Novel antimicrobial strategies to treat multi-drug resistant Staphylococcus aureus infections

Antimicrobial resistance is a major obstacle for the treatment of infectious diseases and currently represents one of the most significant threats to global health. Staphylococcus aureus remains a formidable human pathogen with high mortality rates associated with severe systemic infections. S. aureus has become notorious as a multidrug resistant bacterium, which when combined with its extensive arsenal of virulence factors that exacerbate disease, culminates in an incredibly challenging pathogen to treat clinically. Compounding this major health issue is the lack of antibiotic discovery and development, with only two new classes of antibiotics approved for clinical use in the last 20 years. Combined efforts from the scientific community have reacted to the threat of dwindling treatment options to combat S. aureus disease in several innovative and exciting developments. This review describes current and future antimicrobial strategies aimed at treating staphylococcal colonization and/or disease, examining therapies that show significant promise at the preclinical development stage to approaches that are currently being investigated in clinical trials.     

Neurotrophic Factors Attenuate Glutamate-Induced Accumulation of Peroxides, Elevation of Intracellular Ca2+ Concentration, and Neurotoxicity and Increase Antioxidant Enzyme Activities in Hippocampal Neurons

Abstract: Exposure of cultured rat hippocampal neurons to glutamate resulted in accumulation of cellular peroxides (measured using the dye 2,7-dichlorofluorescein). Peroxide accumulation was prevented by an N -methyl- d -aspartate (NMDA) receptor antagonist and by removal of extracellular Ca²⁺, indicating the involvement of NMDA receptor-induced Ca²⁺ influx in peroxide accumulation. Glutamate-induced reactive oxygen species contributed to loss of Ca²⁺ homeostasis and excitotoxic injury because antioxidants (vitamin E, propyl gallate, and N-tert -butyl-α-phenylnitrone) suppressed glutamate-induced elevation of intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cell death. Basic fibroblast growth factor (bFGF), nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF), but not ciliary neurotrophic factor, each suppressed accumulation of peroxides induced by glutamate and protected neurons against excitotoxicity. bFGF, NGF, and BDNF each increased (to varying degrees) activity levels of superoxide dismutases and glutathione reductase. NGF increased catalase activity, and BDNF increased glutathione peroxidase activity. The ability of the neurotrophic factors to suppress glutamate toxicity and glutamate-induced peroxide accumulation was attenuated by the tyrosine kinase inhibitor genistein, indicating the requirement for tyrosine phosphorylation in the neuroprotective signal transduction mechanism. The data suggest that glutamate toxicity involves peroxide production, which contributes to loss of Ca²⁺ homeostasis, and that induction of antioxidant defense systems is a mechanism underlying the [Ca²⁺]_i-stabilizing and excitoprotective actions of neurotrophic factors.     

Nucleic acid oxidation: an early feature of Alzheimer's disease

Studies of oxidative damage during the progression of Alzheimer's disease (AD) suggest its central role in disease pathogenesis. To investigate levels of nucleic acid oxidation in both early and late stages of AD, levels of multiple base adducts were quantified in nuclear and mitochondrial DNA from the superior and middle temporal gyri (SMTG), inferior parietal lobule (IPL), and cerebellum (CER) of age‐matched normal control subjects, subjects with mild cognitive impairment, preclinical AD, late‐stage AD, and non‐AD neurological disorders (diseased control; DC) using gas chromatography/mass spectrometry. Median levels of multiple DNA adducts in nuclear and mitochondrial DNA were significantly (p ≤ 0.05) elevated in the SMTG, IPL, and CER in multiple stages of AD and in DC subjects. Elevated levels of fapyguanine and fapyadenine in mitochondrial DNA suggest a hypoxic environment early in the progression of AD and in DC subjects. Overall, these data suggest that oxidative damage is an early event not only in the pathogenesis of AD but is also present in neurodegenerative diseases in general.

     

Seasonal Variability of Diazotroph Assemblages Associated with the Rhizosphere of the Salt Marsh Cordgrass, Spartina alterniflora

Nitrogen fixation is the primary N source in the highly productive but N-limited North Inlet, SC, USA salt marsh system. The diverse assemblages of nitrogen-fixing (diazotrophic) bacteria associated with the rhizospheres of the short and tall growth forms of Spartina alterniflora were analyzed at two sites, Crab Haul Creek and Goat Island, which are in different tidal creek drainage systems in this marsh. The sites differed in proximity to the main channel for tidal intrusion and in several edaphic parameters. We hypothesized that either the differing abiotic environmental regimes of the two sites or the variation due to seasonal effects result in differences in the diazotroph assemblage. Rhizosphere samples were collected seasonally during 1999 and 2000. DNA was purified and nifH amplified for denaturing gradient gel electrophoresis (DGGE) analysis of diazotroph assemblage composition. Principal components analysis was used to analyze the binary DGGE band position data. Season strongly influenced assemblage composition and biplots were used to identify bands that significantly affected the seasonal and site-specific clustering. The types of organisms that were most responsive to seasonal or site variability were identified on the basis of DGGE band sequences. Seasonally responsive members of the anaerobic diazotrophs were detected during the winter and postsenescence conditions and may have been responsible for elevated pore water sulfide concentrations. Sequences from a diverse assemblage of Gammaproteobacteria were predominant during growth periods of S. alterniflora. Abiotic environmental parameters strongly influenced both the S. alterniflora and the diazotrophic bacterial assemblages associated with this keystone salt marsh plant species.     

Reaction of Muscimol with 4-Aminobutyrate Aminotransferase

Abstract: The reaction of muscimol as amino donor substrate for GABA transaminase (GABA-T) has been studied using enzyme purified from rabbit brain. Enzyme activity was assayed by measuring the glutamate produced using glutamate dehydrogenase. Kinetic parameters determined at 37°C were for GABA, K _m (app) = 1.92 ± 0.24 m M , specific activity = 7.33 ± 0.27 μmol/min/mg ( k _cat= 13.7s⁻¹), and for muscimol, K _m (app) = 1.27 ± 0.15 m M , specific activity = 0.101 ± 0.009 μmol/min/mg ( k _cat= 0.19s⁻¹). Addition of muscimol to the enzyme caused the spectral changes associated with conversion of the pyridoxaldimine form to the pyridoxamine form, and the first-order rate constant for the reaction showed a dependence on muscimol concentration that followed saturation kinetics, with a K = 1.1 ±0.18 m M and k _max= 0.065 ± 0.004 s⁻¹ (19°C). The rate of spectral change observed on addition of muscimol to ornithine transaminase was extremely slow—at least an order of magnitude slower than that seen with GABA-T.     

Polymorphism in a T-cell receptor variable gene is associated with susceptibility to a juvenile rheumatoid arthritis subset

This report demonstrates a T-cell receptor (Tcr) restriction fragment length polymorphism, defined by a Tcrb-V6.1 gene probe and Bgl II restriction enzyme, to be absolutely correlated with allelic variation in the coding sequence of a Tcrb-V6.1 gene. A pair of non-conservative amino acid substitutions distinguish the Tcrb-V6.1 allelic variants. An association of this Tcrb-V6.1 gene allelic variant with one form of juvenile rheumatoid arthritis (JRA) was established in a cohort of 126 patients. The association was observed in patients possessing the HLA-DQA1*0101 gene. Among HLA-DQA*0101 individuals, 19 of 26 patients (73.1%) carried one particular Tcrb-V6.1 gene allele as opposed to 11 of 33 controls (33%; p<0.005). Haplotypes carrying this HLA gene have previously been shown to confer increased risk for progression of arthritis in JRA. This demonstration of a disease-associated Tcrb-V gene allelic variant has not, to our knowledge, been previously reported and supports the contribution of polymorphism in the Tcr variable region genomic repertoire to human autoimmune disease.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M67511 for V6.1A and M67512 for V6.1B.     

Effects of urine on soil microbial biomass,methanogenesis, nitrification and denitrification in grassland soils

Urine was added under controlled conditions to intact turfs taken from long-term permanent pasture on clay loam and sandy loam soils in South West England. Methane exchanges were small (<+/−0.03 μg CH₄ m^-2 min^-1) and overall absorption equalled or exceeded emission in both soils. On the clay loam, wetting with water or urine increased soil microbial biomass C and N contents by about 20% but there was no specific effect of urine. Urine, however, caused an increase in soil respiration of >50% and the average increase was greater for cow's urine (30.8 mg CO₂ m^-2 min^-1) than for an artificial urine (20.1 mg CO₂ m^-2 min^-1). Emissions of nitric and nitrous oxides following urine application were substantial (on average 0.36 μg NO-N and 29 μg N₂O-N m^-2 min^-1) but short lived (<40 days). The high levels of ammonium found in the urine treated soils (>200 mg NH₄ ⁺-N kg^-1) were nitrified to nitrate over a period of 42 days. Qualitative changes in the soil microbial biomass were evidently not related to biomass size. Relationships between trace gas emissions and soil processes are discussed. ei]Section editor: R Merckx     

Excretion of photosynthetically fixed organic carbon by metalimnetic phytoplankton

The effects of light intensity, oxygen concentration, and pH on the rates of photosynthesis and net excretion by metalimnetic phytoplankton populations of Little Crooked Lake, Indiana, were studied. Photosynthetic rates increased from 1.42 to 3.14 mg C·mg^–1 chlorophylla·hour^–1 within a range of light intensities from 65 to 150E·m^–2·sec^–1, whereas net excretion remained constant at 0.05 mg C·mg^–1 chlorophylla·hour^–1. Bacteria assimilated approximately 50% of the carbon released by the phytoplankton under these conditions. Excreted carbon (organic compounds either assimilated by bacteria or dissolved in the lake water) was produced by phytoplankton at rates of 0.02–0.15 mg C·mg^–1 chlorophylla·hour^–1. These rates were 6%–13% of the photosynthetic rates of the phytoplankton. Both total excretion of carbon and bacterial assimilation of excreted carbon increased at high light intensities whereas net excretion remained fairly constant. Elevated oxygen concentrations in samples incubated at 150E· m^–2·sec^–1 decreased rates of both photosynthesis and net excretion. The photosynthetic rate increased from 3.0 to 5.0 mg C·mg^–1 chlorophylla· hour^–1 as the pH was raised from 7.5 to 8.8. Net excretion within this range decreased slightly. Calculation of total primary production using a numerical model showed that whereas 225.8 g C·m^–2 was photosynthetically fixed between 12 May and 24 August 1982, a maximum of about 9.3 g C·m^–2 was released extracellularly.     

Changes in plasma inhibin A levels during sexual maturation in the female chicken and the effects of active immunization against inhibin alpha-subunit on reproductive hormone profiles and ovarian function

Inhibins and activins are firmly implicated in the control of pituitary FSH secretion and ovarian follicular development in mammals. As in mammals, inhibin A and activin A are expressed in the preovulatory follicles of birds, and a defined ovulation cycle for inhibin A has recently been demonstrated in the laying hen. To investigate further the role of inhibin-related proteins in developing pullets, circulating concentrations of inhibin A, inhibin B, total immunoreactive inhibin alpha-subunit (ir-alpha), activin A, LH, FSH, and progesterone were measured from the juvenile state through to sexual maturity in 22 birds. In the 11 birds assigned to control groups, plasma inhibin A levels were low from 7 to 13 wk of age rising about threefold to a peak at Week 19 after which levels fell slightly to a plateau level characteristic of adult hens. Plasma inhibin A levels were negatively correlated with FSH (r = -0. 33; P: < 0.001) and positively correlated with progesterone (r = 0. 67; P: < 0.001) and ir-alpha (r = 0.53; P: < 0.001). Plasma ir-alpha levels were much higher than inhibin A levels although the relative differences varied with age. Plasma levels of inhibin B and activin A were below assay detection limits at all times. The remaining group of 11 birds was actively immunized (IMM) against a synthetic chicken inhibin alpha-subunit peptide (amino acids 1-26). The IMM generated circulating antibodies that bound native bovine inhibin A but altered neither plasma FSH nor progesterone levels relative to control birds at any stage of development nor the timing of first oviposition in week 19. Apart from a transient decline 1 wk after primary IMM, plasma LH concentrations did not differ from controls. Comparison of the numbers and size-class distribution of ovarian follicles at 29 wk showed an approximate twofold increase in the number of 8- to 9.9-mm-diameter follicles (control; 1.82 +/- 0.44 vs. IMM; 3.91 +/- 0.89; P: < 0.05), a size class that corresponds to follicles that have just joined the preovulatory hierarchy. The numbers of growing follicles in other size-classes and the sizes of hierarchical F(1)-F(7) follicles were not altered by IMM. However, the number of postovulatory follicles increased (control 3.73 +/- 0. 20 vs. IMM 5.55 +/- 0.28; P: < 0.01), and significantly more (P: < 0. 02) immunized hens laid two eggs within a 24-h period on at least one occasion (control 1 of 11 vs. IMM 9 of 11). The IMM increased (P: < 0.05) activin A content of F(1) and F(2) theca layers and decreased (P: < 0.05) activin A content in F(3) and F(4) granulosa layers, raising the possibility of a local intraovarian role of activin in mediating the response to IMM. These findings support a role for inhibin A in regulating the entry of follicles into the preovulatory hierarchy in the chicken, although further studies are required to establish the mechanism by which inhibin IMM increases the rate of follicle selection and ovulation without raising plasma FSH.