Tobramycin-mediated self-assembly of DNA nanostructures for targeted treatment of Pseudomonas aeruginosa-infected lung inflammation.

Pseudomonas aeruginosa (PA) is one of the most common multidrug-resistant pathogens found in clinics, often manifesting as biofilms. However, due to the emergence of superbugs in hospitals and the overuse of antibiotics, the prevention and treatment of PA infections have become increasingly challenging. Utilizing DNA nanostructures for packaging and delivering antibiotics presents an intervention strategy with significant potential. Nevertheless, construction of functional DNA nanostructures with multiple functionalities and enhanced stability in physiological settings remains challenging. In this study, the authors propose a magnesium-free assembly method that utilizes tobramycin (Tob) as a mediator to assemble DNA nanostructures, allowing for the functionalization of DNA nanostructures by combining DNA and antibiotics. Additionally, our study incorporates maleimide-modified DNA into the nanostructures to act as a targeting moiety specifically directed towards the pili of PA. The targeting ability of the constructed functional DNA nanostructure significantly improves the local concentration of Tob, thereby reducing the side effects of antibiotics. Our results demonstrate the successful construction of a maleimide-decorated Tob/DNA nanotube (NTTob-Mal) for the treatment of PA-infected lung inflammation. The stability and biocompatibility of NTTob-Mal are confirmed, highlighting its potential for clinical applications. Furthermore, its specificity in recognizing and adhering to PA has been validated. In vitro experiments have shown its efficacy in inhibiting PA biofilm formation, and in a murine model, NTTob-Mal has exhibited significant therapeutic effectiveness against PA-induced pneumonia. In summary, the proposed antibiotic drug-mediated DNA nanostructure assembly approach holds promise as a novel strategy for targeted treatment of PA infections.

pH-Responsive, Charge-Reversing Layer-by-Layer Nanoparticle Surfaces Enhance Biofilm Penetration and Eradication.

Microbes entrenched within biofilms can withstand 1000-fold higher concentrations of antibiotics, in part due to the viscous extracellular matrix that sequesters and attenuates antimicrobial activity. Nanoparticle (NP)-based therapeutics can aid in delivering higher local concentrations throughout biofilms as compared to free drugs alone, thereby enhancing the efficacy. Canonical design criteria dictate that positively charged nanoparticles can multivalently bind to anionic biofilm components and increase biofilm penetration. However, cationic particles are toxic and are rapidly cleared from circulation in vivo, limiting their use. Therefore, we sought to design pH-responsive NPs that change their surface charge from negative to positive in response to the reduced biofilm pH microenvironment. We synthesized a family of pH-dependent, hydrolyzable polymers and employed the layer-by-layer (LbL) electrostatic assembly method to fabricate biocompatible NPs with these polymers as the outermost surface. The NP charge conversion rate, dictated by polymer hydrophilicity and the side-chain structure, ranged from hours to undetectable within the experimental timeframe. LbL NPs with an increasingly fast charge conversion rate more effectively penetrated through, and accumulated throughout, wildtype (PAO1) and mutant overexpressing biomass (ΔwspF) Pseudomonas aeruginosa biofilms. Finally, tobramycin, an antibiotic known to be trapped by anionic biofilm components, was loaded into the final layer of the LbL NP. There was a 3.2-fold reduction in ΔwspF colony forming units for the fastest charge-converting NP as compared to both the slowest charge converter and free tobramycin. These studies provide a framework for the design of biofilm-penetrating NPs that respond to matrix interactions, ultimately increasing the efficacious delivery of antimicrobials.

A sensitive surface-enhanced Raman spectroscopy detection for gentamicin and tobramycin using γ-Al2O3-modified silver nanoparticles coated with bovine serum albumin as substrate.

Two aminoglycoside antibiotics (AGs), gentamicin (GEN) and tobramycin (TOB), have good antibacterial activity against most pseudomonas aeruginosa and staphylococcus. The molecular structure of these drugs lack chromogenic groups, which brings challenges to their detection. In this project, the detecting methods for GEN and TOB utilizing surface-enhanced Raman spectroscopy (SERS) based on γ-Al2O3-modified silver nanoparticles (AgNPs) coated with bovine serum albumin (BSA) were established. The enhancement factors (EFs) of GEN and TOB were 2.44 × 105 and 2.67 × 106, respectively. The transmission electron microscopy (TEM), X-ray diffraction (XRD), thermogravimetric analysis (TGA) and spectrophotometric techniques were used to characterize the substrate and the combination of the substance with drugs. The pH, the addition amounts for the substrate and coagulant, as well as the mixing time were optimized. On the basis of single factor experiments, a more scientific response surface model was established. The concentrations of GEN and TOB showed good linear relationships with their Raman signals in the ranges of 6.67 × 10-8 - 2.00 × 10-6 and 6.67 × 10-9 - 3.00 × 10-7 mol L-1 respectively. The limits of detection (LODs) were 11.88 and 1.26 nmol L-1 for GEN and TOB, respectively. The methods were used successfully for the samples determination of the two AGs in commercial drugs and meat products.

Albumin-coated pH-responsive dimeric prodrug-based nano-assemblies with high biofilm eradication capacity.

Pseudomonas aeruginosa (PA) biofilms cause many persistent chronic infections in humans, especially in cystic fibrosis (CF) patients. The biofilms form a strong barrier which may inhibit antimicrobial agents from penetrating the biofilms and killing PA bacteria that reside deep within the biofilms. Concomitant therapies based on tobramycin (TOB) and azithromycin (AZM) have demonstrated beneficial effects in CF patients with chronic PA infections. However, the co-delivery of TOB and AZM has rarely been reported. In this study, we constructed a self-assembled pH-sensitive nano-assembly (DPNA) based on a dimeric prodrug (AZM-Cit-TOB) by simply inserting citraconic amide bonds between AZM and TOB. Moreover, the cationic surface of DPNA was further modified with anionic albumin (HSA) via electrostatic interactions to form an electrostatic complex (termed HSA@DPNA) for better biocompatibility. Upon arrival at the infected tissues, the citraconic amide bonds would be cleaved at acidic pH, resulting in the release of TOB and AZM for bacteria killing and biofilm eradication. As expected, HSA@DPNA showed comparable antibacterial abilities against the P. aeruginosa strain PAO1 in both planktonic and biofilm modes of growth compared to the TOB/AZM mixture in vitro. Moreover, HSA@DPNA exhibited excellent therapeutic efficacy on mice with PAO1-induced lung infection compared to the TOB/AZM mixture, and no detectable toxicity to mammalian cells/animals was observed during the therapeutic process. In summary, our study provides a promising method for the co-delivery of AZM and TOB in concomitant therapies against PAO1-related infection.

Thymus vulgaris essential oil + tobramycin within nanostructured archaeolipid carriers: A new approach against Pseudomonas aeruginosa biofilms.

BACKGROUND: Pseudomonas aeruginosa biofilms in the respiratory tract of patients with an excessive inflammatory context are difficult to eradicate. New medicines that simultaneously target biofilms and inflammation should be developed. HYPOTHESIS: Co-delivery of Thymus vulgaris essential oil (EOT) and tobramycin (TB) by nanostructured archaeolipids carriers (NAC) could support nebulization as well as improve EOT and TB antioxidant, anti-inflammatory and antibiofilm activity. METHODS: NAC(EOT+TB) were prepared by loading EOT and TB in NAC having a compritol and miglyol core, covered with a shell of archaeolipids, extracted from the hyperhalophylic archaebacteria Halorubrum tebenquichense, and Tween 80. NAC(EOT+TB) were structurally characterized, including DSC thermograms, Raman spectra, TB release profile, EOT volatilization and in vitro antioxidant activity. In addition, stability upon nebulization, autoclaving and storage were assessed. The antibiofilm activity on P. aeruginosa PAO1 established biofilm of NAC(EOT+TB) and the cytotoxicity on human lung epithelial cells and macrophage were determined, as well as intracellular reactive oxygen species (ROS) production and cytokines release on LPS stimulated cells. RESULTS: NAC(EOT+TB) showed a size of 197 ± 16 nm with PdI of 0.3 ± 0.1 and ζ Potential of -38 ± 3 mV. Structural characterization suggested that EOT was trapped in the compritol-miglyol core and TB was distributed between the surface of nanoparticles and free in solution. NAC(EOT+TB) displayed a dual release profile of TB, a delayed release of EOT and improved EOTs in vitro antioxidant activity. While NAC(EOT+TB) preserved its structural features after nebulization, autoclaving and 18 months of storage, carriers without archaeolipids gelled at room temperature and showed a significant increase of size after the same storage time. Below cytotoxic concentration, NAC(EOT+TB) decreased bacteria viability and enhanced the disruption of established PAO1 biofilms compared to free TB and EOT. Also, the strong entrapment of EOT in NAC(EOT+TB) delayed its volatilization, decreased intracellular ROS production and maintained its anti-inflammatory activity in LPS stimulated cells. CONCLUSION: Combination of EOT + TB within NAC(EOT+TB) result in a stable and nebulizable formulation that enhanced the antioxidant and anti-biofilm activity of free ingredients, improved their ability to decrease intracellular ROS and provided anti-inflammatory activity, at non-cytotoxic concentrations on eukaryotic cells.

Computational Fluid Dynamics (CFD) Guided Spray Drying Recommendations for Improved Aerosol Performance of a Small-Particle Antibiotic Formulation.

PURPOSE: The objective of this study was to implement computational fluid dynamics (CFD) simulations and aerosol characterization experiments to determine best-case spray drying conditions of a tobramycin excipient enhanced growth (Tobi-EEG) formulation for use in a pediatric air-jet dry powder inhaler (DPI). METHODS: An iterative approach was implemented in which sets of spray drying conditions were explored using CFD simulations followed by lead candidate selection, powder production and in vitro aerosol testing. CFD simulations of a small-particle spray dryer were performed to capture droplet drying parameters and surface-averaged temperature and relative humidity (RH) conditions in the powder collection region. In vitro aerosol testing was performed for the selected powders using the pediatric air-jet DPI, cascade impaction, and aerosol transport through a pediatric mouth-throat (MT) model to a tracheal filter. RESULTS: Based on comparisons of CFD simulations and in vitro powder performance, recommended drying conditions for small-particle powders with electrostatic collection include: (i) reducing the CFD-predicted drying parameters of κavg and κmax to values below 3 µm2/ms and 114 µm2/ms, respectively; (ii) maintaining the Collector Surface RH within an elevated range, which for the Tobi-EEG formulation with l-leucine was 20-30 %RH; and (iii) ensuring that particles reaching the collector were fully dried, based on a mass fraction of solute CFD parameter. CONCLUSIONS: Based on the newly recommended spray dryer conditions for small particle aerosols, delivery performance of the lead Tobi-EEG formulation was improved resulting in >60% of the DPI loaded dose passing through the pediatric MT model.

Tobramycin-loaded complexes to prevent and disrupt Pseudomonas aeruginosa biofilms.

Carbohydrate-based materials are increasingly investigated for a range of applications spanning from healthcare to advanced functional materials. Synthetic glycopolymers are particularly attractive as they possess low toxicity and immunogenicity and can be used as multivalent ligands to target sugar-binding proteins (lectins). Here, we utilised RAFT polymerisation to synthesize two families of novel diblock copolymers consisting of a glycopolymers block containing either mannopyranose or galactopyranose pendant units, which was elongated with sodium 2-acrylamido-2-methyl-1-propanesulfonate (AMPS) to generate a polyanionic block. The latter enabled complexation of cationic aminoglycoside antibiotic tobramycin through electrostatic interactions (loading efficiency in the 0.5-6.3 wt% range, depending on the copolymer). The resulting drug vectors were characterized by dynamic light scattering, zeta-potential, and transmission electron microscopy. Tobramycin-loaded complexes were tested for their ability to prevent clustering or disrupt biofilm of the Pseudomonas aeruginosa Gram-negative bacterium responsible for a large proportion of nosocomial infection, especially in immunocompromised patients. P. aeruginosa possesses two specific tetrameric carbohydrate-binding adhesins, LecA (PA-IL, galactose/N-acetyl-D-galactosamine-binding) and LecB (PA-IIL, fucose/mannose-binding), and the cell-associated and extracellular adhesin CdrA (Psl/mannose-binding) thus ideally suited for targeted drug delivery using sugar-decorated tobramycin-loaded complexes here developed. Both aliphatic and aromatic linkers were utilised to link the sugar pendant units to the polyacrylamide polymer backbone to assess the effect of the nature of such linkers on bactericidal/bacteriostatic properties of the complexes. Results showed that tobramycin-loaded complexes efficiently suppressed (40 to 60% of inhibition) in vitro biofilm formation in PAO1-L P. aeruginosa and that preferential targeting of PAO1-L biofilm can be achieved using mannosylated glycopolymer-b-AMPSm.

Tobramycin Supplemented Small-Diameter Vascular Grafts for Local Antibiotic Delivery: A Preliminary Formulation Study.

Peripheral artery occlusive disease is an emerging cardiovascular disease characterized by the blockage of blood vessels in the limbs and is associated with dysfunction, gangrene, amputation, and a high mortality risk. Possible treatments involve by-pass surgery using autologous vessel grafts, because of the lack of suitable synthetic small-diameter vascular prosthesis. One to five percent of patients experience vascular graft infection, with a high risk of haemorrhage, spreading of the infection, amputation and even death. In this work, an infection-proof vascular graft prototype was designed and manufactured by electrospinning 12.5% w/v poly-L-lactic-co-glycolic acid solution in 75% v/v dichloromethane, 23.8% v/v dimethylformamide and 1.2% v/v water, loaded with 0.2% w/wPLGA. Polymer and tobramycin concentrations were selected after viscosity and surface tension and after HPLC-UV encapsulation efficiency (EE%) evaluation, respectively. The final drug-loaded prototype had an EE% of 95.58% ± 3.14%, with smooth fibres in the nanometer range and good porosity; graft wall thickness was 291 ± 20.82 µm and its internal diameter was 2.61 ± 0.05 mm. The graft's antimicrobic activity evaluation through time-kill assays demonstrated a significant and strong antibacterial activity over 5 days against Staphylococcus aureus and Escherichia coli. An indirect cell viability assay on Normal Human Dermal Fibroblasts (NHDF) confirmed the cytocompatibility of the grafts.

Amplified electrochemical antibiotic aptasensing based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework.

A facile and versatile competitive electrochemical aptasensor for tobramycin (TOB) detection is described using electrochemical-deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework (AuNPs/P-MOF) as signal-amplification platform and a DNA probe labeled with methylene blue (MB) at the 3'-end (MB-Probe) as a signal producer. First, F-Probe (short complementary DNA strands of both the aptamer and the MB-Probe label with a sulfhydryl group at the 5'-end) was immobilized on the AuNPs/P-MOF modified electrode as detection probes, which competed with TOB in binding to the aptamer. TOB-aptamer binding resulted in F-Probe remaining unhybridized on the electrode surface, so that a significant current response was generated by hybridizing with MB-Probe instead. The developed strategy showed favorable repeatability, with a relative standard deviation (RSD) of 4.3% computed over five independent assays, and high stability, with only 6.8% degradation after 15 days of storage. Under optimal conditions, the proposed aptamer strategy exhibited a linear detection range from 100 pM to 500 nM with a limit of detection (LOD) of 56 pM (S/N = 3). The electrochemical aptasensor demonstrated remarkable selectivity, and its feasibility for accurate and quantitative detection of TOB in milk samples was confirmed (RSD < 4.5%). Due to its simple design, easy operation, and high sensitivity and selectivity, the proposed method could expect to detect other antibiotics by replacing the aptamers. In summary, this study provides a simple and effective new strategy for electrochemical aptasening based on MOF-based sensing interface. Scheme illustration of label-free competitive electrochemical aptamer-based detection of tobramycin based on electrochemically deposited AuNPs coordinated with PEI-functionalized Fe-based metal-organic framework as signal-amplification platform.

Structural and phylogenetic analyses of resistance to next-generation aminoglycosides conferred by AAC(2') enzymes.

Plazomicin is currently the only next-generation aminoglycoside approved for clinical use that has the potential of evading the effects of widespread enzymatic resistance factors. However, plazomicin is still susceptible to the action of the resistance enzyme AAC(2')-Ia from Providencia stuartii. As the clinical use of plazomicin begins to increase, the spread of resistance factors will undoubtedly accelerate, rendering this aminoglycoside increasingly obsolete. Understanding resistance to plazomicin is an important step to ensure this aminoglycoside remains a viable treatment option for the foreseeable future. Here, we present three crystal structures of AAC(2')-Ia from P. stuartii, two in complex with acetylated aminoglycosides tobramycin and netilmicin, and one in complex with a non-substrate aminoglycoside, amikacin. Together, with our previously reported AAC(2')-Ia-acetylated plazomicin complex, these structures outline AAC(2')-Ia's specificity for a wide range of aminoglycosides. Additionally, our survey of AAC(2')-I homologues highlights the conservation of residues predicted to be involved in aminoglycoside binding, and identifies the presence of plasmid-encoded enzymes in environmental strains that confer resistance to the latest next-generation aminoglycoside. These results forecast the likely spread of plazomicin resistance and highlight the urgency for advancements in next-generation aminoglycoside design.

Tobramycin and Colistin display anti-inflammatory properties in CuFi-1 cystic fibrosis cell line.

Current cystic fibrosis (CF) treatment strategies are primarily focused on oral/inhaled anti-inflammatories and antibiotics, resulting in a considerable treatment burden for CF patients. Therefore, combination treatments consisting of anti-inflammatories with antibiotics could reduce the CF treatment burden. However, there is an imperative need to understand the potential drug-drug interactions of these combination treatments to determine their efficacy. Thus, this study aimed to determine the interactions of the anti-inflammatory agent Ibuprofen with each of the CF-approved inhaled antibiotics (Tobramycin, Colistin and its prodrug colistimethate sodium/Tadim) and anti-bacterial and anti-inflammatory efficacy. Chemical interactions of the Ibuprofen:antibiotic combinations were elucidated using High-Resolution Mass-Spectrometry (HRMS) and 1H NMR. HRMS showed pairing of Ibuprofen and Tobramycin, further confirmed by 1H NMR whilst no pairing was observed for either Ibuprofen:Colistin or Ibuprofen:Tadim combinations. The anti-bacterial activity of the combinations against Pseudomonas aeruginosa showed that neither paired nor non-paired Ibuprofen:antibiotic therapies altered the anti-bacterial activity. The anti-inflammatory efficacy of the combination therapies was next determined at two different concentrations (Low and High) using in vitro models of NuLi-1 (healthy) and CuFi-1 (CF) cell lines. Differential response in the anti-inflammatory efficacy of Ibuprofen:Tobramycin combination was observed between the two concentrations due to changes in the structural conformation of the paired Ibuprofen:Tobramycin complex at High concentration, confirmed by 1H NMR. In contrast, the non-pairing of the Ibuprofen:Colistin and Ibuprofen:Tadim combinations showed a significant decrease in IL-8 secretion at both the concentrations. Importantly, all antibiotics alone showed anti-inflammatory properties, highlighting the inherent anti-inflammatory properties of these antibiotics.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation-induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission "on" signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ-potential and dynamic light-scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold-standard transfection reagent Lipofectamine® 2000.

Spray-dried multidrug particles for pulmonary co-delivery of antibiotics with N-acetylcysteine and curcumin-loaded PLGA-nanoparticles.

Nowadays, the resistance of bacterial biofilms towards the available antibiotics is a severe problem. Therefore, many efforts were devoted to develop new formulations using nanotechnology. We have developed an inhalable microparticle formulation using spray-drying combining multiple drugs: an antibiotic (tobramycin, ciprofloxacin or azithromycin), N-acetylcysteine (NAC), and curcumin (Cur). The use of PLGA nanoparticles (NP) also allowed incorporating curcumin to facilitate spray drying and modify the release of some compounds. The aerosolizable microparticles formulations were characterized in terms of size, morphology, and aerodynamic properties. Biocompatibility when tested on macrophage-like cells was acceptable after 20 h exposure for concentrations up to at least 32 µg/mL. Antibacterial activity of free drugs versus drugs in the multiple drug formulations was evaluated on P. aeruginosa in the same range. When co-delivered the efficacy of tobramycin was enhanced compared to the free drug for the 1 µg/mL concentration. The combinations of azithromycin and ciprofloxacin with NAC and Cur did not show an improved antibacterial activity. Bacteria-triggered cytokine release was not inhibited by free antibiotics, except for TNF-α. In contrast, the application of NAC and the addition of curcumin-loaded PLGA NPs showed a higher potential to inhibit TNF-α, IL-8, and IL-1ß release. Overall, the approach described here allows simultaneous delivery of antibacterial, mucolytic, and anti-inflammatory compounds in a single inhalable formulation and may therefore pave the way for a more efficient therapy of pulmonary infections.

Overcoming ß-Lactam resistance in Pseudomonas aeruginosa using non-canonical tobramycin-based antibiotic adjuvants.

ß-Lactam antibiotics have for long been a mainstay in antimicrobial chemotherapy. However, due to its ubiquitous usage, bacteria have evolved multiple concerted pathways to evade its actions, underscoring the complexity of resistance to this class of drug. Current strategies to mitigate this problem are geared towards developing inhibitors that can shield the ß-lactam core from enzymatic hydrolysis. In reality, a combination of factors including porin loss, overexpressed efflux pumps, expression of ß-lactamases, reduced outer membrane permeability, and target modifications are characteristics of phenotypes that are microbiologically resistant to ß-lactam antibiotics, especially Pseudomonas aeruginosa. Herein, we describe a strategy that may simultaneously address multiple mechanisms of resistance to ß-lactams. A triple combination with ß-lactam/ß-lactamase inhibitors offers better microbiological response against carbapenem-resistant P. aeruginosa than the current standard of care. The observed interactions are also unaffected by efflux pumps. We conclude that a multicomponent combination therapy may be the way forward in addressing the myriads of emerging therapy failure associated with ß-lactam resistance.

A colorimetric nanoprobe based on dynamic aggregation of SDS-capped silver nanoparticles for tobramycin determination in exhaled breath condensate.

A colorimetric nanoprobe was developed for the quantification of tobramycin in exhaled breath condensate (EBC). The nanoprobe consists of silver nanoparticles (Ag NPs) modified with sodium dodecyl sulfate (SDS), which is applied in the presence of sodium metaborate. Characterization of the synthesized SDS-capped Ag NPs by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FT-IR), X-ray powder diffraction (XRD), and energy-dispersive X-ray spectroscopy (EDX) showed that the nanoparticles were well synthesized with nearly uniform size and an average diameter of < 30 nm. Interaction of sodium metaborate with the SDS-capped Ag NPs and tobramycin results in aggregation of the nanoparticles and consequently decreases the absorbance intensity, leading to the production of a new absorbance peak and a color change from yellow to purple. The absorbance intensity was recorded at λmax = 400 nm and 522 nm and λ522/λ400 was used as the analytical signal. The experimental parameters were investigated and optimized using a multivariate optimization method (central composite design). The current nanoprobe gives a linear response for tobramycin from 1.0 to 50.0 ng mL-1 with a detection limit of 0.5 ng mL-1. The intra- and inter-day relative standard deviations for five replicated analyses of 10.0 ng mL-1 tobramycin are 2.8% and 4.2%, respectively. Graphical abstractSchematic representation of SDS-capped silver nanoparticles's response to tobramycin in the presence of sodium metaborate.

Antibacterial activity of iron oxide, iron nitride, and tobramycin conjugated nanoparticles against Pseudomonas aeruginosa biofilms.

BACKGROUND: Novel methods are necessary to reduce morbidity and mortality of patients suffering from infections with Pseudomonas aeruginosa. Being the most common infectious species of the Pseudomonas genus, P. aeruginosa is the primary Gram-negative etiology responsible for nosocomial infections. Due to the ubiquity and high adaptability of this species, an effective universal treatment method for P. aeruginosa infection still eludes investigators, despite the extensive research in this area. RESULTS: We report bacterial inhibition by iron-oxide (nominally magnetite) nanoparticles (NPs) alone, having a mean hydrodynamic diameter of ~ 16 nm, as well as alginate-capped iron-oxide NPs. Alginate capping increased the average hydrodynamic diameter to ~ 230 nm. We also investigated alginate-capped iron-oxide NP-drug conjugates, with a practically unchanged hydrodynamic diameter of ~ 232 nm. Susceptibility and minimum inhibitory concentration (MIC) of the NPs, NP-tobramycin conjugates, and tobramycin alone were determined in the PAO1 bacterial colonies. Investigations into susceptibility using the disk diffusion method were done after 3 days of biofilm growth and after 60 days of growth. MIC of all compounds of interest was determined after 60-days of growth, to ensure thorough establishment of biofilm colonies. CONCLUSIONS: Positive inhibition is reported for uncapped and alginate-capped iron-oxide NPs, and the corresponding MICs are presented. We report zero susceptibility to iron-oxide NPs capped with polyethylene glycol, suggesting that the capping agent plays a major role in enabling bactericidal ability in of the nanocomposite. Our findings suggest that the alginate-coated nanocomposites investigated in this study have the potential to overcome the bacterial biofilm barrier. Magnetic field application increases the action, likely via enhanced diffusion of the iron-oxide NPs and NP-drug conjugates through mucin and alginate barriers, which are characteristic of cystic-fibrosis respiratory infections. We demonstrate that iron-oxide NPs coated with alginate, as well as alginate-coated magnetite-tobramycin conjugates inhibit P. aeruginosa growth and biofilm formation in established colonies. We have also determined that susceptibility to tobramycin decreases for longer culture times. However, susceptibility to the iron-oxide NP compounds did not demonstrate any comparable decrease with increasing culture time. These findings imply that iron-oxide NPs are promising lower-cost alternatives to silver NPs in antibacterial coatings, solutions, and drugs, as well as other applications in which microbial abolition or infestation prevention is sought.

Revisiting the Catalytic Cycle and Kinetic Mechanism of Aminoglycoside O-Nucleotidyltransferase(2″): A Structural and Kinetic Study.

Aminoglycoside antibiotics have lost much of their effectiveness due to widespread resistance, primarily via covalent modification. One of the most ubiquitous enzymes responsible for aminoglycoside resistance is aminoglycoside O-nucleotidyltransferase(2″), which catalyzes a nucleotidylation reaction. Due to its clinical importance, much research has focused on dissecting the mechanism of action, some of it dating back more than 30 years. Here, we present structural data for catalytically informative states of the enzyme, i.e., ANT(2″) in complex with adenosine monophosphate (AMP) and tobramycin (inactive-intermediate state) and in complex with adenylyl-2″-tobramycin, pyrophosphate, and Mn2+(product-bound state). These two structures in conjunction with our previously reported structure of ANT(2″)'s substrate-bound complex capture clinical states along ANT(2″)'s reaction coordinate. Additionally, isothermal titration calorimetry (ITC)-based studies are presented that assess the order of substrate binding and product release. Combined, these results outline a kinetic mechanism for ANT(2″) that contradicts what has been previously reported. Specifically, we show that the release of adenylated aminoglycoside precedes pyrophosphate. Furthermore, the ternary complex structures provide additional details on the catalytic mechanism, which reveals extensive similarities to the evolutionarily related DNA polymerase-ß superfamily.

Screening, Post-SELEX Optimization and Application of DNA Aptamers Specific for Tobramycin.

Tobramycin (TOB) is an aminoglycoside antibiotic. The residue of TOB in animal-derived foods and environment will be harmful to human health, and therefore the specific detection of TOB residue in food and water is of great importance. Herein, through magnetic beads-based SELEX, overall 37 ssDNA aptamers specific for TOB were screened after ten rounds of selection. The affinity and specificity of these aptamers were evaluated, among which No. 32 aptamer (Ap 32) exhibits excellent performance. Then a post-SELEX optimization of Ap 32 was carried out based on rational design, through which a truncated aptamer with the length of 34 nucleotides (Ap 32-2) was identified as the best aptamer for TOB. Finally, the application of the screened aptamer was explored. A colorimetric assay of TOB was established based on the aptamer-modified gold nanoparticles (AuNPs). In the range from 100 to 1400 nM, the absorbance of AuNPs solution at 520 nm was linearly decreased with the increased concentration of TOB. The detection limit was estimated to be 37.9 nM. The assay was applied to detect TOB residue in honey samples.

Comparison of two therapeutic approaches for the management of ventilator-associated pneumonia due to multidrug-resistant Acinetobacter: a randomized clinical trial study.

Management of ventilator-associated pneumonia (VAP) is a puzzling issue for infectious disease specialist. The present clinical trial study was aimed to comparing the effects of injectable colistin plus nebulized colistin and injectable colistin plus nebulized tobramycin on management of patients with VAP due to multidrug-resistant Acinetobacter. VAP patients were randomly divided into two groups (n = 30/each): Group 1 - patients that received intravenous (IV) meropenem, injectable colistin plus nebulized colistin, as a routine treatment, and Group 2 - patients that received IV meropenem, injectable colistin plus nebulized tobramycin. A total of 14 days of therapeutic intervention are required for every case. Follow-up for subjects was performed at five time-points: days 1, 3, 5, 7, and 14 after intervention. Also, a mean of creatinine levels of patients was determined in five times. In the present study, the clinical pulmonary infection score (CPIS) was determined on the basis of points assigned for various clinically manifestations of VAP. Based on our statistical analysis, there was no significant difference between CPIS and creatinine level in both Groups 1 and 2 (p > .05). CPIS and other clinical investigation appeared effectiveness of the treatment with injected colistin plus nebulized tobramycin; on the other hand, the results of present clinical trial showed that aforementioned therapeutic approach can be used as an alternative treatment for the management of infection in VAP patients.

Intelligent Platform for Simultaneous Detection of Multiple Aminoglycosides Based on a Ratiometric Paper-Based Device with Digital Fluorescence Detector Readout.

A digital fluorescence detector (DFD), a handheld fluorescence detection device, can convert the fluorescence signal of samples into the corresponding fluorescer concentration. Herein, by adopting a DFD as the readout, a novel intelligent platform was developed based on a ratiometric paper-based device (RPD) for multiple aminoglycoside detection. There are five layers and four parallel channels contained in the designed RPD, functioning as reagent storage, fluidic path control and signal processing, respectively. The rationale of this design lies in the fact that aptamer/graphitic carbon nitride nanosheet (Apt/g-C3N4 NS) modified layers can catalyze o-phenylenediamine to fluorescent 2,3-diaminophenazine (DAP) in the presence of H2O2. When Apt was removed from nanosheets via the Apt-target reaction, the peroxidase-like activity would be decreased, thus decreasing the production of DAP. All the changes of the fluorescence DAP signal can be read out using a portable DFD. Based on the DFD signal change related to the concentration of the target, a quantitative reaction platform was established. Furthermore, the sample flow and Apt-target reaction time can be reasonably regulated using the H2O2-cleavable hydrophobic compound modified layer placed between the target recognition region and detection region. Then, the practicality of this platform was verified through realizing sensitive analysis of streptomycin, tobramycin, and kanamycin simultaneously. Overall, with merits including portability and ease of operation, the platform shows great potential in on-site simultaneous detection of multiple targets, especially in resource-limited settings.