Confocal microscopy of <i>P</i>. <i>aeruginosa</i> biofilms grown on flow cell.

<p>The formed biofilms were treated for 24 h with HU, HA and M-HA at 40 <b>μ</b>g/ml final concentration. Each panel shows the maximum Z-projection and the orthogonal views for each stack.</p

Disassembling the existing <i>P</i>. <i>aeruginosa</i> biofilms by adding HU, HA and M-HA.

<p><i>P</i>. <i>aeruginosa</i> bacteria were allowed to form biofilms in peg plates for 24 h, the medium was removed and fresh medium with different concentrations of radical scavenger compounds were changed every 24 hours over three days (Days 1, 2 and 3). The percentage of biofilm biomass production is represented for each day. The results are the means ± SD of three-five replicates from one representative of two independent experiments. A Student's t-test was performed (*, <i>P</i> < 0.05; versus non-treated biofilms). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl- hydroxylamine.</p

<i>P</i>. <i>aeruginosa</i> viability according to the LIVE/DEAD assay after treating with HU, HA, and M-HA.

<p>The bacteria were grown with HU (7.6 μg/ml), HA (8.3 μg/ml), or M-HA (6.7 μg/ml) for 3 and 24 hours and stained with LIVE/DEAD assay. Live cells were green (SYTO 9 dye) and dead cells were red (propidium iodide dye) under a fluorescent microscope. Magnification, x 1000. HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.</p

Dose-response curves for HU, HA and M-HA treatments of murine macrophages.

<p>The level of growth alteration for J774 macrophages that were treated with different doses of radical scavenger compounds at 72 hours post-treatment. Cell viability was measured by using an MTT assay. Values represent the means ± standard deviation (SD) of triplicate cultures. The data are representative of one of at least three independent experiments. (*, <i>P</i> < 0.05 <i>vs</i>. non-treated cells). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.</p

Biofilm parameters of wild-type <i>P</i>. <i>aeruginosa</i> treated with 40 μg/ml HA, HU and M-HA.

<p>Biomass values indicate the amount of living cells inside the biofilm. Values represent the mean ± SD of three independent experiments. Asterisk denotes significant differences compared to non-treated biofilm (p<0.05, Student’s <i>t</i>-test).</p><p>Biofilm parameters of wild-type <i>P</i>. <i>aeruginosa</i> treated with 40 μg/ml HA, HU and M-HA.</p

The synergistic effect of ciprofloxacin and M-HA on the reduction in <i>P</i>. <i>aeruginosa</i> biofilm formation.

<p><i>P</i>. <i>aeruginosa</i> bacteria were allowed to form biofilms in peg plates for 24 h, the medium was removed and fresh medium with different concentrations of ciprofloxacin with/without M-HA were added. Biofilm formation (crystal violet stain) was evaluated 24 hours later. The biofilm biomass production percentage is represented here. The results are the means ± SD of three-five replicates from one representative of two independent experiments. A Student's t-test was performed (*, <i>P</i> < 0.05; **, <i>P</i> < 0.005 versus ciprofloxacin treated biofilms). CPX, ciprofloxacin; and M-HA, methyl-hydroxylamine.</p

TNF-α and IL-12 production as triggered by BCG-infected macrophages that were treated with different doses of HU, HA and M-HA.

<p>J774 macrophages were infected with BCG and treated with different concentrations of radical scavenger compounds, and TNF-α and IL-12 levels were measured 24 hours post-infection. The results represent the means ± SD of triplicate preparations with one representative of two independent experiments. A Mann-Whitney test was performed (*, <i>P</i> < 0.01; versus non-treated macrophages (control)). HU, hydroxyurea; HA, hydroxylamine; and M-HA, methyl-hydroxylamine.</p

Data_Sheet_1_3D spatial organization and improved antibiotic treatment of a Pseudomonas aeruginosa–Staphylococcus aureus wound biofilm by nanoparticle enzyme delivery.PDF

Chronic wounds infected by Pseudomonas aeruginosa and Staphylococcus aureus are a relevant health problem worldwide because these pathogens grow embedded in a network of polysaccharides, proteins, lipids, and extracellular DNA, named biofilm, that hinders the transport of antibiotics and increases their antimicrobial tolerance. It is necessary to investigate therapies that improve the penetrability and efficacy of antibiotics. In this context, our main objectives were to study the relationship between P. aeruginosa and S. aureus and how their relationship can affect the antimicrobial treatment and investigate whether functionalized silver nanoparticles can improve the antibiotic therapy. We used an optimized in vitro wound model that mimics an in vivo wound to co-culture P. aeruginosa and S. aureus biofilm. The in vitro wound biofilm was treated with antimicrobial combinatory therapies composed of antibiotics (gentamycin and ciprofloxacin) and biofilm-dispersing free or silver nanoparticles functionalized with enzymes (α-amylase, cellulase, DNase I, or proteinase K) to study their antibiofilm efficacy. The interaction and colocalization of P. aeruginosa and S. aureus in a wound-like biofilm were examined and detailed characterized by confocal and electronic microscopy. We demonstrated that antibiotic monotherapy is inefficient as it differentially affects the two bacterial species in the mixed biofilm, driving P. aeruginosa to overcome S. aureus when using ciprofloxacin and the contrary when using gentamicin. In contrast, dual-antibiotic therapy efficiently reduces both species while maintaining a balanced population. In addition, DNase I nanoparticle treatment had a potent antibiofilm effect, decreasing P. aeruginosa and S. aureus viability to 0.017 and 7.7%, respectively, in combined antibiotics. The results showed that using nanoparticles functionalized with DNase I enhanced the antimicrobial treatment, decreasing the bacterial viability more than using the antibiotics alone. The enzymes α-amylase and cellulase showed some antibiofilm effect but were less effective compared to the DNase I treatment. Proteinase K showed insignificant antibiofilm effect. Finally, we proposed a three-dimensional colocalization model consisting of S. aureus aggregates within the biofilm structure, which could be associated with the low efficacy of antibiofilm treatments on bacteria. Thus, designing a clinical treatment that combines antibiofilm enzymes and antibiotics may be essential to eliminating chronic wound infections.</p

Hydroxylamine Derivatives as a New Paradigm in the Search of Antibacterial Agents

Serious infections caused by bacteria that are resistant to commonly used antibiotics have become a major global healthcare problem in the 21st century. Multidrug-resistant bacteria causing severe infections mainly grow in complex bacterial communities known as biofilms, in which bacterial resistance to antibacterial agents and to the host immune system is strengthened. As drug resistance is becoming a threatening problem, it is necessary to develop new antimicrobial agents with novel mechanisms of action. Here, we designed and synthesized a small library of N-substituted hydroxylamine (N-HA) compounds with antibacterial activity. These compounds, acting as radical scavengers, inhibit the bacterial ribonucleotide reductase (RNR) enzyme. RNR enzyme is essential for bacterial proliferation during infection, as it provides the building blocks for DNA synthesis and repair. We demonstrate the broad antimicrobial effect of several drug candidates against a variety of Gram-positive and Gram-negative bacteria, together with low toxicity toward eukaryotic cells. Furthermore, the most promising compounds can reduce the biomass of an established biofilm on Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. This study settles the starting point to develop new N-hydroxylamine compounds as potential effective antibacterial agents to fight against drug-resistant pathogenic bacteria