Evaluation of combination therapy for Burkholderia cenocepacia lung infection in different in vitro and in vivo models

Burkholderia cenocepacia is an opportunistic pathogen responsible for life-threatening infections in cystic fibrosis patients. B. cenocepacia is extremely resistant towards antibiotics and therapy is complicated by its ability to form biofilms. We investigated the efficacy of an alternative antimicrobial strategy for B. cenocepacia lung infections using in vitro and in vivo models. A screening of the NIH Clinical Collection 1&2 was performed against B. cenocepacia biofilms formed in 96-well microtiter plates in the presence of tobramycin to identify repurposing candidates with potentiator activity. The efficacy of selected hits was evaluated in a three-dimensional (3D) organotypic human lung epithelial cell culture model. The in vivo effect was evaluated in the invertebrate Galleria mellonella and in a murine B. cenocepacia lung infection model. The screening resulted in 60 hits that potentiated the activity of tobramycin against B. cenocepacia biofilms, including four imidazoles of which econazole and miconazole were selected for further investigation. However, a potentiator effect was not observed in the 3D organotypic human lung epithelial cell culture model. Combination treatment was also not able to increase survival of infected G. mellonella. Also in mice, there was no added value for the combination treatment. Although potentiators of tobramycin with activity against biofilms of B. cenocepacia were identified in a repurposing screen, the in vitro activity could not be confirmed nor in a more sophisticated in vitro model, neither in vivo. This stresses the importance of validating hits resulting from in vitro studies in physiologically relevant model systems

Antimicrobial efficacy against Pseudomonas aeruginosa biofilm formation in a three-dimensional lung epithelial model and the influence of fetal bovine serum

In vitro models that mimic in vivo host-pathogen interactions are needed to evaluate candidate drugs that inhibit bacterial virulence traits. We established a new approach to study Pseudomonas aeruginosa biofilm susceptibility on biotic surfaces, using a three-dimensional (3-D) lung epithelial cell model. P. aeruginosa formed antibiotic resistant biofilms on 3-D cells without affecting cell viability. The biofilm-inhibitory activity of antibiotics and/or the anti-biofilm peptide DJK-5 were evaluated on 3-D cells compared to a plastic surface, in medium with and without fetal bovine serum (FBS). In both media, aminoglycosides were more efficacious in the 3-D cell model. In serum-free medium, most antibiotics (except polymyxins) showed enhanced efficacy when 3-D cells were present. In medium with FBS, colistin was less efficacious in the 3-D cell model. DJK-5 exerted potent inhibition of P. aeruginosa association with both substrates, only in serum-free medium. DJK-5 showed stronger inhibitory activity against P. aeruginosa associated with plastic compared to 3-D cells. The combined addition of tobramycin and DJK-5 exhibited more potent ability to inhibit P. aeruginosa association with both substrates. In conclusion, lung epithelial cells influence the efficacy of most antimicrobials against P. aeruginosa biofilm formation, which in turn depends on the presence or absence of FBS

Fabrication of microporous coatings on titanium implants with improved mechanical, antibacterial and cell-interactive properties

The success of an orthopedic implant therapy depends on successful bone integration and the prevention of microbial infections. In this work, plasma electrolytic oxidation (PEO) was performed to deposit TiO2 coatings enriched with Ca, P, and Ag on titanium to improve its surface properties and antibacterial blasts efficacy while maintaining normal biological functions and thus to enhance the performance of orthopedic implants. After PEO treatment, the surface of Ti was converted to anatase and rutile TiO2, hydroxyapatite, and calcium titanate phases. The presence of these crystalline phases was further increased with an increased Ag content in the coatings. The developed coatings also exhibited a more porous morphology with an improved surface wettability, roughness, microhardness, and frictional coefficient. In vitro antibacterial assays indicated that the Ag-doped coatings can significantly prevent the growth of both Staphylococcus aureus and Escherichia coli by releasing Ag+ ions, and the ability to prevent these bacteria was enhanced by increasing the Ag content in the coatings, resulting in a maximal 6-log reduction of E. coli and a maximal 5-log reduction of S. aureus after 24 h of incubation. Moreover, the in vitro cytocompatibility evaluation of the coatings showed that the osteoblast (MC3T3) cell integration on the PEO-based coatings was greatly improved compared to untreated Ti and no notable impact on their cytocompatibility was observed on increasing the amount of Ag in the coating. In conclusion, the coating with favorable physicochemical and mechanical properties along with controlled silver ion release can offer an excellent antibacterial performance and osteocompatibility and can thus become a prospective coating strategy to face current challenges in orthopedics

In Vitro Inhibition of Streptococcus mutans Biofilm Formation on Hydroxyapatite by Subinhibitory Concentrations of Anthraquinones

We report that certain anthraquinones (AQs) reduce Streptococcus mutans biofilm formation on hydroxyapatite at concentrations below the MIC. Although AQs are known to generate reactive oxygen species, the latter do not underlie the observed effect. Our results suggest that AQs inhibit S. mutans biofilm formation by causing membrane perturbation

Genomewide screening for genes involved in biofilm formation and miconazole susceptibility in Saccharomyces cerevisiae

Infections related to fungal biofilms are difficult to treat due to the reduced susceptibility of sessile cells to most antifungal agents. Previous research has shown that 1-10% of sessile Candida cells survive treatment with high doses of miconazole (a fungicidal imidazole). The aim of this study was to identify genes involved in fungal biofilm formation and to unravel the mechanisms of resistance of these biofilms to miconazole. To this end, a screening of a Saccharomyces cerevisiae deletion mutant bank was carried out. Our results revealed that genes involved in peroxisomal transport and the biogenesis of the respiratory chain complex IV play an essential role in biofilm formation. On the other hand, genes involved in transcription and peroxisomal and mitochondrial organization seem to highly influence the susceptibility to miconazole of yeast biofilms. Additionally, our data confirm previous findings on genes involved in biofilm formation and in general stress responses. Our data suggest the involvement of peroxisomes in biofilm formation and miconazole resistance in fungal biofilms

Antibiofilm activities of borneol-citral-loaded pickering emulsions against Pseudomonas aeruginosa and Staphylococcus aureus in physiologically relevant chronic infection models

Phytochemicals are promising antibacterials for the development of novel antibiofilm drugs, but their antibiofilm activity in physiologically relevant model systems is poorly characterized. As the host microenvironment can interfere with the activity of the phytochemicals, mimicking the complex environment found in biofilm associated infections is essential to predict the clinical potential of novel phytochemical-based antimicrobials. In the present study, we examined the antibiofilm activity of borneol, citral, and combinations of both as well as their Pickering emulsions against Staphylococcus aureus and Pseudomonas aeruginosa in an in vivo-like synthetic cystic fibrosis medium (SCFM2) model, an in vitro wound model (consisting of an artificial dermis and blood components at physiological levels), and an in vivo Galleria mellonella model. The Pickering emulsions demonstrated an enhanced biofilm inhibitory activity compared to both citral and the borneol/citral combination, reducing the minimum biofilm inhibitory concentration (MBIC) values up to 2 to 4 times against P. aeruginosa PAO1 and 2 to 8 times against S. aureus P8-AE1 in SCMF2. In addition, citral, the combination borneol/citral, and their Pickering emulsions can completely eliminate the established biofilm of S. aureus P8-AE1. The effectiveness of Pickering emulsions was also demonstrated in the wound model with a reduction of up to 4.8 log units in biofilm formation by S. aureus Mu50. Furthermore, citral and Pickering emulsions exhibited a significant degree of protection against S. aureus infection in the G. mellonella model. The present findings reveal the potential of citral- or borneol/citral-based Pickering emulsions as a type of alternative antibiofilm candidate to control pathogenicity in chronic infection. IMPORTANCE There is clearly an urgent need for novel formulations with antimicrobial and antibiofilm activity, but while there are plenty of studies investigating them using simple in vitro systems, there is a lack of studies in which (combinations of) phytochemicals are evaluated in relevant models that closely resemble the in vivo situation. Here, we examined the antibiofilm activity of borneol, citral, and their combination as well as Pickering emulsions (stabilized by solid particles) of these compounds. Activity was tested against Staphylococcus aureus and Pseudomonas aeruginosa in in vitro models mimicking cystic fibrosis sputum and wounds as well as in an in vivo Galleria mellonella model. The Pickering emulsions showed drastically increased antibiofilm activity compared to that of the compounds as such in both in vitro models and protected G. mellonella larvae from S. aureus-induced killing. Our data show that Pickering emulsions from phytochemicals are potentially useful for treating specific biofilm-related chronic infections