Activities of Combinations of Antistaphylococcal Antibiotics with Fusidic Acid against Staphylococcal Biofilms in In Vitro Static and Dynamic Models.

Staphylococcal biofilms are a major cause of therapeutic failure, especially when caused by multiresistant strains. Oral fusidic acid is currently being redeveloped in the United States for skin, skin structure, and orthopedic infections, in which biofilms play a major role. The aim of this study was to examine the activity of fusidic acid alone or combined with other antistaphylococcal drugs against biofilms made by a reference strain and five clinical isolates of Staphylococcus aureus or Staphylococcus epidermidis in in vitro static and dynamic models (microtiter plates and a CDC reactor) exposed to clinically relevant concentrations. In microtiter plates, antibiotics alone were poorly active, with marked differences among strains. At concentrations mimicking the free-drug human maximum concentration of drug in serum (Cmax), the combination of fusidic acid with linezolid, daptomycin, or vancomycin resulted in increased activity against 4 to 5 strains, while the combination with doxycycline, rifampin, or moxifloxacin increased activity against 1 to 3 strains only. In the CDC reactor, biofilms were grown under constant flow and antibiotic concentrations decreased over time according to human elimination rates. A bactericidal effect was obtained when fusidic acid was combined with daptomycin or linezolid, but not with vancomycin. The higher tolerance of biofilms to antibiotics in the CDC reactor is probably attributable to the more complex architecture they adopt when growing under constant flow. Because biofilms grown in the CDC reactor are considered more similar to those developing in vivo, the data support further testing of combinations of fusidic acid with daptomycin or linezolid in models pertinent to chronic skin, skin structure, or orthopedic infections.

Anidulafungin increases the antibacterial activity of tigecycline in polymicrobial Candida albicans/Staphylococcus aureus biofilms on intraperitoneally implanted foreign bodies.

Objectives: We aimed to establish a novel murine intra-abdominal foreign body infection model to study the activity of anidulafungin and tigecycline against dual species Candida albicans/Staphylococcus aureus biofilms. Methods: In vitro and in vivo single and dual species biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well plates or implanted intraperitoneally in BALB/c mice. The effect of tigecycline and anidulafungin alone and in combination was tested using clinically relevant concentrations. Scanning electron microscopy was used to visualize the mature biofilm structure developed intraperitoneally. Flow cytometry was used to determine the immunological response upon infection. Immunoblot analysis allowed us to determine the effect of anidulafungin on poly-ß-(1,6)-N-acetylglucosamine in in vitro-grown S. aureus biofilms. Results: We determined the MIC, MBC and in vitro susceptibility profile for anidulafungin and tigecycline against C. albicans and S. aureus in mixed and single species biofilms. We demonstrated that anidulafungin acts synergistically when combined with tigecycline against in vivo intra-abdominal biofilms. Moreover, we reveal that anidulafungin reduces the abundance of S. aureus poly-ß-(1,6)-N-acetylglucosamine. The influx of neutrophils is much increased when infected with mixed biofilms compared with single species biofilms. Conclusions: Currently, treatment of intra-abdominal infections, in particular polymicrobial catheter-associated peritonitis, is ineffective. To the best of our knowledge, this is the first study that provides insight into new possible options for treatment of C. albicans/S. aureus biofilms present in the abdominal cavity.

Comparison of the antibiotic activities of Daptomycin, Vancomycin, and the investigational Fluoroquinolone Delafloxacin against biofilms from Staphylococcus aureus clinical isolates.

Biofilm-related infections remain a scourge. In an in vitro model of biofilms using Staphylococcus aureus reference strains, delafloxacin and daptomycin were found to be the most active among the antibiotics from 8 different pharmacological classes (J. Bauer, W. Siala, P. M. Tulkens, and F. Van Bambeke, Antimicrob. Agents Chemother. 57:2726-2737, 2013, doi:10.1128/AAC.00181-13). In this study, we compared delafloxacin to daptomycin and vancomycin using biofilms produced by 7 clinical strains (S. aureus epidemic clones CC5 and CC8) in order to rationalize the differences observed between the antibiotics and strains. The effects of the antibiotics on bacterial viability (resazurin reduction assay) and biomass (crystal violet staining) were measured and correlated with the proportion of polysaccharides in the matrix, the local microenvironmental pH (micro-pH), and the antibiotic penetration in the biofilm. At clinically meaningful concentrations, delafloxacin, daptomycin, and vancomycin caused a ≥25% reduction in viability against the biofilms formed by 5, 4, and 3 strains, respectively. The antibiotic penetration within the biofilms ranged from 0.6 to 52% for delafloxacin, 0.2 to 10% for daptomycin, and 0.2 to 1% for vancomycin; for delafloxacin, this was inversely related to the polysaccharide proportion in the matrix. Six biofilms were acidic, explaining the high potency of delafloxacin (lower MICs at acidic pH). Norspermidine and norspermine (disassembling the biofilm matrix) drastically increased delafloxacin potency and efficacy (50% reduction in viability for 6 biofilms at clinically meaningful concentrations) in direct correlation with its increased penetration within the biofilm, while they only modestly improved daptomycin efficacy (50% reduction in viability for 2 biofilms) and penetration, and they showed marginal effects with vancomycin. Delafloxacin potency and efficacy against biofilms are benefited by its penetration into the matrix and the local acidic micro-pH.

A combined pharmacodynamic quantitative and qualitative model reveals the potent activity of daptomycin and delafloxacin against Staphylococcus aureus biofilms.

Biofilms are associated with persistence of Staphylococcus aureus infections and therapeutic failures. Our aim was to set up a pharmacodynamic model comparing antibiotic activities against biofilms and examining in parallel their effects on viability and biofilm mass. Biofilms of S. aureus ATCC 25923 (methicillin-sensitive S. aureus [MSSA]) or ATCC 33591 (methicillin-resistant S. aureus [MRSA]) were obtained by culture in 96-well plates for 6 h/24 h. Antibiotic activities were assessed after 24/48 h of exposure to concentrations ranging from 0.5 to 512 times the MIC. Biofilm mass and bacterial viability were quantified using crystal violet and the redox indicator resazurin. Biofilms stained with Live/Dead probes were observed by using confocal microscopy. Concentration-effect curves fitted sigmoidal regressions, with a 50% reduction toward both matrix and viability obtained at sub-MIC or low multiples of MICs against young biofilms for all antibiotics tested. Against mature biofilms, maximal efficacies and potencies were reduced, with none of the antibiotics being able to completely destroy the matrix. Delafloxacin and daptomycin were the most potent, reducing viability by more than 50% at clinically achievable concentrations against both strains, as well as reducing biofilm depth, as observed in confocal microscopy. Rifampin, tigecycline, and moxifloxacin were effective against mature MRSA biofilms, while oxacillin demonstrated activity against MSSA. Fusidic acid, vancomycin, and linezolid were less potent overall. Antibiotic activity depends on biofilm maturity and bacterial strain. The pharmacodynamic model developed allows ranking of antibiotics with respect to efficacy and potency at clinically achievable concentrations and highlights the potential utility of daptomycin and delafloxacin for the treatment of biofilm-related infections.

The NADPH-dependent thioredoxin system constitutes a functional backup for cytosolic glutathione reductase in Arabidopsis.

Tight control of cellular redox homeostasis is essential for protection against oxidative damage and for maintenance of normal metabolism as well as redox signaling events. Under oxidative stress conditions, the tripeptide glutathione can switch from its reduced form (GSH) to oxidized glutathione disulfide (GSSG), and thus, forms an important cellular redox buffer. GSSG is normally reduced to GSH by 2 glutathione reductase (GR) isoforms encoded in the Arabidopsis genome, cytosolic GR1 and GR2 dual-targeted to chloroplasts and mitochondria. Measurements of total GR activity in leaf extracts of wild-type and 2 gr1 deletion mutants revealed that approximately 65% of the total GR activity is attributed to GR1, whereas approximately 35% is contributed by GR2. Despite the lack of a large share in total GR activity, gr1 mutants do not show any informative phenotype, even under stress conditions, and thus, the physiological impact of GR1 remains obscure. To elucidate its role in plants, glutathione-specific redox-sensitive GFP was used to dynamically measure the glutathione redox potential (E(GSH)) in the cytosol. Using this tool, it is shown that E(GSH) in gr1 mutants is significantly shifted toward more oxidizing conditions. Surprisingly, dynamic reduction of GSSG formed during induced oxidative stress in gr1 mutants is still possible, although significantly delayed compared with wild-type plants. We infer that there is functional redundancy in this critical pathway. Integrated biochemical and genetic assays identify the NADPH-dependent thioredoxin system as a backup system for GR1. Deletion of both, NADPH-dependent thioredoxin reductase A and GR1, prevents survival due to a pollen lethal phenotype.

Activity of Moxifloxacin Against Biofilms Formed by Clinical Isolates of Staphylococcus aureus Differing by Their Resistant or Persister Character to Fluoroquinolones.

Staphylococcus aureus biofilms are poorly responsive to antibiotics. Underlying reasons include a matrix effect preventing drug access to embedded bacteria, or the presence of dormant bacteria with reduced growth rate. Using 18 clinical isolates previously characterized for their moxifloxacin-resistant and moxifloxacin-persister character in stationary-phase culture, we studied their biofilm production and matrix composition and the anti-biofilm activity of moxifloxacin. Biofilms were grown in microtiter plates and their abundance quantified by crystal violet staining and colony counting; their content in polysaccharides, extracellular DNA and proteins was measured. Moxifloxacin activity was assessed after 24 h of incubation with a broad range of concentrations to establish full concentration-response curves. All clinical isolates produced more biofilm biomass than the reference strain ATCC 25923, the difference being more important for those with high relative persister fractions to moxifloxacin, most of which being also resistant. High biofilm producers expressed icaA to higher levels, enriching the matrix in polysaccharides. Moxifloxacin was less potent against biofilms from clinical isolates than from ATCC 25923, especially against moxifloxacin-resistant isolates with high persister fractions, which was ascribed to a lower concentration of moxifloxacin in these biofilms. Time-kill curves in biofilms revealed the presence of a moxifloxacin-tolerant subpopulation, with low multiplication capacity, whatever the persister character of the isolate. Thus, moxifloxacin activity depends on its local concentration in biofilm, which is reduced in most isolates with high-relative persister fractions due to matrix effects, and insufficient to kill resistant isolates due to their high MIC.

Glutaredoxins and thioredoxins in plants.

During the 70s and 80s two plant thioredoxin systems were identified. The chloroplastic system is composed of a ferredoxin-dependent thioredoxin, with two thioredoxin types (m and f) regulating the activity of enzymes implicated in photosynthetic carbon assimilation. In the cytosol of heterotrophic tissues, an NADP dependent thioredoxin reductase and a thioredoxin (h) were identified. The first plant glutaredoxin was only identified later, in 1994. Our view of plant thioredoxins and glutaredoxins was profoundly modified by the sequencing programs which revealed an unexpected number of genes encoding not only the previously identified disulfide reductases, but also numerous new types. At the same time it became clear that plant genomes encode chloroplastic, cytosolic and mitochondrial peroxiredoxins, suggesting a major role for redoxins in anti-oxidant defense. Efficient proteomics approaches were developed allowing the characterization of numerous thioredoxin target proteins. They are implicated in different aspects of plant life including development and adaptation to environmental changes and stresses. The most important challenge for the next years will probably be to identify in planta which redoxin reduces which target, a question which remains unsolved due to the low specificities of redoxins in vitro and the numerous redundancies which in most cases mask the phenotype of redoxin mutants.

Synergistic activity between an antimicrobial polyacrylamide and daptomycin versus Staphylococcus aureus biofilm.

Antibiotic resistance of bacteria growing in biofilms compared to their planktonic counterparts enhances the difficulty to eradicate biofilm-associated infections. In the last decade, combination antibiotic therapy has emerged as an attractive strategy for treating biofilm infections, even if in most of tolerant biofilms the optimal combinations are still unknown. In this study, an antimicrobial cationic polyacrylamide was used in combination with daptomycin or moxifloxacin against mature biofilms of Staphylococcus aureus clinical isolates to examine a possible improvement of the antibiofilm activity of the two antibiotics. The polymer did not have an effect on moxifloxacin but significantly increased the antibiofilm efficacy of daptomycin. These findings are presumably related to the different mechanism of action of the two drugs. In summary, our data highlighted the ability of polycations to increase daptomycin antibiofilm activity providing a potential strategy to eradicate biofilms in industrial or medical settings.

The antifungal caspofungin increases fluoroquinolone activity against Staphylococcus aureus biofilms by inhibiting N-acetylglucosamine transferase.

Biofilms play a major role in Staphylococcus aureus pathogenicity but respond poorly to antibiotics. Here, we show that the antifungal caspofungin improves the activity of fluoroquinolones (moxifloxacin, delafloxacin) against S. aureus biofilms grown in vitro (96-well plates or catheters) and in vivo (murine model of implanted catheters). The degree of synergy among different clinical isolates is inversely proportional to the expression level of ica operon, the products of which synthesize poly-N-acetyl-glucosamine polymers, a major constituent of biofilm matrix. In vitro, caspofungin inhibits the activity of IcaA, which shares homology with ß-1-3-glucan synthase (caspofungin's pharmacological target in fungi). This inhibition destructures the matrix, reduces the concentration and polymerization of exopolysaccharides in biofilms, and increases fluoroquinolone penetration inside biofilms. Our study identifies a bacterial target for caspofungin and indicates that IcaA inhibitors could potentially be useful in the treatment of biofilm-related infections.

NTR/NRX define a new thioredoxin system in the nucleus of Arabidopsis thaliana cells.

Thioredoxins (TRX) are key components of cellular redox balance, regulating many target proteins through thiol/disulfide exchange reactions. In higher plants, TRX constitute a complex multigenic family whose members have been found in almost all cellular compartments. Although chloroplastic and cytosolic TRX systems have been largely studied, the presence of a nuclear TRX system has been elusive for a long time. Nucleoredoxins (NRX) are potential nuclear TRX found in most eukaryotic organisms. In contrast to mammals, which harbor a unique NRX, angiosperms generally possess multiple NRX organized in three subfamilies. Here, we show that Arabidopsis thaliana has two NRX genes (AtNRX1 and AtNRX2), respectively, belonging to subgroups I and III. While NRX1 harbors typical TRX active sites (WCG/PPC), NRX2 has atypical active sites (WCRPC and WCPPF). Nevertheless, both NRX1 and NRX2 have disulfide reduction capacities, although NRX1 alone can be reduced by the thioredoxin reductase NTRA. We also show that both NRX1 and NRX2 have a dual nuclear/cytosolic localization. Interestingly, we found that NTRA, previously identified as a cytosolic protein, is also partially localized in the nucleus, suggesting that a complete TRX system is functional in the nucleus. We show that NRX1 is mainly found as a dimer in vivo. nrx1 and nrx2 knockout mutant plants exhibit no phenotypic perturbations under standard growth conditions. However, the nrx1 mutant shows a reduced pollen fertility phenotype, suggesting a specific role of NRX1 at the haploid phase.