Susceptibility and intrinsic tolerance of Pseudomonas aeruginosa to selected plant volatile compounds.

AIMS: To examine the causes for variations in sensitivity and intrinsic tolerance of Pseudomonas aeruginosa to plant volatile compounds. METHODS AND RESULTS: Minimum inhibitory concentrations were determined for a selection of volatile phytochemicals against P. aeruginosa using a microdilution assay. Effects on growth were also assessed in 100-ml broth cultures. The two strains of P. aeruginosa included in the study exhibited intrinsic tolerance to all compounds, with the exception of carvacrol and trans-cinnamaldehyde. The protonophore carbonyl cyanide m-chlorophenylhydrazone increased P. aeruginosa sensitivity to all compounds except trans-cinnamaldehyde, implicating an ATP-dependent efflux mechanism in the observed tolerance. Outer membrane integrity following treatment with test compounds was assessed by measuring sensitization to detergents. Only carvacrol caused damage to the outer membrane of P. aeruginosa. CONCLUSIONS: The intrinsic tolerance of P. aeruginosa strains to plant volatile compounds is associated with an active efflux mechanism and the barrier function of the outer membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings offer an explanation for the intrinsic tolerance to plant volatile compounds exhibited by P. aeruginosa. The study also confirms that the outer membrane-permeabilizing action of carvacrol, previously reported for the gram-negative bacteria Escherichia coli and Salmonella, extends to monoterpene-tolerant strains of P. aeruginosa.

Comparison of Bayer Advia Centaur immunoassay results obtained on samples collected in four different Becton Dickinson Vacutainer tubes.

BACKGROUND: Medicines and Healthcare products Regulatory Agency's (MHRA's) Medical Device Alert MDA/2004/048 described bias in some endocrine test results obtained on a few immunoassay platforms, particularly the Bayer Advia Centaur instrument, when using blood specimens collected into Becton Dickinson (BD) Vacutainer SSTII Advance tubes. As users of BD tubes and the Advia Centaur instrument, we addressed our concerns about the quality of the results that we had previously reported by undertaking an independent study. METHOD: We compared the results of 15 immunoassays performed on Bayer Advia Centaur using blood specimens collected into four different BD Vacutainer tubes (plain, old and newly released BD SSTII Advance, and BD PSTII). RESULTS: Compared with plain tubes, old SSTII Advance tube results showed no bias for testosterone, CA15-3, follicle-stimulating hormone and folate assays, but gave a positive bias for cortisol and a negative bias for vitamin-B12. Compared with plain tubes, BD PSTII tubes gave no significant bias for thyroid function tests, prolactin, parathyroid hormone, and CA125, but gave a negative bias for steroid assays, and a positive bias for gonadotrophins. The results obtained using new BD SSTII Advance tubes were generally comparable with those on plain tubes. CONCLUSIONS: Only for cortisol did our findings support the bias described by MHRA. Based on our results, apart from vitamin-B12 and possibly cortisol, there may have been no significant influence on clinical decisions as a result of using the old BD SSTII Advance specimen tubes. New BD SSTII Advance tubes and plain tubes give generally comparable results. BD PSTII tubes should not be used for steroid hormone measurements on the Bayer Advia Centaur instrument.

Effects of a topical essential oil-containing formulation on biofilm-forming coagulase-negative staphylococci.

AIMS: To evaluate the antimicrobial effects of Polytoxinol (PT), a topical essential oil-based formulation, against biofilm positive strains of coagulase-negative staphylococci. METHODS AND RESULTS: Using a microtitre plate assay we measured inhibitory effects for PT against a selection of biofilm-forming clinical isolates of coagulase-negative staphylococci. Susceptibility varied considerably (MIC = 0.6-20 000 ppm). For the most tolerant clinical isolate (Staphylococcus warneri) biofilm growth was inhibited by a 32-fold lower PT concentration than planktonic growth. This inhibition of biofilm development, which was not observed with the other test isolates, was related to an inhibition of the initial phase of S. warneri cell adherence to the polystyrene surface. CONCLUSION: The antimicrobial efficacy of PT was verified against clinical isolates of coagulase-negative staphylococci in vitro. PT was able to inhibit biofilm formation in the most tolerant isolate at sub-inhibitory concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: These observations indicate that an ability to prevent biofilm formation, independently of effects on cell viability may contribute to the in vivo topical efficacy of essential oils.

Interactions between components of the essential oil of Melaleuca alternifolia.

AIMS: This study compared the antimicrobial activity of Melaleuca alternifolia (tea tree) oil with that of some of its components, both individually and in two-component combinations. METHODS AND RESULTS: Minimum inhibitory concentration and time-kill assays revealed that terpinen-4-ol, the principal active component of tea tree oil, was more active on its own than when present in tea tree oil. Combinations of terpinen-4-ol and either gamma-terpinene or p-cymene produced similar activities to tea tree oil. Concentration-dependent reductions in terpinen-4-ol activity and solubility also occurred in the presence of gamma-terpinene. CONCLUSION: Non-oxygenated terpenes in tea tree oil appear to reduce terpinen-4-ol efficacy by lowering its aqueous solubility. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings explain why tea tree oil can be less active in vitro than terpinen-4-ol alone and further suggest that the presence of a non-aqueous phase in tea tree oil formulations may limit the microbial availability of its active components.

The bacterial multiple antibiotic resistant (Mar) phenotype leads to increased tolerance to tea tree oil.

Mutants of Escherichia coil strain AG100 exhibiting the multiple antibiotic resistance (Mar) phenotype demonstrated a greater level of tolerance to tea tree oil (TTO) compared with the parent strain. The ability of TTO to kill all E. coil strains studied was greater at 37 than at 30 degrees C. Growth of parent strain AG100 in the presence of salicylate, which induces the mar operon leading to the Mar phenotype, also increased tolerance to TTO. Escherichia coli Mar mutant YL1 demonstrated greater tolerance to antimicrobial terpenes found in TTO and did not leak K+ as rapidly in the presence of TTO when compared with its parent strain AG100. Attempts to isolate Mar mutants of Staphylococcus aureus using tetracycline gradients proved unsuccessful. However, when grown in the presence of salicylate, S. aureus strain BB255 demonstrated greater tolerance to TTO and did not leak K+ as rapidly in the presence of TTO compared with this strain grown without additions. This evidence demonstrates that bacterial Mar phenotypes increase tolerance to the killing action of TTO. This work also adds indirect evidence that the target of TTO is the cell membrane.

Treating benign colon disorders using laparoscopic colectomy.

Laparoscopic bowel surgery is a recent application of minimally invasive videoscopic techniques. Understanding the anatomy and physiology of the bowel, the background of bowel disorders and their treatment, signs and symptoms of bowel disease, and the patient selection process can help perioperative nurses better care for patients diagnosed with colon polyps, diverticulitis, and inflammatory bowel disease.

The outer membrane of Pseudomonas aeruginosa NCTC 6749 contributes to its tolerance to the essential oil of Melaleuca alternifolia (tea tree oil).

Pseudomonas aeruginosa is less susceptible to the antimicrobial properties of tea tree oil than many bacteria and its tolerance is considered to be due to its outer membrane. Polymyxin B nonapeptide (PMBN), which has no antibacterial action, was used to permeabilize the outer membrane. The addition of PMBN to Ps. aeruginosa NCTC 6749 markedly increased this organism's susceptibility to tea tree oil and to its normally inert hydrocarbons, p-cymene and gamma-terpinene.

The mode of antimicrobial action of the essential oil of Melaleuca alternifolia (tea tree oil).

The essential oil of Melaleuca alternifolia (tea tree) exhibits broad-spectrum antimicrobial activity. Its mode of action against the Gram-negative bacterium Escherichia coli AG100, the Gram-positive bacterium Staphylococcus aureus NCTC 8325, and the yeast Candida albicans has been investigated using a range of methods. We report that exposing these organisms to minimum inhibitory and minimum bactericidal/fungicidal concentrations of tea tree oil inhibited respiration and increased the permeability of bacterial cytoplasmic and yeast plasma membranes as indicated by uptake of propidium iodide. In the case of E. coli and Staph. aureus, tea tree oil also caused potassium ion leakage. Differences in the susceptibility of the test organisms to tea tree oil were also observed and these are interpreted in terms of variations in the rate of monoterpene penetration through cell wall and cell membrane structures. The ability of tea tree oil to disrupt the permeability barrier of cell membrane structures and the accompanying loss of chemiosmotic control is the most likely source of its lethal action at minimum inhibitory levels.

A new mechanism of action of fluoride on streptococci.

Addition of fluoride to the growth medium of Streptococcus sobrinus resulted in a loss of glucan-binding lectin activity. Upon removal of fluoride, the bacteria regained their ability to bind glucan in about one generation. Chloramphenicol prevented recovery of ability to produce the lectin, showing the requirement for protein synthesis. Fluoride also caused a significant reduction in the tendency of the streptococci to form chains of cells, although the spent medium from fluoride-containing growth media did not dechain control cells. The fluoride thus does not activate autolytic enzymes. Importantly, 2-D electrophoresis and SDS-PAGE revealed several proteins were synthesized in the presence of fluoride that were not synthesized in its absence. It seems possible that fluoride places a stress on the bacteria, causing the synthesis of proteins that may play a role in protecting the cells against the stress. Numerous stress proteins are known for bacteria, including those resulting from heat, enzymes and osmotic shocks. The ability of fluoride to cause loss of glucan-binding may be related to its reported beneficial effects on oral health.

An orientation anisotropy in the effects of scaling vertical disparities.

Gårding et al. (Vis Res 1995;35:703-722) proposed a two-stage theory of stereopsis. The first uses horizontal disparities for relief computations after they have been subjected to a process called disparity correction that utilises vertical disparities. The second stage, termed disparity normalisation, is concerned with computing metric representations from the output of stage one. It uses vertical disparities to a much lesser extent, if at all, for small field stimuli. We report two psychophysical experiments that tested whether human vision implements this two-stage theory. They tested the prediction that scaling vertical disparities to simulate different viewing distances to the fixation point should affect the perceived amplitudes of vertically but not horizontally oriented ridges. The first used elliptical half-cylinders and the 'apparently circular cylinder' judgement task of Johnston (Vis Res 1991;31:1351-1360). The second experiment used parabolic ridges and the amplitude judgement task of Buckley and Frisby (Vis Res 1993;33:919-934). Both studies broadly confirmed the anisotropy prediction by finding that large scalings of vertical disparities simulating near distances had a strong effect on the perceived amplitudes of the vertically oriented stimuli but little effect on the horizontal ones. When distances > 25 cm were simulated there were no significant differential effects and various methodological reasons are offered for this departure from expectations.

Tea tree oil causes K+ leakage and inhibits respiration in Escherichia coli.

Concentrations of tea tree oil (TTO) which inhibit or decrease growth of Escherichia coli also inhibit glucose-dependent respiration and stimulate the leakage of intracellular K+. Stationary phase cells are more tolerant to these TTO effects than exponential phase cells.

Pooling of vertical disparities by the human visual system.

Two experiments are described in which the effects of scaling vertical disparities on the perceived amplitudes of dome-shaped surfaces depicted with horizontal disparities were examined. The Mayhew and Longuet-Higgins's theory and the regional-disparity-correction theory of Garding et al predict that scaling should generate a change in perceived depth appropriate to the viewing distance simulated by the scaled vertical disparities. Significant depth changes were observed, by means of a nulling task in which the vertical-disparity-scaling effect was cancelled by the observer choosing a pattern of horizontal disparities that made the dome-shaped surface appear flat. The sizes of the scaling effects were less than those predicted by either theory, suggesting that other cues to fixation distance such as oculomotor information played an appreciable role. In conditions in which 50% of the texture elements were given one value of vertical-disparity scaling and the remaining 50% were left unscaled, the size of the scaling effect on perceived depth could be accounted for by equally weighted pooling of the vertical-disparity information unless the two scalings were very dissimilar, in which case the lower scaling factor tended to dominate. These findings are discussed in terms of a Hough parameter estimation model of the vertical-disparity-pooling process.

Fluoride inhibits the glucan-binding lectin of Streptococcus sobrinus.

The glucan-binding lectins of Streptococcus cricetus AHT and Streptococcus sobrinus 6715 were reversibly inhibited by sodium fluoride. Fluoride was superior to chloride, bromide, iodide and thiocyanate in preventing glucan-mediated aggregation of the bacteria. Fluoride was also an effective inhibitor of the sucrose-dependent adhesion of S. sobrinus to glass surfaces. The inhibition of glucan-binding lectin activities may be one of the mechanisms of action of fluoride in preventing dental disease.

Hyperbaric oxygen as prophylaxis or treatment for radiation myelitis.

This animal study was designed to investigate HBO as a treatment or prophylaxis for radiation myelitis. All animals received identical spinal cord radiation doses of 69 Gy in 10 daily fractions. Group I received no HBO; group II began HBO at the onset of signs of myelitis; group III received HBO with prophylactic intent beginning 6 wk after irradiation; and group IV received both modalities on the same day, but radiation always preceded HBO by at least 4 h. HBO consisted of 90 min oxygen at 2.4 atm abs for 20 daily treatments. Animals were objectively assessed for the loss of certain neurologic reflexes indicative of four levels of myelitis. Although all animals progressed to severe myelitis, group III animals had group-averaged levels of myelitis consistently less than control. The differences were statistically significant for several weeks. Group IV animals progressed to severe myelitis much more rapidly than any other group. Additional study is justified by this trial. Key questions to be answered include the optimal timing of HBO to produce a beneficial rather than detrimental effect.

A critical appraisal of positive cooperativity in oral streptococcal adhesion: Scatchard analyses of adhesion data versus analyses of the spatial arrangement of adhering bacteria.

Positive cooperativity is a mechanism proposed to account for the adhesion of bacteria to surfaces. In this paper, two methods that both claim to assess experimentally cooperative phenomena, viz. Scatchard analysis of adhesion data (using adhesion to vials) and analysis of the spatial arrangement of adhering cells (using a flow chamber), were compared and critically evaluated. Three oral strains were used and the substrata involved were glass (hydrophilic) and silicone-coated glass (hydrophobic) employed with or without a salivary coating. Scatchard analysis and near-neighbour analysis of adhering cells yield similar conclusions with regard to the mechanism of adhesion of the cells, provided that adhering cells are sufficiently immobilized under the experimental conditions. In the case of incomplete immobilization, near-neighbour collection results from sliding of adhering cells rather than from cooperative phenomena. Also, the agreement between the conclusions from both methods seems to be better, the more reversibly the cells adhere. Positive cooperativity can be absent or present on saliva-coated substrata with a distinct effect of the substratum hydrophobicity, despite the presence of an adsorbed film. This suggests that a different pellicle develops on a hydrophobic substratum than on a hydrophilic substratum. This is confirmed by our observation that the amino acid composition of salivary films is different on both types of substratum.

Imaging and manipulating molecules on a zeolite surface with an atomic force microscope.

The adsorption of neutral molecules and ions on the surfaces of zeolites was observed in real time with an atomic force microscope (AFM). Direct imaging of the surface of the zeolite clinoptilolite was possible by using a diluted tert-butyl ammonium chloride solution as a medium. Images of the crystal in different liquids revealed that molecules could be bound to the surface in different ways; neutral molecules of tert-butanol formed an ordered array, whereas tert-butyl ammonium ions formed clusters. These absorbed molecules were not rearranged by the AFM tip when used in an imaging mode. However, when a sufficiently large force was applied, the tip of the AFM could rearrange the tert-butyl ammonium ions on the zeolite surface. This demonstration of molecular manipulation suggests new applications, including biosensors and lithography.

Molecular cloning of the genomes of poliovirus type 3 strains by the cDNA: RNA hybrid method.

The genomes of two neurovirulent strains of poliovirus type 3, wild type P3/Leon/37 and a vaccine revertant P3/119/70, have been cloned in E. coli. The cDNA: RNA hybrid method used was efficient and may have wide applicability for cDNA cloning. Overlapping clones spanning the entire genome were obtained for each strain. These have been used to produce full-length DNA copies of the two genomes each within a single plasmid.

Right-handed alternating DNA conformation: poly(dA-dT) adopts the same dinucleotide repeat with cesium, tetraalkylammonium, and 3 alpha, 5 beta, 17 beta-dipyrrolidinium steroid dimethiodide cations in aqueous solution.

We demonstrate that poly(dA-dT) can adopt two conformations in solution, with the relative proportions dependent on the nature and concentration of the counter ion and cationic ligands. The synthetic DNA exhibits a dinucleotide repeat conformation on addition of CsF and Me4NCl at molar concentrations, with the NMR spectral changes reflecting a common conformational change at one glycosidic torsion angle and one phosphodiester linkage. We also observe the same dinucleotide repeat in the neighbor-exclusion 3 alpha, 17 beta-dipyrrolidin-1'-yl-5 beta- delta 9,11-androstene dimethiodide (3 alpha, 5 beta, 17 beta-dipyrandenium) complex, with the steroid diammonium ligand binding in the groove of the stacked poly(dA-dT) duplex and the complex stabilized through the interaction of one of the charged ends with the backbone phosphate. We demonstrate further that 3 alpha, 5 beta, 17 beta-dipyrandenium bound to poly(dA-dT) at low binding ratios induces a switch to the dinucleotide repeat conformation at adjacent steroid-free duplex regions. This observation contrasts with a previous demonstration that the diastereoisomeric 3 beta, 5 alpha, 17 beta-dipyrandium binds to poly(dA-dT) by partial insertion between unstacked tilted base pairs. The NMR parameters rule out a left-handed alternating DNA structure (Z DNA) for the observed poly(dA-dT) dinucleotide repeat conformation, but right-handed alternating DNA models are under consideration. The facile interconversion of poly(dA-dT) between two conformations, one of which exhibits a dinucleotide repeat and can be induced by ligand binding, may provide a mechanism for the recognition of specific nucleic acid sequences by DNA-binding proteins.