TOF-SIMS imaging of chlorhexidine-digluconate transport in frozen hydrated biofilms of the fungus Candida albicans

The diffusion of the anti-microbial chlorhexidine digluconate (CHG) has been studied in C. albicans biofilms by time-of-flight secondary-ion mass spectrometry (TOF-SIMS). C. albicans has been shown to become resistant to common anti-microbial agents, including CHG, when growing as a biofilm. Mass transport resistance within biofilms has commonly been suggested as a resistance mechanism, but measurement of transport for most anti-microbial agents in biofilms has proven extremely difficult because of the heterogeneity of the biofilms and the difficulty in detecting these agents within an intact biofilm. In this study, TOF-SIMS has been used to study the transport of CHG and glucose in a frozen hydrated biofilm. The TOF-SIMS images reveal a progression of CHG from the top of the biofilm to its base with time. Images suggest that there are channels within the biofilm and show preferential binding of CHG to cellular components of the biofilm. Additionally, both living and dead cells can be identified in the TOF-SIMS images by the sequestration of K⁺ and the presence of cell markers. This study demonstrates that TOF-SIMS has the unique potential to simultaneously observe the presence of an antimicrobial agent, concentration of nutrients, and the viability of the cell population.     

Polyethylene compounds with antimicrobial surface properties

The present study aims at antimicrobial polyethylene surfaces. To achieve this, LLDPE was compounded with the polymeric biocide poly(2-tert-butylaminoethyl) methacrylate TBAM (bulk modification with 1.5-5.0 wt.% of TBAM). Surfaces of these polymer compounds were then subjected to microbial assays. Using standard methods the colony forming units (CFU) for Escherichia coli and Staphylococcus aureus were determined on these surfaces. In all cases, polyethylene surfaces with highly antimicrobial properties were achieved. An average reduction of 10⁴ CFU ml⁻¹ compared to neat LLDPE was achieved. The surfaces of these LLDPE/TBAM compounds were assessed by electrokinetic (zeta potential) measurements. The results indicate a relation between the antimicrobial activity and the zeta-potential of the polymer compounds. Moreover, the antimicrobial compounds were investigated towards biofilm formation. Compared to pristine LLDPE, the surfaces of the polymer compounds showed less adhering biofilm after a testing period of 16 weeks.     

Secondary metabolites produced by fungi derived from a microbial mat encountered in an iron-rich natural spring

A collection of fungal isolates was obtained from a complex microbial mat, which occupied an iron-rich freshwater spring that feeds into Clear Creek, Golden, Colorado, USA. Two of the fungal isolates, a Glomeromycete (possibly Entrophospora sp.) and a Dothideomycete (possibly Phaeosphaeria sp.), were investigated for bioactive secondary metabolites. In total, six new compounds consisting of clearanols A-E (5, 6, 10-12) and disulochrin (7) were purified and their structures were determined. Disulochrin exhibited modest antibacterial activity against methicillin-resistant Staphylococcus aureus, whereas clearanol C showed weak inhibitory activity against Candida albicans biofilm formation.     

Rheological properties of viscoelastic biofilm extracellular polymeric substances and comparison to the behavior of calcium alginate gels

In this publication we present a detailed study of viscoelastic biofilms of Pseudomonas aeruginosa. Sample solutions were extracted from biofilm layers grown on Pseudomonas isolation agar. This aqueous solutions of extracellular polymeric substances exhibit weak elastic effects caused by entanglements and a small number of permanent junction points formed by calcium ions. The cross-linking mechanisms are confirmed by the Cox–Merz rule and dynamic frequency sweep tests, which result in an average lifetime of junction points of the order of 17 ms. The experimental data reveal 3.4×10¹⁷ elastically effective chains per liter of solution and no significant temperature effects in the regime between 2 and 24 °C. This result coincides pretty well with the concentration of dissolved polymer chains (2.9×10¹⁷ molecules/l). Upon addition of calcium ions, one observes the formation of stable supermolecular networks with permanent junction points. These cross-linking points did not show thermal fluctuations in time zones between 10 ms and several hours. The entanglement density of these gels is of the same order as observed in the non-cross-linked sol state (entrapped entanglements). In spite of the different molecular composition alginate gels show the same type of cross-linking mechanism as gels of extracellular polymeric substances.     

Fiber-optic sensors for the estimation of oxygen gradients within biofilms on metals

This paper presents an optical approach to measure the oxygen gradients in biofilms grown on stainless-steel 316L. The sensor is constructed by immobilizing an oxygen-quenchable fluorophore at the tip of an optical fiber. The response time and reversibility of fiber-optic sensor have been analyzed. By mounting the optical fiber using a computerized micromanipulator, the vertical profiles of oxygen are measured with 10 μm depth resolution across biofilms on metal coupons. Steep concentration gradients of oxygen have been observed within the biofilm, both perpendicular and parallel to the metal surface. Optical response of the biofilm grown metal surface is found to be a consistent measure of the oxygen profiles within biofilms.     

A study on the interactions of Aurein 2.5 with bacterial membranes

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH₂) is an uncharacterised antimicrobial peptide. At an air/water interface, it exhibited strong surface activity (maximal surface pressure 25 mN m⁻¹) and molecular areas consistent with the adoption of α-helical structure orientated either perpendicular (1.72 nm² molecule⁻¹) or parallel (3.6 nm² molecule⁻¹) to the interface. Aurein 2.5 was strongly antibacterial, exhibiting a minimum inhibitory concentration (MIC) of 30 μM against Bacillus subtilis and Escherichia coli. The peptide induced maximal surface pressure changes of 9 mN m⁻¹ and 5 mN m⁻¹, respectively, in monolayers mimicking membranes of these organisms whilst compression isotherm analysis of these monolayers showed ΔG_Mix > 0, indicating destabilisation by Aurein 2.5. These combined data suggested that toxicity of the peptide to these organisms may involve membrane invasion via the use of oblique orientated α-helical structure. The peptide induced strong, comparable maximal surface changes in monolayers of DOPG (7.5 mN m⁻¹) and DOPE monolayers (6 mN m⁻¹) suggesting that the membrane interactions of Aurein 2.5 were driven by amphiphilicity rather than electrostatic interaction. Based on these data, it was suggested that the differing ability of Aurein 2.5 to insert into membranes of B. subtilis and E. coli was probably related to membrane-based factors such as differences in lipid packing characteristics. The peptide was active against both sessile E. coli and Staphylococcus aureus with an MIC of 125 μM. The broad-spectrum antibacterial activity and non-specific modes of membrane action used by Aurein 2.5 suggested use as an anti-biofilm agent such as in the decontamination of medical devices.     

Antimicrobial activity of collagen material with thymol addition for potential application as wound dressing

Recently, special attention has been paid to the development of active wound dressing materials based on biopolymers. Collagen is a natural polymer, which meets the requirements of modern materials for medical applications. However, despite its unique properties, collagen has no antimicrobial activity. In this work thymol was incorporated into collagen films to meet antimicrobial properties of the material. Thymol is a naturally occurring phenolic compound recognized as an antimicrobial agent. Collagen/thymol thin films were obtained through solvent evaporation using collagen solutions containing different amounts of thymol. The structure of the obtained materials was studied using FTIR-ATR spectroscopy. The inhibition ability on the growth of several strains of microorganisms was tested. The standard ISO 22196:2007 was used to define the bactericidal properties of the material. The growth of the following bacteria on the collagen/thymol films was studied: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis, Enterobacter aerogenes, Candida albicans. The results showed that the growth of Staphylococcus aureus was the most inhibited compared to the other tested strains. Collagen/thymol material is more efficient against pathogens through direct contact compared to the diffusion of thymol from the material. In general, the thymol addition inhibits biofilm formation on the collagen surface.     

Antimicrobial and anti-biofilm activity of tannic acid against Staphylococcus aureus

Tannic acid, a rich of natural and process-derived phenolic compound, has been shown to be an effective antagonist against viruses and bacteria. In this study, we determined the antimicrobial activity and mechanisms of tannic acid against Staphylococcus aureus with emphasis on inhibiting effect on biofilm formation. Based on the results of time-kill assay, binding ability assay, lysozyme susceptibility assay and the transmission electron microscope, we tentatively speculated that peptidoglycan might be the target of the process that tannic acid destroy the integrity of cell wall, moreover, tannic acid could reduce the biofilm formation at sub-MIC concentrations. These results manifested that natural product tannic acid could serve as a potentially effective candidate for development of novel strategies to treat methicillin-resistant S. aureus infections.