Shoghik Hakobyan

Surface-initiated polymer brushes in the biomedical field : applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings

Surface-initiated polymer brushes in the biomedical field : applications in membrane science, biosensing, cell culture, regenerative medicine and antibacterial coatings

The gallium(III)–salicylidene acylhydrazide complex shows synergistic anti-biofilm effect and inhibits toxin production by Pseudomonas aeruginosa

Bacterial biofilms cause a range of problems in many areas and especially in health care. Biofilms are difficult to eradicate with traditional antibiotics and consequently there is a need for alternative ways to prevent and/or remove bacterial biofilms. Furthermore, the emergence of antibiotic resistance in bacteria creates a challenge to find new types of antibiotics with a lower evolutionary pressure for resistance development. One route to develop such drugs is to target the so called virulence factors, i.e. bacterial systems used when bacteria infect a host cell. This study investigates synergy effects between Ga(III) ions, previously reported to suppress biofilm formation and growth in bacteria, and salicylidene acylhydrazides (hydrazones) that have been proposed as antivirulence drugs targeting the type three secretion system used by several Gram-negative pathogens, including Pseudomonas aerugionosa, during bacterial infection of host cells. A library of hydrazones was screened for: Fe(III) binding, enhanced anti-biofilm effect with Ga(III) on P. aeruginosa, and low cytotoxicity to mammalian cells. The metal coordination for the most promising ligand, 2-Oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (ME0163) with Ga(III) was investigated using extended X-ray absorption fine structure spectroscopy as well as density functional theory. The results showed that Ga(III) chelates the hydrazone with 5- and 6-membered chelating rings, and that the Ga(III)-ME0163 complex enhanced the antibiofilm effect of Ga(III) while suppressing the type three secretion system in P. aeruginosa. The latter effect was not observed for the hydrazone alone and was similar for Ga(III)-citrate and Ga(III)-ME0163 complexes, indicating that the inhibition of virulence was caused by Ga(III).

Proton and gallium(III) binding properties of a biologically active salicylidene acylhydrazide

Bacterial biofilm formation causes a range of problems in our society, especially in health care. Salicylidene acylhydrazides (hydrazones) are promising antivirulence drugs targeting secretion systems used during bacterial infection of host cells. When mixed with the gallium ion they become especially potent as bacterial and biofilm growth-suppressing agents, although the mechanisms through which this occurs are not fully understood. At the base of this uncertainty lies the nature of hydrazone-metal interactions. This study addresses this issue by resolving the equilibrium speciation of hydrazone-gallium aqueous solutions. The protonation constants of the target 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (ME0163) hydrazone species and of its 2,4,6-trihydroxybenzaldehyde and oxamic acid hydrazide building blocks were determined by UV-visible spectrophotometry to achieve this goal. These studies show that the hydrazone is an excessively strong complexing agent for gallium and that its antivirulence properties are predominantly ascribed to monomeric 1:1Ga-ME0163 complexes of various Ga hydrolysis and ME0163 protonation states. The chelation of Ga(III) to the hydrazone also increased the stability of the compounds against acid-induced hydrolysis, making this group of compounds very interesting for biological applications where the Fe-antagonist action of both Ga(III) and the hydrazone can be combined for enhanced biological effect.

Detecting Bacterial Surface Organelles on Single Cells Using Optical Tweezers

Bacterial cells display a diverse array of surface organelles that are important for a range of processes such as intercellular communication, motility and adhesion leading to biofilm formation, infections, and bacterial spread. More specifically, attachment to host cells by Gram-negative bacteria are mediated by adhesion pili, which are nanometers wide and micrometers long fibrous organelles. Since these pili are significantly thinner than the wavelength of visible light, they cannot be detected using standard light microscopy techniques. At present, there is no fast and simple method available to investigate if a single cell expresses pili while keeping the cell alive for further studies. In this study, we present a method to determine the presence of pili on a single bacterium. The protocol involves imaging the bacterium to measure its size, followed by predicting the fluid drag based on its size using an analytical model, and thereafter oscillating the sample while a single bacterium is trapped by an optical tweezer to measure its effective fluid drag. Comparison between the predicted and the measured fluid drag thereby indicate the presence of pili. Herein, we verify the method using polymer coated silica microspheres and Escherichia coli bacteria expressing adhesion pili. Our protocol can in real time and within seconds assist single cell studies by distinguishing between piliated and nonpiliated bacteria.

Characterization of clinically relevant model bacterial strains of Pseudomonas aeruginosa for anti-biofilm testing of materials

There is a great interest in developing novel anti-biofilm materials in order to decrease medical device-associated bacterial infections causing morbidity and high healthcare costs. However, the testing of novel materials is often done using bacterial lab strains that may not exhibit the same phenotype as clinically relevant strains infecting medical devices. Furthermore, no consensus of strain selection exists in the field, making results very difficult to compare between studies. In this work, 19 clinical isolates of Pseudomonas aeruginosa originating from intubated patients in an intensive care unit have been characterized and compared to the lab reference strain PAO1 and a rmlC lipopolysaccharide mutant of PAO1. The adhesion and biofilm formation was monitored, as well as cell properties such as hydrophobicity, zeta potential and motility. Two groups of isolates were observed: one with high adhesion to polymer surfaces and one with low adhesion (the latter including PAO1). Furthermore, detailed biofilm assays in a flow system were performed using five characteristic isolates from the two groups. Confocal microscopy showed that the adhesion and biofilm formation of four of these five strains could be reduced dramatically on zwitterionic surface coatings. However, one isolate with pronounced swarming colonized and formed biofilm also on the antifouling surface. We demonstrate that the biofilm properties of clinical isolates can differ greatly from that of a standard lab strain and propose two clinical model strains for testing of materials designed for prevention of biofilm formation in the respiratory tract. The methodology used could beneficially be applied for screening of other collections of pathogens to identify suitable model strains for in vitro biofilm testing. STATEMENT OF SIGNIFICANCE Medical-device associated infections present a great challenge in health care. Therefore, much research is undertaken to prevent bacterial colonization of new types of biomaterials. The work described here characterizes, tests and presents a number of clinically relevant bacterial model strains for assessing biofilm formation by Pseudomonas aeruginosa. Such model strains are of importance as they may provide better predictability of lab testing protocols with respect to how well materials would perform in an infection situation in a patient. Furthermore, this study uses the strains to test the performance of polymer surfaces designed to repel bacterial adhesion and it is shown that the biofilm formation for four out of the five tested bacterial strains was reduced.