Novel strategies to combat gram-negative bacterial pathogens using bacteriophage proteins

Dissertação de mestrado em Bioengenharia

Structural and enzymatic characterization of ABgp46, a novel phage endolysin with broad anti-Gram-negative bacterial activity

The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0-10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 hours. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (> 5 log) and more than 4 logs P. aeruginosa and Salmonella Typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine.

The role of marine anaerobic bacteria and archaea in bioenergy production

The development of products from marine bioresources is gaining importance in the biotechnology sector. The global market for Marine Biotechnology products and processes was, in 2010, estimated at 2.8 billion with a cumulative annual growth rate of 510% (Børresen et al., Marine biotechnology: a new vision and strategy for Europe. Marine Board Position Paper 15. Beernem: Marine Board-ESF, 2010). Marine Biotechnology has the potential to make significant contributions towards the sustainable supply of food and energy, the solution of climate change and environmental degradation issues, and the human health. Besides the creation of jobs and wealth, it will contribute to the development of a greener economy. Thus, huge expectations anticipate the global development of marine biotechnology. The marine environment represents more than 70% of the Earths surface and includes the largest ranges of temperature, light and pressure encountered by life. These diverse marine environments still remain largely unexplored, in comparison with terrestrial habitats. Notwithstanding, efforts are being done by the scientific community to widespread the knowledge on oceans microbial life. For example, the J. Craig Venter Institute, in collaboration with the University of California, San Diego (UCSD), and Scripps Institution of Oceanography have built a state-of-the-art computational resource along with software tools to catalogue and interpret microbial life in the worlds oceans. The potential application of the marine biotechnology in the bioenergy sector is wide and, certainly, will evolve far beyond the current interest in marine algae. This chapter revises the current knowledge on marine anaerobic bacteria and archaea with a role in bio-hydrogen production, syngas fermentation and bio-electrochemical processes, three examples of bioenergy production routes.

Laccase immobilization on bacterial nanocellulose membranes: antimicrobial, kinetic and stability properties

This work studied the physical immobilization of a commercial laccase on bacterial nanocellulose (BNC) aiming to identify the laccase antibacterial properties suitable for wound dressings. Physico-chemical analysis demonstrates that the BNC structure is manly formed by pure crystalline I cellulose. The pH optimum and activation energy of free laccase depends on the substrate employed corresponding to pH 6, 7, 3 and 57, 22, 48 kJ mol1 for 2,6-dimethylphenol (DMP), catechol and 2,2 -azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), respectively. The Michaelis-Menten constant (Km) value for the immobilized laccase (0.77 mM) was found to be almost double of that of the free enzyme (0.42 mM). However, the specific activities of immobilized and free laccase are similar suggesting that the cage-like structure of BNC allows entrapped laccase to maintain some flexibility and favour substrate accessibility. The results clearly show the antimicrobial effect of laccase in Gram-positive (92%) and Gram-negative (26%) bacteria and cytotoxicity acceptable for wound dressing applications.

Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: the effect of pH, dextran sulfate and probe concentration

Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300 nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria.

Prediction of drug targets in human pathogens

The identification of new and druggable targets in bacteria is a critical endeavour in pharmaceutical research of novel antibiotics to fight infectious agents. The rapid emergence of resistant bacteria makes today's antibiotics more and more ineffective, consequently increasing the need for new pharmacological targets and novel classes of antibacterial drugs. A new model that combines the singular value decomposition technique with biological filters comprised of a set of protein properties associated with bacterial drug targets and similarity to protein-coding essential genes of E. coli has been developed to predict potential drug targets in the Enterobacteriaceae family [1]. This model identified 99 potential target proteins amongst the studied bacterial family, exhibiting eight different functions that suggest that the disruption of the activities of these proteins is critical for cells. Out of these candidates, one was selected for target confirmation. To find target modulators, receptor-based pharmacophore hypotheses were built and used in the screening of a virtual library of compounds. Postscreening filters were based on physicochemical and topological similarity to known Gram-negative antibiotics and applied to the retrieved compounds. Screening hits passing all filters were docked into the proteins catalytic groove and 15 of the most promising compounds were purchased from their chemical vendors to be experimentally tested in vitro. To the best of our knowledge, this is the first attempt to rationalize the search of compounds to probe the relevance of this candidate as a new pharmacological target.

Comparison of oleate conversion under microaerophilic and anaerobic conditions

Excessive accumulation of Long Chain Fatty Acids (LCFA) in methanogenic bioreactors is the cause of process failure associated to a severe decrease in methane production. In particular, fast and persistent accumulation of palmitate is critical and still not elucidated. Aerobes or facultative anaerobes were detected in those reactors, raising new questions on LCFA biodegradation. To get insight into the influence of oxygen, two bioreactors were operated under microaerophilic and anaerobic conditions, with oleate at 1 and 4 gCOD/(L d). Palmitate accumulated up to 2 and 16 gCOD/L in the anaerobic and microaerophilic reactor, respectively, which shows the importance of oxygen in this conversion. A second experiment was designed to understand the dynamics of oleate to palmitate conversion. A CSTR and a PFR were assembled in series and fed with oleate under microaerophilic conditions. HRT from 6 to 24 h were applied in the CSTR, and 14 to 52 min in the PFR. In the PFR a biofilm was formed where palmitate accounted for 82% of total LCFA. Pseudomonas was the predominant genus (42 %) in this biofilm, highlighting the role of aerobic and facultative anaerobic bacteria in LCFA bioconversion.

Bacterial vaginosis biofilms: challenges to current therapies and emerging solutions

Bacterial vaginosis (BV) is the most common genital tract infection in women during their reproductive years and it has been associated with serious health complications, such as preterm delivery and acquisition or transmission of several sexually transmitted agents. BV is characterized by a reduction of beneficial lactobacilli and a significant increase in number of anaerobic bacteria, including Gardnerella vaginalis, Atopobium vaginae, Mobiluncus spp., Bacteroides spp. and Prevotella spp.. Being polymicrobial in nature, BV etiology remains unclear. However, it is certain that BV involves the presence of a thick vaginal multi-species biofilm, where G. vaginalis is the predominant species. Similar to what happens in many other biofilm-related infections, standard antibiotics, like metronidazole, are unable to fully eradicate the vaginal biofilm, which can explain the high recurrence rates of BV. Furthermore, antibiotic therapy can also cause a negative impact on the healthy vaginal microflora. These issues sparked the interest in developing alternative therapeutic strategies. This review provides a quick synopsis of the currently approved and available antibiotics for BV treatment while presenting an overview of novel strategies that are being explored for the treatment of this disorder, with special focus on natural compounds that are able to overcome biofilm-associated antibiotic resistance.

Development of elastin-like recombinamer films with antimicrobial activity

In the present work we explored the ABP-CM4 peptide properties from Bombyx mori for the creation of biopolymers with broad antimicrobial activity. An antimicrobial recombinant protein-based polymer (rPBP) was designed by cloning the DNA sequence coding for ABP-CM4 in frame with the N-terminus of the elastin-like recombinamer consisting of 200 repetitions of the pentamer VPAVG, here named A200. The new rPBP, named CM4-A200, was purified via a simplified nonchromatographic method, making use of the thermoresponsive behavior of the A200 polymer. ABP-CM4 peptide was also purified through the incorporation of a formic acid cleavage site between the peptide and the A200 sequence. In soluble state the antimicrobial activity of both CM4-A200 polymer and ABP-CM4 peptide was poorly effective. However, when the CM4-A200 polymer was processed into free-standing films high antimicrobial activity against Gram-positive and Gram-negative bacteria, yeasts and filamentous fungi was observed. The antimicrobial activity of CM4-A200 was dependent on the physical contact of cells with the film surface. Furthermore, CM4-A200 films did not reveal a cytotoxic effect against both normal human skin fibroblasts and human keratinocytes. Finally, we have developed an optimized ex vivo assay with pig skin demonstrating the antimicrobial properties of the CM4-A200 cast films for skin applications.

Conversion of Cn-unsaturated into Cn-2-saturated LCFA can occur uncoupled from methanogenesis in anaerobic bioreactors

Fat, oils, and grease present in complex wastewater can be readily converted to methane, but the energy potential of these compounds is not always recyclable, due to incomplete degradation of long chain fatty acids (LCFA) released during lipids hydrolysis. Oleate (C18:1) is generally the dominant LCFA in lipid-containing wastewater, and its conversion in anaerobic bioreactors results in palmitate (C16:0) accumulation. The reason why oleate is continuously converted to palmitate without further degradation via β-oxidation is still unknown. In this work, the influence of methanogenic activity in the initial conversion steps of unsaturated LCFA was studied in 10 bioreactors continuously operated with saturated or unsaturated C16- and C18-LCFA, in the presence or absence of the methanogenic inhibitor bromoethanesulfonate (BrES). Saturated Cn-2-LCFA accumulated both in the presence and absence of BrES during the degradation of unsaturated Cn-LCFA, and represented more than 50\% of total LCFA. In the presence of BrES further conversion of saturated intermediates did not proceed, not even when prolonged batch incubation was applied. As the initial steps of unsaturated LCFA degradation proceed uncoupled from methanogenesis, accumulation of saturated LCFA can be expected. Analysis of the active microbial communities suggests a role for facultative anaerobic bacteria in the initial steps of unsaturated LCFA biodegradation. Understanding this role is now imperative to optimize methane production from LCFA.

The influence of biofilm formation by Gardnerella vaginalis and other anaerobes on bacterial vaginosis

Bacterial vaginosis (BV) is the worldwide leading vaginal disorder in women of reproductive age. BV is characterized by the replacement of beneficial lactobacilli and the augmentation of anaerobic bacteria. Gardnerella vaginalis is a predominant bacterial species, however, BV is also associated with other numerous anaerobes, such as Atopobium vaginae, Mobiluncus mulieris, Prevotella bivia, Fusobacterium nucleatum and Peptoniphilus sp.. Currently, the role of G. vaginalis in the etiology of BV remains a matter of controversy. It is however known that, in BV patients, a biofilm is usually formed on the vaginal epithelium and G. vaginalis is typically the predominant species. So, the current paradigm is that the establishment of a biofilm plays a key role in the pathogenesis of BV. This review provides background on the influence of biofilm formation by G. vaginalis and other anaerobes in the polymicrobial etiology of BV, through its initial adhesion until biofilm formation and discusses the commensal and synergic interactions established between them to understand the phenotypic shift of G. vaginalis' biofilms into BV establishment.

Dissolution enhancement of active pharmaceutical ingredients by therapeutic deep eutectic systems

A therapeutic deep eutectic system (THEDES) is here defined as a deep eutectic solvent (DES) having an active pharmaceutical ingredient (API) as one of the components. In this work, THEDESs are proposed as enhanced transporters and delivery vehicles for bioactive molecules. THEDESs based on choline chloride (ChCl) or menthol conjugated with three different APIs, namely acetylsalicylic acid (AA), benzoic acid (BA) and phenylacetic acid (PA), were synthesized and characterized for thermal behaviour, structural features, dissolution rate and antibacterial activity. Differential scanning calorimetry and polarized optical microscopy showed that ChCl:PA (1:1), ChCl:AA (1:1), menthol:AA (3:1), menthol:BA (3:1), menthol:PA (2:1) and menthol:PA (3:1) were liquid at room temperature. Dissolution studies in PBS led to increased dissolution rates for the APIs when in the form of THEDES, compared to the API alone. The increase in dissolution rate was particularly noticeable for menthol-based THEDES. Antibacterial activity was assessed using both Gram-positive and Gram-negative model organisms. The results show that all the THEDESs retain the antibacterial activity of the API. Overall, our results highlight the great potential of THEDES as dissolution enhancers in the development of novel and more effective drug delivery systems.

Incorporation of antimicrobial peptides on functionalized cotton gauzes for medical applications

A large group of low molecular weight natural compounds that exhibit antimicrobial activity has been isolated from animals and plants during the past two decades. Among them, peptides are the most widespread resulting in a new generation of antimicrobial agents with higher specific activity. In the present study we have developed a new strategy to obtain antimicrobial wound-dressings based on the incorporation of antimicrobial peptides into polyelectrolyte multilayer films built by the alternate deposition of polycation (chitosan) and polyanion (alginic acid sodium salt) over cotton gauzes. Energy dispersive X ray microanalysis technique was used to determine if antimicrobial peptides penetrated within the films. FTIR analysis was performed to assess the chemical linkages, and antimicrobial assays were performed with two strains: Staphylococcus aureus (Gram-positive bacterium) and Klebsiella pneumonia (Gram-negative bacterium). Results showed that all antimicrobial peptides used in this work have provided a higher antimicrobial effect (in the range of 4 log–6 log reduction) for both microorganisms, in comparison with the controls, and are non-cytotoxic to normal human dermal fibroblasts at the concentrations tested.