Targeting the nonmevalonate pathway in Burkholderia cenocepacia increases susceptibility to certain β-lactam antibiotics

The nonmevalonate pathway is the sole pathway for isoprenoid biosynthesis in Burkholderia cenocepacia and is possibly a novel target for the development of antibacterial chemotherapy. The goals of the present study were to evaluate the essentiality of dxr, the second gene of the nonmevalonate pathway, in B. cenocepacia and to determine whether interfering with the nonmevalonate pathway increases susceptibility toward antibiotics. To this end, a rhamnose-inducible conditional dxr knockdown mutant of B. cenocepacia strain K56-2 (B. cenocepacia K56-2dxr) was constructed, using a plasmid which enables the delivery of a rhamnose-inducible promoter in the chromosome. Expression of dxr is essential for bacterial growth; the growth defect observed in the dxr mutant could be complemented by expressing dxr in trans under the control of a constitutive promoter, but not by providing 2C-methyl-D-erythritol-4-phosphate, the reaction product of DXR (1-deoxy-D-xylulose 5-phosphate reductoisomerase). B. cenocepacia K56-2dxr showed markedly increased susceptibility to the beta-lactam antibiotics aztreonam, ceftazidime, and cefotaxime, while susceptibility to other antibiotics was not (or was much less) affected; this increased susceptibility could also be complemented by in trans expression of dxr. A similarly increased susceptibility was observed when antibiotics were combined with FR900098, a known DXR inhibitor. Our data confirm that the nonmevalonate pathway is essential in B. cenocepacia and suggest that combining potent DXR inhibitors with selected beta-lactam antibiotics is a useful strategy to combat B. cenocepacia infections

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

Stimulation of superoxide production increases fungicidal action of miconazole against Candida albicans biofilms

We performed a whole-transcriptome analysis of miconazole-treated Candida albicans biofilms, using RNA-sequencing. Our aim was to identify molecular pathways employed by biofilm cells of this pathogen to resist action of the commonly used antifungal miconazole. As expected, genes involved in sterol biosynthesis and genes encoding drug efflux pumps were highly induced in biofilm cells upon miconazole treatment. Other processes were affected as well, including the electron transport chain (ETC), of which eight components were transcriptionally downregulated. Within a diverse set of 17 inhibitors/inducers of the transcriptionally affected pathways, the ETC inhibitors acted most synergistically with miconazole against C. albicans biofilm cells. Synergy was not observed for planktonically growing C. albicans cultures or when biofilms were treated in oxygen-deprived conditions, pointing to a biofilm-specific oxygen-dependent tolerance mechanism. In line, a correlation between miconazole's fungicidal action against C. albicans biofilm cells and the levels of superoxide radicals was observed, and confirmed both genetically and pharmacologically using a triple superoxide dismutase mutant and a superoxide dismutase inhibitor N-N'-diethyldithiocarbamate, respectively. Consequently, ETC inhibitors that result in mitochondrial dysfunction and affect production of reactive oxygen species can increase miconazole's fungicidal activity against C. albicans biofilm cells

Innovative approaches to treat Staphylococcus aureus biofilm-related infections

Many bacterial infections in humans and animals are caused by bacteria residing in biofilms, complex communities of attached organisms embedded in an extracellular matrix. One of the key properties of microorganisms residing in a biofilm is decreased susceptibility towards antimicrobial agents. This decreased susceptibility, together with conventional mechanisms leading to antimicrobial resistance, makes biofilm-related infections increasingly difficult to treat and alternative antibiofilm strategies are urgently required. In this review, we present three such strategies to combat biofilm-related infections with the important human pathogen Staphylococcus aureus: (i) targeting the bacterial communication system with quorum sensing (QS) inhibitors, (ii) a 'Trojan Horse' strategy to disturb iron metabolism by using gallium-based therapeutics and (iii) the use of 'non-antibiotics' with antibiofilm activity identified through screening of repurposing libraries

Search for potentiators of miconazole resulting in increased activity against Candida albicans biofilms

Candida albicans is the major cause of fungal infections in human, affecting even healthy individuals as it easily colonizes oral and vaginal mucosae where it progresses as a biofilm. The limited amount of successful anti-biofilm therapy options has led to failure of treatment, emergence of drug resistance and disease recurrence. To improve current treatments, we are looking for potentiators of the azole-type antifungal drug miconazole. On one hand, we randomly screened 1600 compounds of a drug repositioning library in combination with a sub-inhibitory concentration of miconazole for increased activity against mature C. albicans biofilms. Hits of the screen were molecules with diverse medical applications, including several artemisinins, used to treat malaria. On the other hand, we performed a comprehensive transcriptome analysis (using RNA-sequencing) of mature C. albicans biofilms treated with miconazole to reveal tolerance mechanisms induced upon application of this drug. Combination of an inhibitor of such tolerance pathway with miconazole could potentially lead to increased activity against C. albicans biofilms. Many of the differentially expressed genes were related to known defence mechanisms against azoles such as induction of drug efflux pumps. Interestingly, also new potential tolerance targets were identified. For the most promising hits from the screen and for selected inhibitors of miconazole-induced biological processes, we performed checkerboard analyses and calculated the fractional inhibitory concentration index, thereby revealing several synergistic combinations. We conclude that transcriptome analysis of drug-treated microbial populations can successfully identify tolerance targets for potentiation. However, randomly screening bio-active compound libraries seemed more efficient in this study.status: publishe

Infection of 3D lung epithelial cells.

<p>Left image showing an uninfected control where the microcarrier bead scaffolds are covered with A549 human lung epithelial cells, right image shows the 3D lung epithelial cells 17 h <i>p</i>.<i>i</i>., green dots (white arrows) indicate intracellular growth of <i>B</i>. <i>cenocepacia</i> K56-2, while the large green structure (blue arrow) suggests that biofilm-like structures are formed. Magnification is 300x. Scale bar is 400 μm.</p

<i>G</i>. <i>mellonella</i> survival assay.

<p>Percentage survival of <i>G</i>. <i>mellonella</i> in uninfected control groups (NI) and infected groups (I) 24, 48, and 72 h after administration of the different treatments (tobramycin (TOB, 512 μg/ml), econazole (ECO, 50 μM), miconazole (MICO, 50 μM) or the combinations) and/or infection. (Data shown are average, n = 6, error bars indicate SEM).</p