Biomedical subjects

S P Denyer

Publications and source records attributed to S P Denyer.

At least 19 recordsLinked to original sources

PubMed2006

Alcohol ethoxylates mediate their bacteriostatic effect by altering the cell membrane of Escherichia coli NCTC 8196.

The minimum inhibitory concentration (MIC) of a homologous series of alcohol ethoxylates with the same head group size (E6) but differing in the number of carbon atoms in their 'tail group' from 10 to 16 was determined for Staphylococcus aureus NCTC 4163 and Escherichia coli NCTC 8196 using a turbidimetric assay. All the surfactants tested demonstrated bacteriostatic activity against both organisms. A tetrazolium assay showed that C14E6 and C16E6 had little effect on the membrane-bound dehydrogenase enzyme activity of E. coli NCTC 8196 compared with C10E6 and C12E6. C10E6 caused leakage both of K(+) and nucleotides in a concentration-dependent manner above its MIC of 0.2 mM. C12E6 caused some leakage at concentrations below its MIC (0.12 mM).

Alcohols↗

PubMed2006

In vivo assessment of the osteointegrative potential of phosphatidylserine-based coatings.

The successful implantation of titanium-based implants for orthopaedic and dental applications is often hindered because of their mobility, which arises because of a lack of direct binding of the metal surface to the mineral phase of the surrounding bone. Ceramic coatings, although ensuring the integration of the implant within the tissue, are unstable and carry risks of delamination and of failure. Recently, a novel biomimetic approach has been developed where porous titanium implants are coated with calcium-binding phospholipids able to catalyse the nucleation of discrete apatite crystals after only 30 min incubation in simulated body fluids. The present work assesses the osteointegrative potential of this new class of coatings in an in vivo rabbit model and compares its performance with those of bare porous titanium and hydroxyapatite-coated titanium. The data obtained show that phosphatidylserine-based coatings, whilst resorbing, drive the growing bone into apposition with the metal surface. This is in contrast to the case of bare titanium.

Animals↗

PubMed2006

When does it hurt? Pain during flexible cystoscopy in men.

INTRODUCTION: In this observational study, pain was measured continuously in men undergoing flexible cystoscopy, in order to help to identify which parts of the procedure were the most painful. PATIENTS AND METHODS: Men who were to undergo flexible cystoscopy were recruited. Pain was measured continuously throughout flexible cystoscopy by asking the subject to squeeze a pressure-sensing rubber bulb in proportion to the amount of discomfort experienced giving a score of 0-10. RESULTS: The most painful part of the procedure was as the cystoscope passed through the membranous urethra with a median pain score of 2.82. The initial lidocaine administration gives a median pain score of 0.84. The other parts of the cystoscopy produced median scores of between 0.14 and 0.33. The difference in the pain scores was significant (p = 0.03). There was no statistically significant difference in the pain scores between those who were having their first cystoscopy and those who were having repeat cystoscopy. The subjects' age did not influence the degree of pain experienced. CONCLUSION: This study gives further understanding of how pain is experienced during flexible cystoscopy. It may help explain why previous studies have not reached a clear consensus on the value of lidocaine during flexible cystoscopy.

Aged↗

PubMed2004

The synergistic effect of EDTA/antimicrobial combinations on Pseudomonas aeruginosa.

AIMS: To demonstrate that the nonlinear concentration-dependent inhibition of Pseudomonas aeruginosa to EDTA can be used to successfully model and predict the potentiation of antimicrobials by EDTA. METHODS AND RESULTS: A model used successfully to describe the concentration-dependent inhibition of bacterial growth caused by many antimicrobials was unable to describe the inhibition of P. aeruginosa by EDTA. Examination of the inhibition profiles for EDTA against P. aeruginosa revealed a biphasic inhibitory pattern suggesting different mechanisms of action at different concentrations. A modelled, two-stage inhibitory process was shown to fit the observations. This model was then used to examine the effect of combining EDTA with other antimicrobials. The apparent synergy of mixtures of EDTA with quaternary ammonium surfactants (QAC) and specific antibiotics was successfully modelled. Minimum inhibitory concentrations (MIC) of the QAC and that of oxacillin and cefamandole were reduced by a factor of 3-10, whereas ampicillin was reduced by a factor of 70 from an MIC of 1524 to 21 mg l(-1) in the presence of 500 mg l(-1) of EDTA. CONCLUSIONS: A nonlinear concentration-dependent inhibition of P. aeruginosa by EDTA gives rise to apparent observation of synergy with other antimicrobials. SIGNIFICANCE AND IMPACT OF THE STUDY: This is a further example where the current methodology for the examination of antimicrobial synergy (the summed fractional inhibitory concentrations) leads to false conclusions.

Ampicillin↗

PubMed2004

Biofilm formation and changes in bacterial cell surface hydrophobicity during growth in a CAPD model system.

Peritonitis is a frequent complication of continuous ambulatory peritoneal dialysis (CAPD), with patients suffering recurrent attacks. The microorganisms most frequently implicated in the infection are the skin microflora, in particular, the coagulase-negative staphylococci such as Staphylococcus epidermidis. These microorganisms gain access to the peritoneal cavity via the in-dwelling silicone rubber catheter in the abdominal wall and often persist as biofilms on the surface of the catheter. The surface characteristics of S. epidermidis were monitored during growth in a CAPD in-vitro model together with their ability to adhere to silicone rubber substrata. Fresh dialysis fluid exerted an injurious effect on the cells leading to a decrease in cell numbers but during the simulated dialysis period the cells adapted to the applied stresses. Over a 96-h period in the model both a clinical isolate and a skin isolate of S. epidermidis adopted a more hydrophobic phenotype. The data presented here show that the bacteria grown in this in-vivo reflective CAPD model continually adapt to their environment and become more tolerant to the stresses imposed. The adapted cells were seen to colonise silicone rubber substrata.

Bacterial Adhesion↗

PubMed2003

Theory of antimicrobial combinations: biocide mixtures - synergy or addition?

AIMS: To demonstrate the effect that non-linear dose responses have on the appearance of synergy in mixtures of antimicrobials. METHODS AND RESULTS: A mathematical model, which allows the prediction of the efficacy of mixtures of antimicrobials with non-linear dose responses, was produced. The efficacy of antimicrobial mixtures that would be classified as synergistic by time-kill methodology was shown to be a natural consequence of combining antimicrobials with non-linear dose responses. CONCLUSIONS: The effectiveness of admixtures of biocides and other antimicrobials with non-linear dose responses can be predicted. If the dose response (or dilution coefficient) of any biocidal component, in a mixture, is other than one, then the time-kill methodology used to ascertain the existence of synergy in antimicrobial combinations is flawed. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model developed allows the prediction of the efficacy of antimicrobial combinations. Combinations of known antimicrobials, which reduce the time taken to achieve a specified level of microbial inactivation, can be easily assessed once the kinetic profile of each component has been obtained. Most patented cases of antimicrobial synergy have not taken into account the possible effect of non-linear dose responses of the component materials. That much of the earlier literature can now be predicted, suggests that future cases will require more thorough proof of the alleged synergy.

Cresols↗

PubMed2003

Membrane damage to bacteria caused by single and combined biocides.

AIMS: To examine the effect on the leakage of low molecular weight cytoplasmic constituents from Staphylococcus aureus using phenolics singly and in combination, and to see if the observations could be modelled using a non-linear dose response. METHODS AND RESULTS: The rate of potassium, phosphate and adenosine triphosphate leakage was examined in the presence of chlorocresol and m-cresol. Individually, leakage was observed only at long contact times or high concentrations. Combined at these ineffective concentrations, the cytoplasmic pool of all constituents studied was released within minutes. Both chlorocresol and m-cresol were shown to have non-linear dose responses. A rate model for the combinations, which takes account of these non-linear responses, accurately predicted the observations. CONCLUSIONS: Antimicrobials, which when used alone exhibit a non-linear dose response, will also give a non-linear dose response in combination. The simple linear-additive model ignores the concept of the dilution coefficient and will always describe the phenomenon of synergy for combinations where one or more of the components has a dilution coefficient greater than one. This has been borne out by examination of the purported prime lesion of chlorocresol and m-cresol, alone and in combination. SIGNIFICANCE AND IMPACT OF THE STUDY: Studies aimed at producing synergistic mixtures of antimicrobials, which ignore the non-linear additive effect, may waste valuable research effort looking for a physiological explanation for an apparent synergy, where none, in-fact, exists. Patents granted on the basis of analyses using the linear-additive model for combinations of compounds with non-linear dose responses may no longer be supportable.

Adenosine Triphosphate↗

PubMed2002

A rapid method for assessing the suitability of quenching agents for individual biocides as well as combinations.

AIMS: To develop a novel, rapid method for testing the ability of quenching agents to neutralize disinfectants. METHODS AND RESULTS: Tests were performed to determine the suitability of different neutralizers for a range of disinfectants, using a new method based on the Bioscreen optical density analyser. Results showed that during disinfection tests, efficacy could be over-estimated due to poor, or no, neutralization of the disinfectant after a specified time of exposure to the bacteria. The failure to distinguish adequately between bacteriostatic and bactericidal effects can lead to false results during disinfectant testing. Experiments also showed that dilution of the disinfectant, following exposure to the bacteria, was not always sufficient to stop the activity of the disinfectant for chemicals with low dilution coefficients. CONCLUSIONS: The quench test proved to be very quick and easy to perform, with results being available within 18 h. Using the Bioscreen, the test is automated and determines whether dilution into a particular neutralizer is able to inactivate a disinfectant within 30 s. SIGNIFICANCE AND IMPACT OF THE STUDY: This new approach allows the efficacy of quenching agents to be determined, prior to undertaking each disinfection study, and can help in the development of more suitable quenching solutions. The test has also been used to find suitable neutralizers for mixtures of disinfectants which might be used during studies on synergistic biocide combinations.

Colony Count, Microbial↗

PubMed2002

Cellular impermeability and uptake of biocides and antibiotics in Gram-negative bacteria.

The principal targets for antibacterial agents reside at the cytoplasm and cytoplasmic membrane, damage to other structures often arising from initial events at these loci. The Gram-negative bacteria offer a complex barrier system to biocides and antibiotics, regulating, and sometimes preventing, their passage to target regions. Routes of entry differ between hydrophobic and hydrophilic agents, often with a structure dependency; specialized uptake mechanisms are exploited and portage transport can occur for pro-drug antibacterials. Uptake isotherms offer insight into the sorption process and can sometimes shed light on biocide mechanisms of action. The multi-component barrier system of Gram-negative bacteria offers opportunities for phenotypic resistance development where partitioning or exclusion minimizes the delivery of an antibacterial agent to the target site. Active efflux processes are recognized as increasingly relevant mechanisms for resistance, potentially offering routes to biocide:antibiotic cross-resistance. These mechanisms may be targeted directly in an attempt to compromise their role in microbial survival.

Anti-Bacterial Agents↗

PubMed2002

Cellular impermeability and uptake of biocides and antibiotics in gram-negative bacteria.

The principal targets for antibacterial agents reside at the cytoplasm and cytoplasmic membrane, damage to other structures often arising from initial events at these loci. The gram-negative bacteria offer a complex barrier system to biocides and antibiotics, regulating, and sometimes preventing, their passage to target regions. Routes of entry differ between hydrophobic and hydrophilic agents, often with a structure dependency; specialized uptake mechanisms are exploited and portage transport can occur for pro-drug antibacterials. Uptake isotherms offer insight into the sorption process and can sometimes shed light on biocide mechanisms of action. The multi-component barrier system of gram-negative bacteria offers opportunities for phenotypic resistance development where partitioning or exclusion minimizes the delivery of an antibacterial agent to the target site. Active efflux processes are recognized as increasingly relevant mechanisms for resistance, potentially offering routes to biocide:antibiotic cross-resistance. These mechanisms may be targeted directly in an attempt to compromise their role in microbial survival.

Anti-Bacterial Agents↗

PubMed2001

Ocular biomaterials and implants.

The maintenance of vision is a key determinant of healthy ageing. This has been facilitated over recent decades by the development of a wide range of implants and biomedical devices to correct the functional deficiencies of disease, age and ocular trauma. This brief overview provides an insight into the structure of this unique organ, the major physiological functions of the component tissues and the present state of the art with respect to modern ocular implants. The review focuses primarily on the existing limitations of existing ocular biomaterials used in the fabrication of contact lenses, intraocular lenses, glaucoma filtration implants, keratoprostheses, intracorneal implants, scleral buckles and viscoelastic replacement agents. The challenge of improving ocular compatibility and ensuring the longevity of indwelling ocular devices is addressed along with the need to improve the physicochemical and mechanical properties of existing ocular biomaterials.

Animals↗

PubMed2001

A comparison of the use of an ATP-based bioluminescent assay and image analysis for the assessment of bacterial adhesion to standard HEMA and biomimetic soft contact lenses.

The aim of this study was to investigate in vitro adhesion of clinically relevant bacteria to standard HEMA and novel biomimetic soft contact lenses (SCL) using bioluminescent ATP assay and image analysis. Unworn SCL were incubated with Pseudomonas aeruginosa, Staphylococcus epidermidis or Serratia marcescens suspended in sterile phosphate buffered saline (PBS). The level of bacterial adhesion after 1, 2, 4, 6 and 18h, was assessed using both image analysis and a bioluminescent ATP assay. Species differences in the overall level of adhesion to the different types of lens were observed using both measurement techniques. Generally bacterial adhesion was shown to peak at 4-6 h, then decline to a much lower level by 18 h. After 4 h, adhesion of all species of bacteria to the biomimetic SCL (omafilcon A) was found to be significantly lower than to the standard HEMA SCL (polymacon) (p<0.05. Student's t-test, n = 4). Both these techniques demonstrated that novel biomimetic SCL materials exhibit significantly lower bacterial adhesion in vitro compared to standard HEMA SCL materials. SCL manufactured with these novel biomimetic materials may reduce the risk of infection.

Adenosine Triphosphate↗

PubMed2001

Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms.

To cause an infection, bacteriophages must penetrate the alginate exopolysaccharide of Pseudomonas aeruginosa to reach the bacterial surface. Despite a lack of intrinsic motility, phage were shown to diffuse through alginate gels at alginate concentrations up to 8% (wt/vol) and to bring about a 2-log reduction in the cell numbers in 20-day-old biofilms of P. aeruginosa. The inability of alginate to act as a more effective diffusional barrier suggests that phage may cause a reduction in the viscosity of the exopolysaccharide. Samples (n = 5) of commercial alginate and purified cystic fibrosis (CF) alginate were incubated with 2 x 10(8) purified phage per ml for 24 h at 37 degrees C. After incubation the samples and controls were subjected to rheological analysis with a Carrimed controlled stress rheometer. The viscosities of phage-treated samples were reduced by up to 40% compared to those of controls incubated in the absence of phage. The experiment was repeated by using phage concentrations of 10(10) and 10(12) phage per ml and samples taken for analysis at intervals up to 4 h. The results indicated that there was a time- and concentration-dependent reduction in viscosity of up to 40% compared to the viscosities of the controls. Commercial and purified CF alginate samples, both phage treated and untreated, were subjected to gel filtration chromatography by using Sephacryl High Resolution S-400 medium in order to obtain evidence of degradation. The results demonstrated that alginate treated with phage had a lower molecular weight than untreated alginate. The data suggest that bacteriophage migration through P. aeruginosa biofilms may be facilitated by a reduction in alginate viscosity brought about by enzymic degradation and that the source of the enzyme may be the bacterial host itself.

Alginates↗

PubMed2000

A cell kinetic analysis of human umbilical vein endothelial cells.

Cultures of normal human cells 'age' and become senescent in vitro due to a continuously declining mitotic fraction. Although endothelial cells represent a tissue of major relevance in the development of age-related vascular disease, the rate at which these cells senesce has never been systematically measured in culture. Accordingly the population kinetics of human vascular endothelial cells (HUVECs) serially passaged in vitro has been studied in order to determine (i) the rate of decline in the growth fraction; (ii) the rate of increase of the senescent fraction and (iii) the relationship between changes in these parameters and the baseline rate of apoptosis. Immunocytochemical visualisation of the growth fraction using antisera to the proliferation marker pKi67 showed a rate of decline in the growth fraction of 4.43+/-0.31% per population doubling. This was not accompanied by any change in cell cycle time as assessed using time lapse video microscopy. The number of senescent cells within the population increased at a rate of 6.47+/-0.3% as assessed by senescence associated beta-galactosidase activity. The baseline rate of apoptosis as measured by TUNEL remained essentially unchanged (0.31+/-0.07%) during this process. These data show (i) that senescence and apoptosis are unrelated processes in HUVEC and (ii) that senescent cells rapidly and progressively accumulate in dividing populations of endothelial cells. The physiological relevance of these observations is discussed.

Apoptosis↗

PubMed1999

Effect of the urine conditioning film on ureteral stent encrustation and characterization of its protein composition.

The goal of this study was to characterize the protein composition of the conditioning film deposited onto the surface of ureteral stents during in vivo implantation and to relate its presence to the precipitation of calcium crystals. The protein pattern of the conditioning film of implanted nonencrusted and encrusted urological stents was assessed by SDS-PAGE and Western blot of the desorbed species. The results obtained highlighted different electrophoresis profiles between nonencrusted and encrusted stents. Western blot showed the ubiquitous presence of albumin, while Tamm-Horsfall Protein and alpha1-microglobulin adsorption was limited to nonencrusted devices. By an in vitro dynamic model in which artificial urine was flowed through the lumen of control and retrieved nonencrusted stents, we demonstrated that the organic layer remarkably enhanced crystal precipitation and aggregation events on the surface.

Adsorption↗

PubMed1999

A standard strain of human ocular keratocytes.

The ability of an injured cornea to regenerate from deep tissue trauma is largely due to wound healing processes mediated by the surviving stromal keratocytes. Despite the importance of the wound healing process, and the ease with which keratocytes can be grown in tissue culture, a standardised strain of the cells has never been made available. Accordingly, this study reports a strain of human embryonic keratocytes, designated EK1.BR as a research tool for the ophthalmic community. EK1.BR has been characterised with respect to life-span, fraction of dividing cells and maintenance of a keratocyte phenotype in culture. It is hoped that these cells will prove useful in the in vitro study of stromal wound healing and the characterisation of keratocyte gene expression.

Aged↗

PubMed1999

A novel image-analysis technique for measurement of bacterial cell surface tension.

Cell-surface hydrophobicity is different for Staphylococcus epidermidis cells grown under different environmental conditions; this might influence attachment and colonization of surfaces. Although a wide variety of techniques has been employed to measure bacterial surface hydrophobicity, including contact angle determinations, adherence to hydrocarbons, hydrophobic-interaction chromatography and salt aggregation, many of these either require large numbers of cells or do not yield comparable quantitative data. This study describes a novel, quantitative method for the determination of bacterial surface tension on the basis of image analysis of cell-cell interactions. S. epidermidis (strains 900 and 901) were suspended in different concentrations of propanol of known surface tension and examined by bright-field microscopy linked via a charge-couple device (CCD) camera to an image analyser. Frames were chosen randomly and the data recorded as a ratio of count/percentage coverage for each frame. The results showed that for strains 900 and 901 this ratio was maximum at surface tensions of 67 and 61 mN m(-1) respectively. At these values of minimal interaction the surface tension of the liquid was equal to the bacterial cell surface tension. The results were in close agreement with those obtained from contact angles. The advantage of surface tension measurements is that, irrespective of the method used, the results generated are quantitative values and are therefore directly comparable. The method reported is reliable, reproducible and is of particular value because the number of cells required is, typically, at least two orders of magnitude lower than is required for commonly used alternative methods.

Ammonium Sulfate↗

PubMed1998

Possible role of alpha-1-microglobulin in mediating bacterial attachment to model surfaces.

Urine proteins in the molecular weight range of 9-137 kDa deposit to an equal extent from pooled human urine onto glass (12.7 +/- 1.9 micrograms/cm) and polystyrene (11.8 +/- 1.8 micrograms/cm). Selective desorption of the proteins was achieved by washing with water or water/isopropanol mixtures. Irrespective of the washing process, proteins of molecular weight greater than 90 kDa remained associated with both surfaces while water washings alone removed most low molecular weight material. A 29 kDa protein, alpha-1-microglobulin, was removed from glass by water washing but required a 30% (v/v) isopropanol wash to desorb from polystyrene, implying attachment via hydrophobic bonding. The adhesion to polystyrene surfaces of Pseudomonas aeruginosa B4, a clinical isolate from a urinary tract infection (UTI), was strongly associated with the presence of alpha-1-microglobulin, which may be acting as a mediator of bacterial adhesion.

Adsorption↗