Evaluation of novel compounds as anti-bacterial or anti-virulence agents.

Antimicrobial resistance is a global threat, leading to an alarming increase in the prevalence of bacterial infections that can no longer be treated with available antibiotics. The World Health Organization estimates that by 2050 up to 10 million deaths per year could be associated with antimicrobial resistance, which would equal the annual number of cancer deaths worldwide. To overcome this emerging crisis, novel anti-bacterial compounds are urgently needed. There are two possible approaches in the fight against bacterial infections: a) targeting structures within bacterial cells, similar to existing antibiotics; and/or b) targeting virulence factors rather than bacterial growth. Here, for the first time, we provide a comprehensive overview of the key steps in the evaluation of potential new anti-bacterial and/or anti-virulence compounds. The methods described in this review include: a) in silico methods for the evaluation of novel compounds; b) anti-bacterial assays (MIC, MBC, Time-kill); b) anti-virulence assays (anti-biofilm, anti-quorum sensing, anti-adhesion); and c) evaluation of safety aspects (cytotoxicity assay and Ames test). Overall, we provide a detailed description of the methods that are an essential tool for chemists, computational chemists, microbiologists, and toxicologists in the evaluation of potential novel antimicrobial compounds. These methods are cost-effective and have high predictive value. They are widely used in preclinical studies to identify new molecular candidates, for further investigation in animal and human trials.

Targeting outer membrane protein A (OmpA) - inhibitory effect of 2'-hydroxychalcone derivatives on Acinetobacter baumannii and Candida albicans dual-species biofilm formation.

Biofilm production facilitates microbial colonization of wounds and catheters. Acinetobacter baumannii produces high levels of biofilm and causes difficult-to-treat nosocomial infections. Candida albicans is another strong biofilm producer which may facilitate A. baumannii adhesion by providing hyphae-mediated OmpA-binding sites. Here we tested the potential of 2'-hydroxychalcones to inhibit dual-species biofilm production of A. baumannii and Candida spp., and further predicted the mechanism of structure-related difference in activity. The results suggest that 2'-hydroxychalcones exhibit potent activity against Candida spp./A. baumannii dual-species biofilm production. Particularly active was trifluoromethyl-substituted derivative (p-CF3), which decreased C. albicans/A. baumannii biomass produced on vein-indwelling parts of the central venous catheterization set by up to 99%. Further, higher OmpA-binding affinity was also calculated for p-CF3, which together with demonstrated significant ompA-downregulating activity, suggests that superior antibiofilm activity of this chalcone against the tested dual-species community of A. baumannii is mediated through the OmpA.

In vitro colistin susceptibility of pandrug-resistant Ac. baumannii is restored in the presence of selenium nanoparticles.

AIMS: To investigate the synergistic activity of colistin and selenium nanoparticles (SeNPs) against pandrug-resistant (PDR) Ac. baumannii. METHODS AND RESULTS: Chequerboard and time-kill assays were employed to explore the potential synergistic interactions between colistin and SeNPs against Ac. baumannii isolates (8), previously determined as colistin-resistant (MIC range 16-256 µg ml-1 ). Also, whole-genome sequencing (WGS) and gene expression analyses were used to elucidate the mechanisms of colistin resistance. Exceptionally strong synergistic activity (FICI range 0.004-0.035) of colistin and SeNPs against colistin-resistant isolates was revealed. Colistin (0.5 or 1 µg ml-1 ) used in combination with SeNPs (0.5 µg ml-1 ) was able to reduce initial inoculum during the first 4 h of incubation, in contrast to colistin (0.5, 1 or 2 µg ml-1 ) alone. CONCLUSIONS: These findings propose colistin/SeNPs combination as a new option to fight PDR Ac. baumannii, the therapeutic possibilities of which should be proved in future in vivo studies. SIGNIFICANCE AND IMPACT OF STUDY: Here we present the first evidence of synergy between colistin and selenium compounds against bacteria in general. Also, WGS and gene expression analyses provide some new insights into Ac. baumannii colistin resistance mechanisms.

Synthesis and characterization of a collagen-based composite material containing selenium nanoparticles.

Multidrug-resistant bacterial strains represent an emerging global health threat and a great obstacle for bone tissue engineering. One of the major components of the extracellular matrix of the bone is a collagen protein, while selenium is an element that has antimicrobial potential, and is also important for bone metabolism and bone health. Here we represent the incorporation of selenium nanoparticles (SeNPs) synthesized by the green chemical reduction method into collagen gels to produce a composite material, collagen/SeNPs, with antimicrobial properties. The samples were comprehensively characterized by zeta potential measurements, dynamic light scattering inductively coupled plasma-mass spectrometry (ICP-MS), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), optical microscopy, field-emission scanning electron microscopy (FE-SEM), and differential scanning calorimetry The cytotoxicity of the SeNPS, as well as collagen/SeNPs, was tested on the MRC-5 cells. It was revealed that collagen/SeNPS expressed a lower cytotoxic effect. Collagen/SeNPs showed significant antibacterial activity against all tested Gram-positive strains, the major causative agents of orthopedic infections as well as Candida albicans. Furthermore, three-dimensional ß-tricalcium phosphate (3D-TCP) scaffolds were fabricated by a well-established 3D printing (lithography) method, and afterward preliminary coated by newly-synthesized SeNPs or collagen/SeNPs. In addition, uncoated 3D-TCP scaffolds as well as coated by collagen/SeNPs were subjected to biofilm formation. The production of Staphylococcus aureus biofilm on coated scaffolds by collagen/SeNPs was significantly reduced compared to the uncoated ones.

Methoxy-Substituted Hydroxychalcone Reduces Biofilm Production, Adhesion and Surface Motility of Acinetobacter baumannii by Inhibiting ompA Gene Expression.

An increasing lack of available therapeutic options against Acinetobacter baumannii urged researchers to seek alternative ways to fight this extremely resistant nosocomial pathogen. Targeting its virulence appears to be a promising strategy, as it offers considerably reduced selection of resistant mutants. In this study, we tested antibiofilm potential of four synthetic chalcone derivatives against A. baumannii. Compound that showed the greatest activity was selected for further evaluation of its antivirulence properties. Real-time PCR was used to evaluate mRNA expression of biofilm-associated virulence factor genes (ompA, bap, abaI) in treated A. baumannii strains. Also, we examined virulence properties related to the expression of these genes, such as fibronectin- and collagen-mediated adhesion, surface motility, and quorum-sensing activity. The results revealed that the expression of all tested genes is downregulated together with the reduction of adhesion and motility. The conclusion is that 2'-hydroxy-2-methoxychalcone exhibits antivirulence activity against A. baumannii by inhibiting the expression of ompA and bap genes, which is reflected in reduced biofilm formation, adhesion, and surface motility.

Comparative Study of the Antimicrobial Activity of Selenium Nanoparticles With Different Surface Chemistry and Structure.

Although selenium nanoparticles (SeNPs) have gained attention in the scientific community mostly through investigation of their anticancer activity, a great potential of this nanomaterial was recognized recently regarding its antimicrobial activity. The particle form, size, and surface chemistry have been recognized as crucial parameters determining the interaction of nanomaterials with biological entities. Furthermore, considering a narrow boundary between beneficial and toxic effects for selenium per se, it is clear that investigations of biomedical applications of SeNPs are very demanding and must be done with great precautions. The goal of this work is to evaluate the effects of SeNPs surface chemistry and structure on antimicrobial activity against several common bacterial strains, including Staphylococcus aureus (ATCC 6538), Enterococcus faecalis (ATCC 29212), Bacillus subtilis (ATCC 6633), and Kocuria rhizophila (ATCC 9341), as well as Escherichia coli (ATCC 8739), Salmonella Abony (NCTC 6017), Klebsiella pneumoniae (NCIMB 9111) and Pseudomonas aeruginosa (ATCC 9027), and the standard yeast strain Candida albicans (ATCC 10231). Three types of SeNPs were synthesized by chemical reduction approach using different stabilizers and reducing agents: (i) bovine serum albumin (BSA) + ascorbic acid, (ii) chitosan + ascorbic acid, and (iii) with glucose. A thorough physicochemical characterization of the obtained SeNPs was performed to determine the effects of varying synthesis parameters on their morphology, size, structure, and surface chemistry. All SeNPs were amorphous, with spherical morphology and size in the range 70-300 nm. However, the SeNPs obtained under different synthesis conditions, i.e. by using different stabilizers as well as reducing agents, exhibited different antimicrobial activity as well as cytotoxicity which are crucial for their applications. In this paper, the antimicrobial screening of the selected systems is presented, which was determined by the broth microdilution method, and inhibitory influence on the production of monomicrobial and dual-species biofilm was evaluated. The potential mechanism of action of different systems is proposed. Additionally, the cytotoxicity of SeNPs was examined on the MRC-5 cell line, in the same concentration interval as for antimicrobial testing. It was shown that formulation SeNPs-BSA expressed a significantly lower cytotoxic effect than the other two formulations.

Antimicrobial activity of novel chalcones and modulation of virulence factors in hospital strains of Acinetobacter baumannii and Pseudomonas aeruginosa.

Acinetobacter baumannii and Pseudomonas aeruginosa are frequent multiresistant nosocomial pathogens that cause wound and pulmonary infections in hospitalized patients. As being increasingly resistant to most clinically available antibiotics, there is a constant need for exploration of new substances that could kill them or inhibit their growth, or alternatively inhibit some of their essential virulence factors. Chalcones are chemical compounds with well-documented antimicrobial potential. The aim of this study was to examine effectiveness of four newly-synthesized chalcones against the multiresistant clinical strains of A. baumannii and P. aeruginosa. Antibacterial activity of chalcones was investigated with broth-microdilution test and time-dependent killing assay. Synergistic effects of tested compounds with antibiotics (meropenem, amikacin and ciprofloxacin) were determined by checkerboard assay. The effects of chalcones on expression of virulence factors in P. aeruginosa (pyocyanin production, swimming and swarming motility) and A. baumannii (twitching and surface-associated motility), along with their biofilm production, were also examined. The obtained results indicate substantial antimicrobial activity of the tested chalcones (MICs = 100-175 µg/mL) and several synergistic interactions with antibiotics, as well as notable reduction in expression of all investigated virulence factors. These promising results may constitute a good basis for further research.