Leslie Hugh Glyn Morton

CONSIDERATION OF SOME IMPLICATIONS OF THE RESISTANCE OF BIOFILMS TO BIOCIDES

Abstract Biofilms are considered as the growth of cells at a surface with the production of extracellular polymeric substances. Biofilm formation has serious implications in industrial, environmental, public health and medical situations. There is now a large body of evidence and thus concern that microorganisms within biofilms (including certain pathogenic microorganisms) are less susceptible to the activity of biocides than their planktonic counterparts. The mechanisms of bacterial resistance to some antibacterial agents such as many antibiotics are reasonably well-understood. The extensive exopolysaccharide polymer associated with biofilms is considered as a potential barrier which can hinder or prevent biocides from reaching target organisms within the biofilm, but the actual physical barrier properties of the biofilm may be of less importance and it now seems that perhaps the intrinsic (phenotypic) resistance to biocides, shown by cells within a biofilm, is of primary concern to researchers in the field. Nutrient limitation and reduced growth rates resulting from the position of bacteria within a biofilm are considered to influence the physiology of bacteria which in turn alters their sensitivity to biocides. When these phenomena are coupled with the ability of both Gram-positive and Gram-negative bacteria to communicate with each other in a cell-density-dependent or growth phase manner via diffusible communication molecules, it is becoming obvious that individual biofilm organisms may be able to behave as sociable, collective communities to regulate their gene expression in order to control various physiological processes and responses, in aid of the ‘common good’. Since most antimicrobial agents have been developed as a consequence of their activities against relatively fast-growing planktonic organisms, this approach is unlikely to be entirely suitable for the development of biocides against sessile biofilm organisms. Thus, the generation of biofilms which are representative of their true nature in the environment and which are suitable for the evaluation of the effects of biocides is essential in biocide research and development but it is not an easy task, as every application involving problematic biofilm formation will probably represent a unique niche which will require individual solutions. Disruption of a biofilm prior to assessing the viability of individual members by conventional microbiological methods is not considered ideal for the evaluation of biocides. There is a need for the development of non-disruptive methods for the measurement of biofilm formation.

Biofilms in biodeterioration — a review

Abstract This review defines the various types of biodeterioration processes and discusses the role that microbial films play in the biodeterioration of a number of materials of economic importance. A review of the way in which biofilms may form and attach to surfaces is presented and the occurrence and nature of biofilms is considered. Included in this review is an account of biodeterioration problems associated with water distribution systems, biocorrosion, plastics, hydrocarbons, paints and coatings and buildings and monuments. The micro-organisms involved include bacteria, fungi and algae, which form members of the biofilm communities responsible for the biodeterioration problems described.

Investigations into the ability of an oblique α‐helical template to provide the basis for design of an antimicrobial anionic amphiphilic peptide

AP1 (GEQGALAQFGEWL) was shown by theoretical analysis to be an anionic oblique‐orientated α‐helix former. The peptide exhibited a monolayer surface area of 1.42 nm2, implying possession of α‐helical structure at an air/water interface, and Fourier transform infrared spectroscopy (FTIR) showed the peptide to be α‐helical (100%) in the presence of vesicle mimics of Escherichia coli membranes. FTIR lipid‐phase transition analysis showed the peptide to induce large decreases in the fluidity of these E. coli membrane mimics, and Langmuir–Blodgett trough analysis found the peptide to induce large surface pressure changes in monolayer mimics of E. coli membranes (4.6 mN·m−1). Analysis of compression isotherms based on mixing enthalpy (ΔH) and the Gibbs free energy of mixing (ΔGMix) predicted that these monolayers were thermodynamically stable (ΔH and ΔGMix each negative) but were destabilized by the presence of the peptide (ΔH and ΔGMix each positive). The peptide was found to have a minimum lethal concentration of 3 mm against E. coli and was seen to cause lysis of erythrocytes at 5 mm. In combination, these data clearly show that AP1 functions as an anionic α‐helical antimicrobial peptide and suggest that both its tilted peptide characteristics and the composition of its target membrane are important determinants of its efficacy of action.

The dependence of Legionella pneumophila on other aquatic bacteria for survival on R2A medium

Biofilms were grown in a continous culture model biofilm system. Bacteria isolated from these biofilms were shown to prolong the viability of Legionella pneumophila when inoculated onto R2A medium. Heat-killed and cell-free extracts of the same bacteria were not able to support the survival of L. pneumophila on R2A. This suggests that the non-Legionellaceae play a role as yet undefined in the survival of L. pneumophila.

The role of C-terminal amidation in the membrane interactions of the anionic antimicrobial peptide, maximin H5.

Maximin H5 is an anionic antimicrobial peptide from amphibians, which carries a C-terminal amide moiety, and was found to be moderately haemolytic (20%). The α-helicity of the peptide was 42% in the presence of lipid mimics of erythrocyte membranes and was found able to penetrate (10.8 mN m(-1)) and lyse these model membranes (64 %). In contrast, the deaminated peptide exhibited lower levels of haemolysis (12%) and α-helicity (16%) along with a reduced ability to penetrate (7.8 m Nm(-1)) and lyse (55%) lipid mimics of erythrocyte membranes. Taken with molecular dynamic simulations and theoretical analysis, these data suggest that native maximin H5 primarily exerts its haemolytic action via the formation of an oblique orientated α-helical structure and tilted membrane insertion. However, the C-terminal deamination of maximin H5 induces a loss of tilted α-helical structure, which abolishes the ability of the peptides N-terminal and C-terminal regions to H-bond and leads to a loss in haemolytic ability. Taken in combination, these observations strongly suggest that the C-terminal amide moiety carried by maximin H5 is required to stabilise the adoption of membrane interactive tilted structure by the peptide. Consistent with previous reports, these data show that the efficacy of interaction and specificity of maximin H5 for membranes can be attenuated by sequence modification and may assist in the development of variants of the peptide with the potential to serve as anti-infectives.

A study on the interactions of Aurein 2.5 with bacterial membranes.

Aurein 2.5 (GLFDIVKKVVGAFGSL-NH(2)) is an uncharacterised antimicrobial peptide. At an air/water interface, it exhibited strong surface activity (maximal surface pressure 25mNm(-1)) and molecular areas consistent with the adoption of alpha-helical structure orientated either perpendicular (1.72nm(2)molecule(-1)) or parallel (3.6nm(2)molecule(-1)) to the interface. Aurein 2.5 was strongly antibacterial, exhibiting a minimum inhibitory concentration (MIC) of 30microM against Bacillus subtilis and Escherichia coli. The peptide induced maximal surface pressure changes of 9mNm(-1) and 5mNm(-1), respectively, in monolayers mimicking membranes of these organisms whilst compression isotherm analysis of these monolayers showed DeltaG(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 alpha-helical structure. The peptide induced strong, comparable maximal surface changes in monolayers of DOPG (7.5mNm(-1)) and DOPE monolayers (6mNm(-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 125microM. 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.

A novel form of bacterial resistance to the action of eukaryotic host defense peptides, the use of a lipid receptor

Host defense peptides show great potential for development as new antimicrobial agents with novel mechanisms of action. However, a small number of resistance mechanisms to their action are known, and here, we report a novel bacterial resistance mechanism mediated by a lipid receptor. Maximin H5 from Bombina maxima bound anionic and zwitterionic membranes with low affinity (Kd > 225 μM) while showing a strong ability to lyse (>55%) and penetrate (π > 6.0 mN m(-1)) these membranes. However, the peptide bound Escherichia coli and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE) membranes with higher affinity (Kd < 65 μM) and showed a very low ability for bilayer lysis (<8%) and partitioning (π > 1.0 mN m(-1)). Increasing levels of membrane DMPE correlated with enhanced binding by the peptide (R(2) = 0.96) but inversely correlated with its lytic ability (R(2) = 0.98). Taken with molecular dynamic simulations, these results suggest that maximin H5 possesses membranolytic activity, primarily involving bilayer insertion of its strongly hydrophobic N-terminal region. However, this region was predicted to form multiple hydrogen bonds with phosphate and ammonium groups within PE head-groups, which in concert with charge-charge interactions anchor the peptide to the surface of E. coli membranes, inhibiting its membranolytic action.

Role of molecular architecture on the relative efficacy of aurein 2.5 and modelin 5

In order to gain an insight into the mechanism of antimicrobial peptide action, aurein 2.5 and modelin-5 were studied. When tested against Staphylococcus aureus, aurein 2.5 showed approximately 5-fold greater efficacy even though the higher net positive charge and higher helix stability shown by modelin-5 would have predicated modelin-5 to be the more effective antimicrobial. However, in the presence of S. aureus membrane mimics, aurein 2.5 showed greater helical content (75% helical) relative to modelin-5 (51% helical) indicative of increase in membrane association. This was supported by monolayer data showing that aurein 2.5 (6.6mNm(-1)) generated greater pressure changes than modelin-5 (5.3mNm(-1)). Peptide monolayers indicted that modelin-5 formed a helix horizontal to the plane of an asymmetric interface which would be supported by the even distribution of charge and hydrophobicity along the helical long axis and facilitate lysis by non-specific membrane binding. In contrast, a groove structure observed on the surface of aurein 2.5 was predicted to be the cause of enhanced lipid binding (K(d)=75μM) relative to modelin-5 (K(d)=118μM) and the balance of hydrophobicity along the aurein 2.5 long axis supported deep penetration into the membrane in a tilt formation. This oblique orientation generates greater lytic efficacy in high anionic lipid (71%) compared to modelin-5 (32%).

Effect of Amidation on the Antimicrobial Peptide Aurein 2.5 from Australian Southern Bell Frogs

Aurein 2.5 is a naturally C-terminally amidated amphibian antimicrobial peptide. C-terminal amidation can increase efficacy and hence a comparison was made between aurein 2.5-CONH2 and its nonamidated analogue. Amidation of the C-terminal carboxyl of aurein 2.5 enhanced antimicrobial activity 2.5- fold against Klebsiella pneumonia. Our results demonstrate that both peptide analogues had high surface activities (23 mN m-1for aurein 2.5-COOH and 26 mN m-1 aurein 2.5-CONH2). Circular dichroism measurements suggest that the helical content of the amidated form, in the presence of trifluoroethanol, was significantly enhanced (33.66 % for aurein 2.5-COOH and 60.89 % aurein 2.5-CONH2). The interaction of aurein 2.5 with bacterial cell membrane mimics was investigated using Langmuir monolayers. Aurein 2.5-CONH2 induced stable surface pressure changes in monolayers formed from K. pneumonia (circa 4.7 mN m-1), however, lower surface pressure changes were observed for aurein 2.5- COOH (circa 3.8 mN m-1). The data shows that in the case of aurein 2.5, amidation is able to enhance antibacterial activity and it is proposed that the increase in effectiveness is due to stabilization of the α-helical structure at the membrane interface.

Laboratory and field studies on thin paint films

An account is presented of some aspects and results of a collaborative project currently being undertaken as part of the European Collaborative Action COST 520 programme in Working Group 3. The project involves the response of biocide- and non-biocide-containing thin paint films to microbial colonisation under laboratory and field conditions. From the results of exposure studies at four sites, two in the UK and two in Norway, it was found that some microorganisms were common to both locations. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI TOF MS) was used in a novel way to establish characteristic mass spectral fingerprints of different fungal genera and different species of the same fungal genus. The results show that databases can be produced which provide convincing evidence for the application of this technique in taxonomy. Scanning electron microscopy was used to visualise Aureobasidium pullulans colonising the paint film. Evidence of hyphal penetration and disruption of the paint binder is suggested. Surface roughness measurement was used to investigate the effect of exposure on the surface topography of the paint. It was found that changes in the surface roughness increased over the duration of the experiment. Fourier transform infrared spectroscopy was used in the reflectance mode to detect chemical changes in the surface of the paint film. It was found that no positional or absorbance changes in the spectrum of the paint film were detected as a result of inoculating the film in a vermiculite bed system. However, some spectra did suggest that surface changes had occurred as a result of a reduction in diffuse scattering from the surface.