Bacterial biofilms affect many areas of human activity including food processing, transportation, public infrastructure, and most importantly healthcare. This study addresses the prevention of biofilms and shows that the surface charge of an abiotic substrate influences bacterial motility as well as the morphology and physiology of the biofilm. Grafting-from polymerisation was used to create polymer brush surfaces with different characteristics, and the development of Pseudomonas aeruginosa biofilms was followed using confocal microscopy. Interestingly, two types of biofilms developed on these surfaces: mushroom structures with high levels of cyclic diguanylate (c-di-GMP) were found on negatively charged poly (3-sulphopropylmethacrylate) (SPM) and zwitterionic poly (2-(methacryloyloxy)ethyl)dimethyl-3-sulphoproyl) ammonium hydroxide) (MEDSAH), while flat biofilms developed on glass, positively charged poly (2-(methacryloyloxy)-ethyl trimethyl ammonium chloride) (METAC), protein-repellent poly oligo(ethylene glycol methyl ether methacrylate) (POEGMA) and hydrophobic polymethylmethacrylate (PMMA). The results show that of all the surfaces studied, overall the negatively charged polymer brushes were most efficient in reducing bacterial adhesion and biofilm formation. However, the increased level of regulatory c-di-GMP in mushroom structures suggests that bacteria are capable of a quick physiological response when exposed to surfaces with varying physicochemical characteristics enabling some bacterial colonization also on negatively charged surfaces.

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.

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).

Development of antibiotic resistance in bacteria causes major challenges for our society and has prompted a great need for new and alternative treatment methods for infection. One promising approach is to target bacterial virulence using for example salicylidene acylhydrazides (hydrazones). Hydrazones coordinate metal ions such as Fe(III) and Ga(III) through a five-membered and a six-membered chelation ring. One suggested mode of action is via restricting bacterial Fe uptake. Thus, it was hypothesized that the chelating strength of these substances could be used to predict their biological activity on bacterial cells. This was investigated by comparing Ga chelation strength of two hydrazone complexes, as well as bacterial Ga uptake, biofilm formation, and virulence in the form of production and secretion of a toxin (ExoS) by Pseudomonas aeruginosa. Equilibrium constants for deprotonation and Ga(III) binding of the hydrazone N′-(5-chloro-2-hydroxy-3-methylbenzylidene)-2,4-dihydroxybenzhydrazide (ME0329), with anti-virulence effect against P. aeruginosa, were determined and compared to bacterial siderophores and the previously described Ga(III) 2-oxo-2-[N-(2,4,6-trihydroxy-benzylidene)-hydrazino]-acetamide (Ga-ME0163) and Ga-citrate complexes. In comparison with these two complexes, it was shown that the uptake of Ga(III) was higher from the Ga-ME0329 complex. The results further show that the Ga-ME0329 complex reduced ExoS expression and secretion to a higher extent than Ga-citrate, Ga-ME0163 or the non-coordinated hydrazone. However, the effect against biofilm formation by P. aeruginosa, by the ME0329 complex, was similar to Ga-citrate and lower than what has been reported for Ga-ME0163.