Impact of azithromycin, doxycycline and redox-active small molecules on amoxicillin-induced Chlamydia pneumoniae persistence.

Amoxicillin is recommended as primary treatment for community-acquired bacterial pneumonia (CABP). 5-10% of CABP cases are caused by Chlamydia pneumoniae, an obligate intracellular bacterium which responds to beta-lactam antibiotics by converting to a persistent phenotype. To support rational pharmacotherapy of C. pneumoniae infections, we investigated how clinically relevant concentrations of azithromycin and doxycycline affect amoxicillin induced C. pneumoniae persistence. Given the known role of redox state alterations in the action of bactericidal antibiotics and widespread use of redox-active dietary supplements when experiencing respiratory symptoms, we also studied how redox active compounds affect the studied antibiotic treatments. Our data demonstrate that clinically applied amoxicillin concentrations (10 and 25 mg/l) fail to eradicate C. pneumoniae infection in respiratory epithelial cells. Transmission electron microscopy (TEM) of amoxicillin-treated C. pneumoniae infected cells reveal aberrant bacterial morphology characteristic of chlamydial stress response. Amoxicillin was also found to significantly limit the antichlamydial effect of azithromycin or doxycycline. However, based on quantitative culture and quantitative PCR data, azithromycin was superior to doxycycline in C. pneumoniae eradication either as monotherapy or in combination with amoxicillin. Amoxicillin was also found to decrease respiratory epithelial cell glutathione (GSH) levels, whereas redox-active dibenzocyclooctadiene lignans increased C. pneumoniae load in amoxicillin-treated cultures up to two-fold. These data highlight the impact of relative administration time on the efficacy of antichlamydial antibiotics and indicate unfavorable interactions between amoxicillin and redox-active small molecules.

Repurposing the Sphingosine-1-Phosphate Receptor Modulator Etrasimod as an Antibacterial Agent Against Gram-Positive Bacteria.

New classes of antibiotics are urgently needed in the fight against multidrug-resistant bacteria. Drug repurposing has emerged as an alternative approach to accelerate antimicrobial research and development. In this study, we screened a library of sphingosine-1-phosphate receptor (S1PR) modulators against Staphylococcus aureus and identified five active compounds. Among them, etrasimod (APD334), an investigational drug for the treatment of ulcerative colitis, displayed the best inhibitory activity against S. aureus when growing as free-floating planktonic cells and within biofilms. In follow-up studies, etrasimod showed bactericidal activity and drastic reduction of viable bacteria within 1 h of exposure. It also displayed a potent activity against other Gram-positive bacteria, including penicillin- and methicillin-resistant S. aureus strains, S. epidermidis, and Enterococcus faecalis, with a minimum inhibitory concentration (MIC) ranging from 5 to 10 µM (2.3-4.6 µg/mL). However, no inhibition of viability was observed against Gram-negative bacteria Acinetobacter baumannii, Escherichia coli, and Pseudomonas aeruginosa, showing that etrasimod preferably acts against Gram-positive bacteria. On the other hand, etrasimod was shown to inhibit quorum sensing (QS) signaling in Chromobacterium violaceum, suggesting that it may block the biofilm formation by targeting QS in certain Gram-negative bacteria. Furthermore, etrasimod displayed a synergistic effect with gentamicin against S. aureus, thus showing potential to be used in antibiotic combination therapy. Finally, no in vitro toxicity toward mammalian cells was observed. In conclusion, our study reports for the first time the potential of etrasimod as a repurposed antibacterial compound against Gram-positive bacteria.

Alterations in Bacterial Metabolism Contribute to the Lifespan Extension Exerted by Guarana in Caenorhabditis elegans.

Guarana (Paullinia cupana) is a widely consumed nutraceutical with various health benefits supported by scientific evidence. However, its indirect health impacts through the gut microbiota have not been studied. Caenorhabditis elegans is a useful model to study both the direct and indirect effects of nutraceuticals, as the intimate association of the worm with the metabolites produced by Escherichia coli is a prototypic simplified model of our gut microbiota. We prepared an ethanoic extract of guarana seeds and assessed its antioxidant capacity in vitro, with a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, and in vivo, utilizing C. elegans. Additionally, we studied the impact of this extract on C. elegans lifespan, utilizing both viable and non-viable E. coli, and assessed the impact of guarana on E. coli folate production. The extract showed high antioxidant capacity, and it extended worm lifespan. However, the antioxidant and life-extending effects did not correlate in terms of the extract concentration. The extract-induced life extension was also less significant when utilizing dead E. coli, which may indicate that the effects of guarana on the worms work partly through modifications on E. coli metabolism. Following this observation, guarana was found to decrease E. coli folate production, revealing one possible route for its beneficial effects.

Screening of natural compounds identifies ferutinin as an antibacterial and anti-biofilm compound.

Antibacterial screenings are most commonly targeted at planktonic bacteria but less effort is dedicated to the exploration of agents acting on biofilms. Here, a natural compounds library was screened against Staphylococcus aureus using a 384-well plate platform to identify compounds preventing biofilm formation. Five structurally diverse hits were selected for follow-up studies: honokiol, tschimganidin, ferutinin, oridonin and deoxyshikonin. The compounds were evaluated against different bacterial species for their capacity to prevent and disrupt biofilms. The development of resistance and cytotoxicity were also investigated. Ferutinin displayed the best antibacterial activity, with a minimum inhibitory, bactericidal and biofilm preventive concentration of 25 µM against S. aureus. It efficiently disrupted pre-formed biofilms (over 5-log reduction of viable cells) and reduced biofilm formation on a catheter in the presence of neutrophils. This work provides new information on the antibacterial activity of five natural compounds and identified ferutinin as a promising candidate against S. aureus biofilms.

Interlaboratory study for the evaluation of three microtiter plate-based biofilm quantification methods.

Microtiter plate methods are commonly used for biofilm assessment. However, results obtained with these methods have often been difficult to reproduce. Hence, it is important to obtain a better understanding of the repeatability and reproducibility of these methods. An interlaboratory study was performed in five different laboratories to evaluate the reproducibility and responsiveness of three methods to quantify Staphylococcus aureus biofilm formation in 96-well microtiter plates: crystal violet, resazurin, and plate counts. An inter-lab protocol was developed for the study. The protocol was separated into three steps: biofilm growth, biofilm challenge, biofilm assessment. For control experiments participants performed the growth and assessment steps only. For treatment experiments, all three steps were performed and the efficacy of sodium hypochlorite (NaOCl) in killing S. aureus biofilms was evaluated. In control experiments, on the log10-scale, the reproducibility SD (SR) was 0.44 for crystal violet, 0.53 for resazurin, and 0.92 for the plate counts. In the treatment experiments, plate counts had the best responsiveness to different levels of efficacy and also the best reproducibility with respect to responsiveness (Slope/SR = 1.02), making it the more reliable method to use in an antimicrobial efficacy test. This study showed that the microtiter plate is a versatile and easy-to-use biofilm reactor, which exhibits good repeatability and reproducibility for different types of assessment methods, as long as a suitable experimental design and statistical analysis is applied.

Surfaceome and Exoproteome Dynamics in Dual-Species Pseudomonas aeruginosa and Staphylococcus aureus Biofilms.

Bacterial biofilms are an important underlying cause for chronic infections. By switching into the biofilm state, bacteria can evade host defenses and withstand antibiotic chemotherapy. Despite the fact that biofilms at clinical and environmental settings are mostly composed of multiple microbial species, biofilm research has largely been focused on single-species biofilms. In this study, we investigated the interaction between two clinically relevant bacterial pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) by label-free quantitative proteomics focusing on proteins associated with the bacterial cell surfaces (surfaceome) and proteins exported/released to the extracellular space (exoproteome). The changes observed in the surfaceome and exoproteome of P. aeruginosa pointed toward higher motility and lower pigment production when co-cultured with S. aureus. In S. aureus, lower abundances of proteins related to cell wall biosynthesis and cell division, suggesting increased persistence, were observed in the dual-species biofilm. Complementary phenotypic analyses confirmed the higher motility and the lower pigment production in P. aeruginosa when co-cultured with S. aureus. Higher antimicrobial tolerance associated with the co-culture setting was additionally observed in both species. To the best of our knowledge, this study is among the first systematic explorations providing insights into the dynamics of both the surfaceome and exoproteome of S. aureus and P. aeruginosa dual-species biofilms.

Synthesis and Biological Evaluation of Fingolimod Derivatives as Antibacterial Agents.

We recently identified fingolimod as a potent antibiofilm compound by screening FDA-approved drugs. To study if the antibacterial activity of fingolimod could be further improved and to explore in-depth structure-activity relationships, we synthesized 28 novel fingolimod derivatives and evaluated their efficacy against Staphylococcus aureus grown in planktonic/single cell and biofilms. The most effective derivatives were tested on preformed S. aureus biofilms and against Gram-negative bacteria Acinetobacter baumannii and Pseudomonas aeruginosa, using fingolimod as the reference compound. Seven derivatives were more effective against S. aureus, while five other derivatives showed improved activity against P. aeruginosa and/or A. baumannii, with no apparent change in cytotoxicity on human cells. The most interesting derivatives, compounds 43 and 55, displayed a broader spectrum of antibacterial activity, possibly exerted by the change of the para-hydrocarbon chain to a meta position for 43 and by an additional hydroxyl group for 55.

Combined Effect of Naturally-Derived Biofilm Inhibitors and Differentiated HL-60 Cells in the Prevention of Staphylococcus aureus Biofilm Formation.

Nosocomial diseases represent a huge health and economic burden. A significant portion is associated with the use of medical devices, with 80% of these infections being caused by a bacterial biofilm. The insertion of a foreign material usually elicits inflammation, which can result in hampered antimicrobial capacity of the host immunity due to the effort of immune cells being directed to degrade the material. The ineffective clearance by immune cells is a perfect opportunity for bacteria to attach and form a biofilm. In this study, we analyzed the antibiofilm capacity of three naturally derived biofilm inhibitors when combined with immune cells in order to assess their applicability in implantable titanium devices and low-density polyethylene (LDPE) endotracheal tubes. To this end, we used a system based on the coculture of HL-60 cells differentiated into polymorphonuclear leukocytes (PMNs) and Staphylococcus aureus (laboratory and clinical strains) on titanium, as well as LDPE surfaces. Out of the three inhibitors, the one coded DHA1 showed the highest potential to be incorporated into implantable devices, as it displayed a combined activity with the immune cells, preventing bacterial attachment on the titanium and LDPE. The other two inhibitors seemed to also be good candidates for incorporation into LDPE endotracheal tubes.

3D-Printed Drug Delivery Systems: The Effects of Drug Incorporation Methods on Their Release and Antibacterial Efficiency.

Additive manufacturing technologies have been widely used in the medical field. More specifically, fused filament fabrication (FFF) 3D-printing technology has been thoroughly investigated to produce drug delivery systems. Recently, few researchers have explored the possibility of directly 3D printing such systems without the need for producing a filament which is usually the feedstock material for the printer. This was possible via direct feeding of a mixture consisting of the carrier polymer and the required drug. However, as this direct feeding approach shows limited homogenizing abilities, it is vital to investigate the effect of the pre-mixing step on the quality of the 3D printed products. Our study investigates the two commonly used mixing approaches-solvent casting and powder mixing. For this purpose, polycaprolactone (PCL) was used as the main polymer under investigation and gentamicin sulfate (GS) was selected as a reference. The produced systems' efficacy was investigated for bacterial and biofilm prevention. Our data show that the solvent casting approach offers improved drug distribution within the polymeric matrix, as was observed from micro-computed topography and scanning electron microscopy visualization. Moreover, this approach shows a higher drug release rate and thus improved antibacterial efficacy. However, there were no differences among the tested approaches in terms of thermal and mechanical properties.

Strategies to Prevent Biofilm Infections on Biomaterials: Effect of Novel Naturally-Derived Biofilm Inhibitors on a Competitive Colonization Model of Titanium by Staphylococcus aureus and SaOS-2 Cells.

Biofilm-mediated infection is a major cause of bone prosthesis failure. The lack of molecules able to act in biofilms has driven research aimed at identifying new anti-biofilm agents via chemical screens. However, to be able to accommodate a large number of compounds, the testing conditions of these screenings end up being typically far from the clinical scenario. In this study, we assess the potential applicability of three previously discovered anti-biofilm compounds to be part of implanted medical devices by testing them on in vitro systems that more closely resemble the clinical scenario. To that end, we used a competition model based on the co-culture of SaOS-2 mammalian cells and Staphylococcus aureus (collection and clinical strains) on a titanium surface, as well as titanium pre-conditioned with high serum protein concentration. Additionally, we studied whether these compounds enhance the previously proven protective effect of pre-incubating titanium with SaOS-2 cells. Out of the three, DHA1 was the one with the highest potential, showing a preventive effect on bacterial adherence in all tested conditions, making it the most promising agent for incorporation into bone implants. This study emphasizes and demonstrates the importance of using meaningful experimental models, where potential antimicrobials ought to be tested for the protection of biomaterials in translational applications.

Antioxidant and Antiaging Effects of Licorice on the Caenorhabditis elegans Model.

The causative relationship between oxidative stress and aging remains controversial, but it is a fact that many of the pathologies of age-related diseases are associated with oxidative stress. Phytochemicals may reduce damage from oxidative stress; the intake of these through diet could represent a strategy to lessen their pathological consequences. The popular and widely consumed licorice (Glycyrrhiza glabra) is a rich source of potential antioxidants. The aim of this study was to investigate whether licorice increases the oxidative stress resistance and lifespan of the animal model Caenorhabditis elegans. Licorice roots ethanolic extract showed in vitro antioxidant activity, with an IC50 of 51.17 µg/mL using 2,2-diphenyl-1-picrylhydrazyl (DPPH) as free radical. C. elegans pretreated with licorice showed an increase of survival rate when exposed to the oxidant juglone, being this increase up to ∼33.56%. This pretreated population also showed an increase in lifespan of 14.28% at a concentration of 250 µg/mL. In conclusion, we suggest that licorice has a high antioxidant capability both in vitro and in vivo and that this activity may explain the observed extension of lifespan.

Viola cornuta and Viola x wittrockiana: Phenolic compounds, antioxidant and neuroprotective activities on Caenorhabditis elegans.

Different Viola species are known for their traditional use as analgesic, antitussive, febrifuge, hipnotic, analgesic and anti-inflammatory medicinal agents. Additionally, they are considered edible flowers in certain cultures. Thus, the aim of this work was to characterize the phenolic composition and to assess the neuroprotective properties of Viola cornuta and Viola x wittrockiana using in vitro and in vivo methodologies with Caenorhabditis elegans as model. The identification of the phenolic compounds was carried out with a LC-DAD-ESI/MSn. The antioxidant activity of the extracts was determined in vitro using Folin- Ciocalteu, DPPH and FRAP assays and in vivo with a juglone-induced oxidative stress in C. elegans. The neuroprotective properties were evaluated measuring the ability to inhibit CNS enzymes (MAO A, AChE), and the capability to avoid paralyzing the C. elegans CL4176, an Alzheimer disease model. The phenolic content was higher in V. x wittrockiana, being quercetin-3-O-(6-O-rhamnosylglucoside)-7-O-rhamnoside the predominant compound in the extract, which also exhibited a stronger antioxidant capacity in vitro and a higher response to lethal oxidative stress on C. elegans than V. cornuta. Only V. x wittrockiana showed inhibitory effect on CNS enzymes, such as acetylcholinesterase and monoamine oxidase A, but both had protective effect against the paralysis of C. elegans. These findings suggest that the studied V. cornuta and V. x wittrockiana could be interesting candidates for age related neurodegenerative disorder associated with oxidative stress.