G. W. Hanlon

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

ABSTRACT 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 × 108 purified phage per ml for 24 h at 37°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 1010 and 1012 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.

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.

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. Students 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.

Preservative efficacy tests in formulated nasal products: reproducibility and factors affecting preservative activity.

Preservative efficacy tests were performed in triplicate on each of three batches of three formulated nasal spray preparations to assess the inter‐ and intra‐batch variation in preservative performance which typically results from these procedures, and to assess the relative importance of factors influencing preservative performance in nasal products.

Virulence traits associated with verocytotoxigenic Escherichia coli O157 recovered from freshwater biofilms

Aim:  To investigate whether epilithic biofilms in freshwater streams in a mixed UK agricultural river catchment harbour Escherichia coli O157, and if so, whether they demonstrate an association with those excreted by grazing farm animals.

Evaluation of environmental scanning electron microscopy for analysis of Proteus mirabilis crystalline biofilms in situ on urinary catheters

Proteus mirabilis is a common cause of catheter-associated urinary tract infections and frequently leads to blockage of catheters due to crystalline biofilm formation. Scanning electron microscopy (SEM) has proven to be a valuable tool in the study of these unusual biofilms, but entails laborious sample preparation that can introduce artefacts, undermining the investigation of biofilm development. In contrast, environmental scanning electron microscopy (ESEM) permits imaging of unprocessed, fully hydrated samples, which may provide much insight into the development of P. mirabilis biofilms. Here, we evaluate the utility of ESEM for the study of P. mirabilis crystalline biofilms in situ, on urinary catheters. In doing so, we compare this to commonly used conventional SEM approaches for sample preparation and imaging. Overall, ESEM provided excellent resolution of biofilms formed on urinary catheters and revealed structures not observed in standard SEM imaging or previously described in other studies of these biofilms. In addition, we show that energy-dispersive X-ray spectroscopy (EDS) may be employed in conjunction with ESEM to provide information regarding the elemental composition of crystalline structures and demonstrate the potential for ESEM in combination with EDS to constitute a useful tool in exploring the mechanisms underpinning crystalline biofilm formation.

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.

A mechanistic investigation into the microbial chiral inversion of 2-arylpropionic acids using deuterated derivatives of 2-phenylpropionic acid

Abstract Cordyceps militaris has been previously shown to invert the chirality of ( r )-2-phenylpropionic acid to its antipode in the absence of any other biotransformation. To investigate the mechanism of this unusual biotransformation. ( r , s )-[2- 2 H 1 ]-2-phenylpropionic acid, ( r , s )-[2- 2 H,3,3,3- 2 H 3 ]-2-phenylpropionic acid, and ( r , s )-[3,3,3- 2 H 3 ]-2-phenylpropionic acid were synthesized and incubated with C. militaris . NMR spectroscopy showed that deuterium exchange of the α-methine proton occurred during the inversion process but there was no exchange of the β-methyl protons. There were no significant differences in the rates of chiral inversion of the three deuterated derivatives and the undeuterated compound, and with all the compounds attaining 84% enantiomeric excess of the ( s )-enantiomer after 48 h incubation. The deuterium exchange is not therefore the rate limiting process in this biotransformation. The recovery profiles demonstrated that there was rapid metabolism of the β-methyl deuterated derivatives which did not occur for the undeuterated 2-phenylpropionic acid or the derivative which was only deuterated at the α-carbon. These studies clearly illustrate that C. militaris is a useful in vitro model of the chiral inversion of 2-arylpropionic acid derivatives in mammalian systems.

Indirect enantiomeric separation of 2-arylpropionic acids and structurally related compounds by reversed phase HPLC

A reversed-phase high-performance liquid chromatographic method, using an organic modifier-phosphate buffered mobile phase, for the determination of the enantiomeric composition of 2-arylpropionic acids and other structurally related compounds in microbial media is described. The method is based on the resolution of diastereoisomeric amides formed from the reaction of the arylpropionic acid with either (-)-(S)-alpha-methylbenzylamine or (-)-(S)-1-(naphthen-1-yl)ethylamine in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl and 1-hydroxybenzotriazole and incorporating an internal standard. The addition of sodium pentanesulphonate to the mobile phase as an ion-pairing agent was necessary to remove unreacted amine to avoid rapid column deterioration. The method provides and efficient, rapid and reproducible means of monitoring the microbial chiral inversion of 2-arylpropionic acids and other structurally related molecules.

The influence of critical surface tension and microrugosity on the adhesion of bacteria to polymer monofilaments

Abstract The adhesion of S. aureus (NCIB 10788) and E. coli (NCIB 8196) to monofilaments of polypropylene (PP), polyethylene (PE), nylon (N) and polyvinylidine chloride (PVDC) was determined using [methyl-3H]thymidine radiolabelled cells. Critical surface tensions were determined from Zisman plots and a microrugosity index developed using the waveform line from scanning electron micrographs of the substrate surface. S. aureus adhered in fewer numbers per cm2 to PP than to PE, N or PVDC (P N > PE > PP whereas the microrugosity indices of N and PE were greater than PP. PVDC could not be assigned a satisfactory microrugosity index since the monofilament possessed well-defined score marks on an otherwise ‘smooth’ surface. From critical surface tension measurements bacterial adhesion in vitro may have been expected to be the greatest to PP and least to PVDC: microrugosity of the substrate surface appears therefore to be a contributory factor in determining the extent of bacterial adhesion.