay, J. L'Ecuyer, P. Provencher, M. G. Bergeron, and Canadian Study Group, Can. Med. Assoc. J. 143:895-900, 1990). The beta-lactamase-negative strains formed three distinct groups, with ampicillin MICs of 0.5 to 1, 2 to 4, and greater than or equal to 8 micrograms/ml for groups I, II, and III, respectively. We have investigated the mechanisms of resistance for eight strains originating 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 beta-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 explain resistance. Peptidoglycan synthesis was measured by incorporation of [14C]alanine into trichloroacetic acid-insoluble cell wall material in the presence of chloramphenicol. The growth rate and the rate of peptidoglycan synthesis observed for the transformants of the isogenic set did not correlate with resistance. Whole-cell labeling with 125I-penicillin revealed modifications in penicillin-binding proteins (PBPs) among the transformants. In particular, PBPs 3A and 3B (65 and 63 kDa, respectively) showed a decrease in affinity for beta-lactams in all transformants (groups I, II, and III) and correlated with an increased MIC except in the transformant of group III, which showed higher levels of resistance. Partial purification and proteolytic digestion of 125I-penicillin-labeled PBP 3B led to two types of CnBr peptide profiles on SDS-PAGE, the profiles of the transformed strains from groups I and II being different from those of the control group and group III. Finally, electron microscopy revealed a distinct cell filamentation for the group III transformants. These data clearly indicate that changes in PBPs are a common mechanism that results in a significant level of non-beta-lactamase-mediated beta-lactam resistance in H. influenzae despite serotype, origin of isolation, or geographic distribution.

A new mercury-penicillin V derivative as a probe for ultrastructural localization of penicillin-binding proteins in Escherichia coli.

The precise ultrastructural localization of penicillin-binding protein (PBP)-antibiotic complexes in Escherichia coli JM101, JM101 (pBS96), and JM101(pPH116) was investigated by high-resolution electron microscopy. We used mercury-penicillin V (Hg-pen V) as a heavy-metal-labeled, electron-dense probe for accurately localizing PBPs in situ in single bacterial cells grown to exponential growth phase. Biochemical data derived from susceptibility tests and bacteriolysis experiments revealed no significant differences between Hg-pen V and the parent compound, penicillin V, or between strains. Both antibiotics revealed differences in the binding affinities for PBPs of all strains. Deacylation rates for PBPs were slow despite the relatively low binding affinities of antibiotics. Cells bound most of the Hg-pen V added to cultures, and the antibiotic-PBP complex could readily be seen by electron microscopy of unstained whole mounts as distinct, randomly situated electron-dense particles. Fifty to 60% of the antibiotic was retained by cells during processing for conventional embedding so that thin sections could also be examined. These revealed similar electron-dense particles located predominantly on the plasma membrane and less frequently in the cytoplasm. Particles positioned on the plasma membranes were occasionally shown to protrude into the periplasmic space, thereby reflecting the high resolution of the Hg-pen V probe. Moreover, some particles were observed free in the periplasm, suggesting, for the first time, that a proportion of PBPs may not be restricted to the plasma membrane but may be tightly associated with the peptidoglycan for higher efficiency of peptidoglycan assembly. All controls were devoid of the electron-dense particles. The presence of electron-dense particles in cells of the wild-type JM101, demonstrated that our probe could identify PBPs in naturally occurring strains without inducing PBP overproduction.

The bacterial phosphotransferase system as a potential vehicle for the entry of novel antibiotics.

During the past twenty-nine years, not a single class of antimicrobial agents has been discovered that has led to new approved human drugs. Despite a dramatic increase in the potency of existing classes, the need for new effective antimicrobial agents continues. The bacterial phosphotransferase system (PTS) offers the possibility of providing new opportunities for the discovery of important agents. This system offers a vehicle for entry into infecting bacteria and pathways for the initiation of metabolism of such agents. Antimicrobial agents which would use the PTS may be found which are active on both growing and sessile bacterial forms, and due to the lack of a eukaryotic PTS counterpart, such analogues may be expected to be non-toxic to the animal host.

Influence of growth media on Escherichia coli cell composition and ceftazidime susceptibility.

Cell composition and surface properties of Escherichia coli were modified by using various growth media to investigate the role of yet uncharacterized components in ceftazidime susceptibility. An eightfold dilution of Luria broth was used as the basic growth medium and was supplemented with up to 4% phosphate, 5% glucose, or 12% L-glutamate. Decreases in cephaloridine and ceftazidime susceptibility, of two- and eightfold, respectively, were observed only in the glucose-enriched medium. The outer membrane permeability to ceftazidime and cephaloridine was evaluated by crypticity indices. Indices were unchanged under all growth conditions. Fluorometry of whole cells with 1-N-phenylnaphthylamine showed that glucose does not affect the interaction of this hydrophobic probe with the membranes but showed that elevated concentrations of phosphate or glutamate cause a marked increase in cell hydrophobicity, which, in turn, correlates with an increase in the susceptibility of E. coli to nalidixic acid. Growth in phosphate- or glutamate-enriched media caused an augmentation in major phospholipid species and may explain the increased hydrophobicity and susceptibility of E. coli to nalidixic acid. These data showed that E. coli susceptibility to ceftazidime is not influenced by cell surface hydrophobicity and suggested that the contribution of a nonspecific lipophilic diffusion route for entry of ceftazidime into cells is not likely to occur or is distinct from that of more hydrophobic molecules such as nalidixic acid. Finally, the penicillin-binding proteins of the E. coli cells were also investigated. Penicillin-binding protein 8 was only markedly labeled with 125I-penicillin V in inner membranes extracted from cells grown with glucose. Results of this study suggest that the unexpected change in penicillin-binding protein 8 observed in the presence of glucose may be responsible for the increase in MICs of cephaloridine and ceftazidime.

W-1 solubilization and kinetics of inhibition by cilofungin of Candida albicans (1,3)-beta-D-glucan synthase.

(1,3)-beta-D-Glucan synthase of Candida albicans was rendered soluble by treatment of membrane preparations with the polyoxyethylene ether detergent W-1. Extraction with 0.025% W-1 at 4 degrees C for 24 h effectively solubilized and activated the enzyme. Under these conditions, greater than 85% of the protein in membrane preparations was released, and about 64% of the glucan synthase activity could be recovered in the soluble form. Soluble enzyme activity was stable for more than 12 days at 4 degrees C. Also, glucan synthase activity in the extracted membrane preparations could be activated to achieve more than twice the enzyme activity in the original, unextracted membrane preparations. The soluble glucan synthase had characteristics similar to those of the membrane-bound enzyme. Soluble glucan synthase had an apparent Km of 2.0 mM, and particulate glucan synthase had an apparent Km of 2.5 mM. Kinetics of cilofungin inhibition for both enzyme preparations were noncompetitive, with an apparent Ki of 2.5 microM; both preparations could be inhibited by cilofungin but not by its peptide nucleus or side chain, either alone or in combination. The reaction products from both forms of the enzyme were sensitive to (1,3)-beta-D-glucanase degradation but not to alpha-amylase, alpha-glucosidase, or proteinase K degradation and thus were shown to be beta(1----3) glucan.

Persistence of Pseudomonas aeruginosa during ciprofloxacin therapy of a cystic fibrosis patient: transient resistance to quinolones and protein F-deficiency.

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.

Outer membrane and porin characteristics of Serratia marcescens grown in vitro and in rat intraperitoneal diffusion chambers.

The composition and antibiotic permeability barrier of the outer membrane of Serratia marcescens were assessed in cells grown in vivo and in vitro. Intraperitoneal diffusion chambers implanted in rats were used for the in vivo cultivation of bacteria. Outer membranes isolated from log-phase bacterial cells recovered from these chambers were compared with membranes isolated from cells grown in vitro. Analysis revealed that the suspected 41-kilodalton porin and the OmpA protein were recovered on sodium dodecyl sulfate-polyacrylamide gels in equal quantities. Several high-molecular-weight proteins, thought to be iron starvation induced, appeared in the diffusion chamber-grown cells. The outer membrane permeability barriers to cephaloridine were similar in in vivo- and in vitro-grown cells based on permeability coefficient calculations. The permeability coefficient of cephaloridine in S. marcescens cells (30.3 x 10(-5) to 38.9 x 10(-5) cm s-1) was greater than that obtained for an Escherichia coli strain expressing only porin OmpC but smaller than those obtained for the E. coli wild type and a strain expressing only porin OmpF. Functional characterization of the suspected porin was performed by using the planar lipid bilayer technology. The sodium dodecyl sulfate-0.4 M NaCl-soluble porin from both in vitro- and in vivo-grown cells showed an average single-channel conductance in 1 M KCl of 1.6. A partial amino acid sequence (19 residues) was obtained for the S. marcescens porin. The sequence showed a very high homology to the E. coli OmpC porin. These data identified the S. marcescens outer membrane 41-kilodalton protein as a porin by both functional and amino acid analyses. Also, the methodology used allowed for efficient growth and recovery of diffusion chamber-grown bacterial cells and permitted identification of specific in vivo-induced changes in bacterial cell membrane composition.