Tobramycin (Nebramycin Factor 6): In Vitro Activity Against Pseudomonas aeruginosa

Tobramycin (factor 6 of the nebramycin complex) is a new aminoglycoside antibiotic isolated from Streptomyces tenebrarius which is active against S. aureus, Enterobacteriaceae, and Pseudomonas aeruginosa. Susceptibility to tobramycin of 96 strains of P. aeruginosa, including 45 recent isolates from blood, was studied by using agar and broth dilution methods. The minimum inhibitory concentration (MIC) for 83 of 96 strains was 3.12 mug/ml or lower in Mueller Hinton agar; MIC values were two to eight times lower in Mueller Hinton broth tests. Agar dilution MIC values were generally lower than those obtained in parallel tests with gentamicin. Killing curves obtained from serial sampling of broth cultures showed a 100- to 10,000-fold decline in viability of log-phase organisms within 30 min of exposure to the drug. Two-dimensional agar dilution tests with carbenicillin and tobramycin with 79 strains showed additive or synergistic effects; no antagonism was documented. Seventy-eight of 79 strains were inhibited by a combination of 50 mug of carbenicillin per ml and 1.56 mug of tobramycin per ml, blood levels which seem attainable in man. Tobramycin appears to be a potent, rapidly bactericidal antibiotic against P. aeruginosa and merits clinical evaluation.     

Inhibition of Macrophage Phagocytosis by Pseudomonas aeruginosa Rhamnolipids In Vitro and In Vivo

Patients with cystic fibrosis often have chronic and ultimately lethal pulmonary infections with Pseudomonas aeruginosa. In order to understand why these bacteria resist pulmonary clearance, we have investigated the interaction of P. aeruginosa and phagocytic cells. In an earlier study we reported that sub-lytic concentrations of two glycolipids produced by P. aeruginosa (the mono- and dirhamnolipids) caused structural changes in human monocyte-derived macrophages, and at lower concentrations inhibited the phagocytosis of Staphylococcus epidermidis by these cells. In the present study we demonstrate that rhamnolipids also inhibit the in vitro phagocytosis of both P. aeruginosa and Saccharomyces cerevisiae by thioglycollate-elicited mouse peritoneal macrophages. Using lucifer yellow to label the lysosomal compartments of macrophages, we determined that rhamnolipids interfere with the internalization of attached particles and reduce the level of phagosome-lysosome fusion of internalized targets within macrophages. We also demonstrate that physiologically relevant concentrations of rhamnolipids injected intratracheally into rat lungs inhibited the response of alveolar macrophages to a challenge of zymosan particles in vivo. These studies further demonstrate the profound inhibitory effects of P. aeruginosa rhamnolipids on macrophage function and are consistent with our hypothesis that the in situ production of these rhamnolipids directly contributes to the persistence of this pathogen in cystic fibrosis patient lungs. Received: 15 December 1995 / Accepted: 22 January 1996     

Characterization of Cyclic AMP Efflux from Swine Adipocytes In Vitro

Objective : A variety of cell types transport cyclic AMP (cAMP) to the extracellular fluid; the purpose of this study was to determine if and how this process occurs in adipocytes. Research Methods and Procedures : Adipocytes were isolated from 3-month-old swine and incubated with stimulators of adenylate cyclase for 2 to 120 minutes to promote cAMP synthesis and efflux. Efflux was characterized in the presence of agents that inhibit ATP production, anion transport, intracellular cAMP metabolism, and extracellular cAMP metabolism. Extracellular cAMP was measured by enzyme immunoassay, then corrected for cell lysis by measuring lactate dehydrogenase release. Results : cAMP efflux averaged 24.7 fmol/min/cm² adipocyte surface area, was linear for 2 hours, and was proportional to adipocyte surface area (r = 0.94, p<0.05). Efflux was reduced by ∽35% in cells incubated with 1 4mUM antimycin, an inhibitor of ATP synthesis (p<0.05), and by ～55% in cells incubated with 2 mM probenecid, an anionpecific transport blocker (p<0.05). Extracellular cAMP levels more than doubled by the addition of 1 μM 1,3-dipropyl-8-p-sulfophenylxanthine, a purported inhibitor of extracellular phosphodiesterase. Discussion : Our data demonstrate that cAMP is transported from swine adipocytes by an energy-dependent anion transporter and can be metabolized extracellularly. Future studies will evaluate extracellular cAMP as a potential source of extracellular adenosine, a potent inhibitor of adipocyte lipolysis.     

The Pseudomonas aeruginosa Quorum-Sensing Molecule N-(3-Oxododecanoyl)Homoserine Lactone Contributes to Virulence and Induces Inflammation In Vivo

Pseudomonas aeruginosa has two well-characterized quorum-sensing systems, Las and Rhl. These systems are composed of LuxR-type proteins, LasR and RhlR, and two acyl homoserine lactone (AHL) synthases, LasI and RhlI. LasI catalyzes the synthesis of N-(3-oxododecanoyl)homoserine lactone (3O-C12-HSL), whereas RhlI catalyzes the synthesis of N-butyryl-homoserine lactone. There is little known about the importance of AHLs in vivo and what effects these molecules have on eukaryotic cells. In order to understand the role of AHLs in vivo, we first tested the effects that deletions of the synthase genes in P. aeruginosa had on colonization of the lung. We demonstrate that in an adult mouse acute-pneumonia model, deletion of the lasI gene or both the lasI and rhlI genes greatly diminished the ability of P. aeruginosa to colonize the lung. To determine whether AHLs have a direct effect on the host, we examined the effects of 3O-C12-HSL injected into the skin of mice. In this model, 3O-C(12)-HSL stimulated a significant induction of mRNAs for the cytokines interleukin-1alpha (IL-1alpha) and IL-6 and the chemokines macrophage inflammatory protein 2 (MIP-2), monocyte chemotactic protein 1, MIP-1beta, inducible protein 10, and T-cell activation gene 3. Additionally, dermal injections of 3O-C12-HSL also induced cyclooxygenase 2 (Cox-2) expression. The Cox-2 enzyme is important for the conversion of arachidonic acid to prostaglandins and is associated with edema, inflammatory infiltrate, fever, and pain. We also demonstrate that 3O-C12-HSL activates T cells to produce the inflammatory cytokine gamma interferon and therefore potentially promotes a Th1 environment. Induction of these inflammatory mediators in vivo is potentially responsible for the significant influx of white blood cells and subsequent tissue destruction associated with 3O-C12-HSL dermal injections. Therefore, the quorum-sensing systems of P. aeruginosa contribute to its pathogenesis both by regulating expression of virulence factors (exoenzymes and toxins) and by inducing inflammation.     

Ganglioside Modulation of Cyclic AMP-Dependent Protein Kinase and Cyclic Nucleotide Phosphodiesterase In Vitro

The expression of glial fibrillary acidic protein (GFAP)-mRNA during mouse brain development and in astroglial primary cultures has been investigated by using two approaches: Northern-blot evaluation using a specific cDNA probe, and cell-free translation associated with immunoprecipitation. During brain maturation (4-56 days postnatal), the GFAP-mRNA underwent a biphasic evolution. An increase was observed between birth and day 15 (i.e., during the period of astroglial proliferation), which was followed by a decrease until day 56 (i.e., during astroglial cell differentiation). At older stages (300 days), an increase was observed, which might reflect gliosis. During astroglial in vitro development (7-32 days in culture), the GFAP-mRNA showed similar variations. An increase, observed during the period of astroglial proliferation (7-18 days), was followed by a decrease which occurred in parallel to marked changes in cell shape, cell process outgrowth, and the organization and accumulation of gliofilaments. During the same culture period (7-32 days), alpha-tubulin mRNA, which was used as an internal standard, did not vary significantly. These results show that the increase of the GFAP protein and of gliofilaments observed both in vivo and in vitro during astroglial differentiation cannot be ascribed to an accumulation of the GFAP-mRNA. It might be that more than one mechanism regulates the levels of free and polymerized GFAP and of its encoding mRNA.     

Enzymatic Characterization and In Vivo Function of Five Terminal Oxidases in Pseudomonas aeruginosa

The ubiquitous opportunistic pathogen Pseudomonas aeruginosa has five aerobic terminal oxidases: bo₃-type quinol oxidase (Cyo), cyanide-insensitive oxidase (CIO), aa₃-type cytochrome c oxidase (aa₃), and two cbb₃-type cytochrome c oxidases (cbb₃-1 and cbb₃-2). These terminal oxidases are differentially regulated under various growth conditions and are thought to contribute to the survival of this microorganism in a wide variety of environmental niches. Here, we constructed multiple mutant strains of P. aeruginosa that express only one aerobic terminal oxidase to investigate the enzymatic characteristics and in vivo function of each enzyme. The K_m values of Cyo, CIO, and aa₃ for oxygen were similar and were 1 order of magnitude higher than those of cbb₃-1 and cbb₃-2, indicating that Cyo, CIO, and aa₃ are low-affinity enzymes and that cbb₃-1 and cbb₃-2 are high-affinity enzymes. Although cbb₃-1 and cbb₃-2 exhibited different expression patterns in response to oxygen concentration, they had similar K_m values for oxygen. Both cbb₃-1 and cbb₃-2 utilized cytochrome c₄ as the main electron donor under normal growth conditions. The electron transport chains terminated by cbb₃-1 and cbb₃-2 generate a proton gradient across the cell membrane with similar efficiencies. The electron transport chain of aa₃ had the highest proton translocation efficiency, whereas that of CIO had the lowest efficiency. The enzymatic properties of the terminal oxidases reported here are partially in agreement with their regulatory patterns and may explain the environmental adaptability and versatility of P. aeruginosa.     

In Vitro and In Vivo Characterization of Pyocin

Pyocin, a bacteriocin obtained from lysates of ultraviolet-induced cultures of Pseudomonas aeruginosa was characterized in vitro and in vivo after 1,000-fold purification by chemical, column, and differential centrifugation procedures. Electron micrographs of negatively stained pyocin preparations contained rod-shaped particles which resembled the contractile tail protein of the T-even phages of Escherichia coli. Although two separate and distinct pyocin fractions were eluted from diethylaminoethyl cellulose (pH 7.5) during the purification procedure, the particles appeared identical. In addition, the two fractions exhibited a close correlation between their titers and the particle numbers as observed in the electron microscope. The particles were approximately 20 by 90 mmu with a core diameter of 5 mmu and a sheath length of 50 mmu. Neither intact phage nor ghosts were seen in any of the preparations, although ringlets of two different diameters, which appeared to correspond to the diameters of the sheath and inner core, were observed. Other studies indicated that, although crude preparations were stable to freezing and thawing, purified preparations lost all of their activity under similar treatment. However, the addition of 50% glycerol to purified preparations completely protected activity. Conversely, aged normal human or rabbit sera enhanced the antibacterial activity of pyocin approximately fourfold, although serum albumin and hemoglobin had no effect. In vivo studies indicated that purified pyocin was not lethal for mice when injected intraperitoneally in concentrations of 28,000 to 1,400,000 units (5.6 to 276 mug of protein), nor was 7,200 to 36,000 units dermonecrotic for rabbits.     

Regulation of Pyruvate Decarboxylase In Vitro and In Vivo

Results presented in this paper strongly support the view thatregulation of the key enzyme of alcoholic fermentation, pyruvatedecarboxylase (PDC), is achieved in a number of ways, all associatedwith possible lowering of the cytoplasmic pH during anoxia.These mechanisms include not only the well-known acid pH optimumof PDC, but also long-term, reversible changes in characteristicsof the enzyme established both in vitro and in vivo. Following transfer of desalted extracts from pH 6.0 to 7.4,maximal activity of PDC was decreased, while there was a considerableincrease in the lag before maximal activity was reached. Similarchanges in enzyme characteristics were observed when wheat (Triticumaestivum L. cv. Gamenya) roots and rice (Oryza sativa L. cv.Calrose) coleoptiles were transferred from anoxic to aerobicsolutions, provided PDC was assayed within 10 min of the startof maceration. All of the above changes were usually readilyreversible when extracts were returned to pH 6.0, or when plantswere returned to anoxic solutions. Additional regulation of PDC would be achieved by the S_0.5 forpyruvate which is 0.75 mol m^–3 at pH 6.0, 1.0 mol m^–3at pH 6.8, and 2.5 mol m^–3 at pH 7.4; the latter is wellabove estimates for pyruvate concentrations in the cytoplasmof aerated tissues. We assess that the combined effects of the acid pH optimum,the high S_0.5 at pH 7.4 and the long-term decreases in activityobserved during incubation at pH 7.4 would reduce PDC activityin aerobic cells to at most 7% of the activity in anoxic cells.Possible additional controls for the pathway of alcoholic fermentationare briefly considered. Key words: PDC, regulation, anoxia     

In Vitro and In Vivo Characterization of the Alkaloid Nuciferine

Rationale

The sacred lotus (Nelumbo nucifera) contains many phytochemicals and has a history of human use. To determine which compounds may be responsible for reported psychotropic effects, we used in silico predictions of the identified phytochemicals. Nuciferine, an alkaloid component of Nelumbo nucifera and Nymphaea caerulea, had a predicted molecular profile similar to antipsychotic compounds. Our study characterizes nuciferine using in vitro and in vivo pharmacological assays.

Methods

Nuciferine was first characterized in silico using the similarity ensemble approach, and was followed by further characterization and validation using the Psychoactive Drug Screening Program of the National Institute of Mental Health. Nuciferine was then tested in vivo in the head-twitch response, pre-pulse inhibition, hyperlocomotor activity, and drug discrimination paradigms.

Results

Nuciferine shares a receptor profile similar to aripiprazole-like antipsychotic drugs. Nuciferine was an antagonist at 5-HT_2A, 5-HT_2C, and 5-HT_2B, an inverse agonist at 5-HT₇, a partial agonist at D_2, D₅ and 5-HT₆, an agonist at 5-HT_1A and D₄ receptors, and inhibited the dopamine transporter. In rodent models relevant to antipsychotic drug action, nuciferine blocked head-twitch responses and discriminative stimulus effects of a 5-HT_2A agonist, substituted for clozapine discriminative stimulus, enhanced amphetamine induced locomotor activity, inhibited phencyclidine (PCP)-induced locomotor activity, and rescued PCP-induced disruption of prepulse inhibition without induction of catalepsy.

Conclusions

The molecular profile of nuciferine was similar but not identical to that shared with several approved antipsychotic drugs suggesting that nuciferine has atypical antipsychotic-like actions.     

A 3′,5′ Cyclic AMP (cAMP) Phosphodiesterase Modulates cAMP Levels and Optimizes Competence in Haemophilus influenzae Rd

Changes in intracellular 3′,5′ cyclic AMP (cAMP) concentration regulate the development of natural competence in Haemophilus influenzae. In Escherichia coli, cAMP levels are modulated by a cAMP phosphodiesterase encoded by the cpdA gene. We have used several approaches to demonstrate that the homologous icc gene of H. influenzae encodes a functional cAMP phosphodiesterase and that this gene limits intracellular cAMP and thereby influences competence and other cAMP-dependent processes. In E. coli, expression of cloned icc reduced both cAMP-dependent sugar fermentation and β-galactosidase expression, as has been shown for cpdA. In H. influenzae, an icc null mutation increased cAMP-dependent sugar fermentation and competence development in strains where these processes are limited by mutations reducing cAMP synthesis. When endogenous production of cAMP was eliminated by a cya mutation, an icc strain was 10,000-fold more sensitive to exogenous cAMP than an icc⁺ strain. The icc strain showed moderately elevated competence under noninducing conditions, as expected, but had subnormal competence increases at onset of stationary phase in rich medium, and on transfer to a nutrient-limited medium, suggesting that excessive cAMP may interfere with induction. Consistent with this finding, a cya strain cultured in 1 mM cAMP failed to develop maximal competence on transfer to inducing conditions. Thus, by limiting cAMP levels, the H. influenzae cAMP phosphodiesterase may coordinate its responses to nutritional stress, ensuring optimal competence development.     

Suppression of Virulence of Toxigenic Vibrio cholerae by Anethole through the Cyclic AMP (cAMP)-cAMP Receptor Protein Signaling System

Use of natural compounds as antivirulence drugs could be an alternative therapeutic approach to modify the outcome of bacterial infections, particularly in view of growing resistance to available antimicrobials. Here, we show that sub-bactericidal concentration of anethole, a component of sweet fennel seed, could suppress virulence potential in O1 El Tor biotype strains of toxigenic Vibrio cholerae, the causative agent of the ongoing 7^th cholera pandemic. The expression of cholera toxin (CT) and toxin coregulated pilus (TCP), the major virulence factors of V. cholerae, is controlled through a regulatory cascade involving activation of ToxT with synergistic coupling interaction of ToxR/ToxS with TcpP/TcpH. We present evidence that anethole inhibits in vitro expression of CT and TCP in a toxT-dependent but toxR/toxS-independent manner and through repression of tcpP/tcpH, by using bead-ELISA, western blotting and quantitative real-time RT-PCR assays. The cyclic AMP (cAMP)-cAMP receptor protein (CRP) is a well-studied global signaling system in bacterial pathogens, and this complex is known to suppress expression of tcpP/tcpH in V. cholerae. We find that anethole influences the virulence regulatory cascade by over-expressing cyaA and crp genes. Moreover, suppression of toxigenic V. cholerae-mediated fluid accumulation in ligated ileum of rabbit by anethole demonstrates its potentiality as an antivirulence drug candidate against the diseases caused by toxigenic V. cholerae. Taken altogether, these results revealing a mechanism of virulence inhibition in V. cholerae by the natural compound anethole, may have relevance in designing antivirulence compounds, particularly against multiple antibiotic resistant bacterial pathogens.     

In Vivo,In Vitro,and In Silico Characterization of Peptoids as Antimicrobial Agents

Bacterial resistance to conventional antibiotics is a global threat that has spurred the development of antimicrobial peptides (AMPs) and their mimetics as novel anti-infective agents. While the bioavailability of AMPs is often reduced due to protease activity, the non-natural structure of AMP mimetics renders them robust to proteolytic degradation, thus offering a distinct advantage for their clinical application. We explore the therapeutic potential of N-substituted glycines, or peptoids, as AMP mimics using a multi-faceted approach that includes in silico, in vitro, and in vivo techniques. We report a new QSAR model that we developed based on 27 diverse peptoid sequences, which accurately correlates antimicrobial peptoid structure with antimicrobial activity. We have identified a number of peptoids that have potent, broad-spectrum in vitro activity against multi-drug resistant bacterial strains. Lastly, using a murine model of invasive S. aureus infection, we demonstrate that one of the best candidate peptoids at 4 mg/kg significantly reduces with a two-log order the bacterial counts compared with saline-treated controls. Taken together, our results demonstrate the promising therapeutic potential of peptoids as antimicrobial agents.     

Identification and Characterization of a Periplasmic Aminoacyl-phosphatidylglycerol Hydrolase Responsible for Pseudomonas aeruginosa Lipid Homeostasis

Specific aminoacylation of the phospholipid phosphatidylglycerol (PG) with alanine (or with lysine) was shown to render various organisms less susceptible to antimicrobial agents and environmental stresses. In this study, we make use of the opportunistic pathogen Pseudomonas aeruginosa to decode ORF PA0919-dependent lipid homeostasis. Analysis of the polar lipid content of the deletion mutant ΔPA0919 indicated significantly enlarged levels of alanyl-PG. The resulting phenotype manifested an increased susceptibility to several antimicrobial compounds when compared with the wild type. A pH-dependent PA0919 promoter located within the upstream gene PA0920 was identified. Localization experiments demonstrated that the PA0919 protein is anchored to the periplasmic surface of the inner bacterial membrane. The recombinant overproduction of wild type and several site-directed mutant proteins in the periplasm of Escherichia coli facilitated a detailed in vitro analysis of the enzymatic PA0919 function. A series of artificial substrates (p-nitrophenyl esters of various amino acids/aliphatic acids) indicated enzymatic hydrolysis of the alanine, glycine, or lysine moiety of the respective ester substrates. Our final in vitro activity assay in the presence of radioactively labeled alanyl-PG then revealed hydrolysis of the aminoacyl linkage, resulting in the formation of alanine and PG. Consequently, PA0919 was termed alanyl-PG hydrolase. The elucidated enzymatic activity implies a new regulatory circuit for the appropriate tuning of cellular alanyl-PG concentrations.