Journal: Frontiers in Molecular Biosciences

Article Title: Ultra-High-Precision, in-vivo Pharmacokinetic Measurements Highlight the Need for and a Route Toward More Highly Personalized Medicine

doi: 10.3389/fmolb.2019.00069

Figure Lengend Snippet: Electrochemical, aptamer-based (E-AB) sensors support the real-time measurement of drug concentrations in situ in the body. (A) E-AB sensors consist of a redox-reporter-modified aptamer (a nucleic acid selected for its ability to bind the target of interest) tethered to an interrogating electrode. In the presence of the target molecule a binding-induced conformational change alters the rate of electron transfer from the reporter, (B) altering the peak currents observed when the sensor is interrogated using square wave voltammetry at 30 and 200 Hz. This electrochemical method is highly sensitive to small changes in electron transfer thus making it ideal for the interrogation of E-AB sensors. (C) With a diameter of just 225 μm, the E-AB sensors we employed are narrow enough to emplace inside the jugular vein of live rats, supporting in-vivo measurements. The comparison of (D) E-AB vs. (E) gold standard HPLC-MS pharmacokinetics measured on two independent animals following intramuscular dosing of tobramycin (20 mg/kg) shows that the differences between the two profiles fall well within the range of animal-to-animal variability (see below). (F) To highlight animal-to-animal variability we present here pharmacokinetic data collected on 12 individual female and male rats, with weights spanning 350–500 g, illustrating the precision with which the few-second time resolution of E-AB sensors enables the high-precision tracking of the tobramycin's adsorption, distribution, and excretion kinetics.

Article Snippet: USP grade tobramycin sulfate was purchased from Gold BioTechnology (St. Louis, MO).

Techniques: In Situ, Modification, Binding Assay, In Vivo, Adsorption

Journal: Frontiers in Molecular Biosciences

Article Title: Ultra-High-Precision, in-vivo Pharmacokinetic Measurements Highlight the Need for and a Route Toward More Highly Personalized Medicine

doi: 10.3389/fmolb.2019.00069

Figure Lengend Snippet: Ultra-high-precision E-AB pharmacokinetic measurements following intravenous administration (20 mg/kg) of tobramycin (A) reveal important inter-subject variability in the (B) distribution and (C) elimination phases, as well as the (D) maximum plasma concentration and the (E) area-under-the-curve in male, and female Sprague-Dawley rats. Error bars indicate the standard error determined by performing a least-squares fit of the drug profiles to a two-compartment pharmacokinetic model.

Article Snippet: USP grade tobramycin sulfate was purchased from Gold BioTechnology (St. Louis, MO).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms

doi: 10.1371/journal.pone.0168615

Figure Lengend Snippet: Bacteria alone were grown 48 hours, then treated with the two phages (N) for 0, 4 or 24 hours. They were then treated with the drug (8X MIC, abbreviations as in ) for the duration of treatment. Starting from the time phages were added, the culture was grown 48 hours, so the duration of treatment following antibiotic addition was shorter with the longer phage pretreatments. ( A) Densities of viable P . aeruginosa PA14 at the end of treatment. The horizontal dashed line is the limit of detection (10 2 /mL), and yellow boxes indicate that estimates were below the limit of detection. ( B) Densities of phage at the end of treatment. The bold black line in (B) is the initial density of phage introduced. * Indicates that the 24 hours delay of gentamycin and tobramycin each have statistically significant effects on cell density compared to simultaneous treatment (P< 0.04, when correcting for multiple comparisons; tests of significance were equivalent for a Mann-Whitney U test and a median test using a Fisher’s exact test calculation—parametric tests were not possible because of some censored data). Means and standard errors from data obtained from two independent experiments, with a combined total of 5 replicate cultures. Raw data of these experiments can be found in .

Article Snippet: Bactericidal antibiotics of five classes were selected for use here: ceftazidime (Sigma), ciprofloxacin (AppliChem), colistin (Sigma), gentamicin (Sigma) and tobramycin (Tocris).

Techniques: Bacteria, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms

doi: 10.1371/journal.pone.0168615

Figure Lengend Snippet: Viable cell densities (mean ± standard error, three replicates) in 48 hours, intact biofilm populations of P . aeruginosa PA14 on plastic then treated for 48 hours in various combinations of two phages (NP1, NP3) and/or five antibiotics (ceftazidime, ciprofloxacin, colistin, gentamicin, and tobramycin). Antibiotics were used at 1X and 8X MIC concentrations. Abbreviations are given by the first 3 letters of the drug name, and the number following the abbreviation indicates 1X or 8X MIC. A prefix N- indicates inclusion of both phages. “Con” is the untreated control with no antibiotic or phage added as treatment. A ‘P’ above the bar indicates that the phage titer at 48 hours exceeded the inoculum density by at least a factor of 10. ‘S’ indicates statistical support for synergy, ‘F’ for facilitation. Raw data of these experiments can be found in .

Article Snippet: Bactericidal antibiotics of five classes were selected for use here: ceftazidime (Sigma), ciprofloxacin (AppliChem), colistin (Sigma), gentamicin (Sigma) and tobramycin (Tocris).

Techniques: Control

Journal: PLoS ONE

Article Title: Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms

doi: 10.1371/journal.pone.0168615

Figure Lengend Snippet: Treatment of 8 hours old biofilm population of P . aeruginosa PA14 on human epithelial cells treated with a mixture of NP1 and NP3 phage (N) and 1X MIC concentrations of ceftazidime, ciprofloxacin, colistin, gentamicin and tobramycin. A- Viable cell densities of Pseudomonas estimated in two independent experiments (red, blue) at 12 hours of exposure to the treatments. ‘S’ indicates statistical support for synergy and ‘F’ for facilitation. B- Densities of phage in two independent experiments sampled 12 hours after treatment. The bold black line in B is the density of the mixture of NP1 and NP3 introduced to the biofilm. Mean ± standard error for 3 replicates. Raw data of these experiments can be found in .

Article Snippet: Bactericidal antibiotics of five classes were selected for use here: ceftazidime (Sigma), ciprofloxacin (AppliChem), colistin (Sigma), gentamicin (Sigma) and tobramycin (Tocris).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Characterization of Amikacin Drug Exposure and Nephrotoxicity in an Animal Model

doi: 10.1128/AAC.00859-20

Figure Lengend Snippet: Pharmacokinetic profiles of a single dose of amikacin. n = 4 rats per group. Data are shown as means ± SD.

Article Snippet: For the chromatography assay, amikacin sulfate (USP) powder was purchased from LKT Laboratories (St. Paul, MN), and tobramycin (USP) powder was purchased from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Characterization of Amikacin Drug Exposure and Nephrotoxicity in an Animal Model

doi: 10.1128/AAC.00859-20

Figure Lengend Snippet: Amikacin (AMK) concentrations in renal tissues stratified by outcomes. n = 4 rats per group. Data are shown as means ± SD, P = 0.009.

Article Snippet: For the chromatography assay, amikacin sulfate (USP) powder was purchased from LKT Laboratories (St. Paul, MN), and tobramycin (USP) powder was purchased from Sigma-Aldrich (St. Louis, MO).

Techniques:

Journal: NPJ Biofilms and Microbiomes

Article Title: Echinacoside reduces intracellular c-di-GMP levels and potentiates tobramycin activity against Pseudomonas aeruginosa biofilm aggregates

doi: 10.1038/s41522-025-00673-2

Figure Lengend Snippet: a and b The effect of 16 μg/mL tobramycin (16Tob) ( a ) and 1 μg/mL meropenem (1Mero) ( b ) in combination with different concentrations of echinacoside (10–200 μM E) against pre-established PAO1 aggregates grown in SCFM2 for 6 h. Data are expressed as the mean number of CFU remaining after an additional 18-h incubation (3 independent experiments with 3 technical replicates; error bars indicate standard deviation). Red bars highlighted the successful combination treatment that potentiated the efficacy of antibiotics. * p < 0.05; ** p < 0.01 (one-way ANOVA, with post-hoc Dunnett’s tests for multiple comparisons between 2 groups). c Extra Log reduction achieved by combination treatment of pre-established aggregates of 27 P. aeruginosa strains, compared to treatment with tobramycin alone was shown (3 independent experiments with 3 technical replicates; error bars indicate standard deviation).

Article Snippet: The Minimum inhibitory concentration (MIC) values of tobramycin (MedChemExpress), ciprofloxacin hydrochloride monohydrate (MedChemExpress), ceftazidime (MedChemExpress) and meropenem (Fresenius Kabi, Belgium) were determined in Mueller-Hinton broth (MHB, Neogen, UK) using EUCAST microdilution method.

Techniques: Incubation, Standard Deviation

Journal: NPJ Biofilms and Microbiomes

Article Title: Echinacoside reduces intracellular c-di-GMP levels and potentiates tobramycin activity against Pseudomonas aeruginosa biofilm aggregates

doi: 10.1038/s41522-025-00673-2

Figure Lengend Snippet: a Viability of PA14-infected 3-D A549 cells (as measured with an LDH assay) exposed to different concentrations (25, 50, and 100 μM) of echinacoside, 2 μg/mL tobramycin (2Tob) or the echinacoside/tobramycin combination treatments normalized to untreated/uninfected groups. Red bars highlighted the successful combination treatment that potentiated the efficacy of tobramycin and elevated cell viability. (6 independent experiments with four technical replicates; error bars indicate standard deviation). * p < 0.05; ** p < 0.01 (one-way ANOVA, with post-hoc Dunnett’s tests for multiple comparisons between 2 groups). b Representative transmission light micrographs (upper panel) of 3-D A549 cells attached to/detached from collagen-treated carrier beads and fluorescent micrographs (lower panel) of GFP-tagged PA14 attached to A549 cells. Some typical PA14 aggregates attached to A549 cells with strong fluorescent signals are highlighted with white arrows. Scale bar = 400 μm. c The bacterial load in each uninfected lung with empty agar beads, PA14-infected lung, or PA14-infected lung after exposure to 7.2 µg/mouse tobramycin (Tob), 5.2 µg/mouse echinacoside, or the echinacoside/tobramycin combination treatments. The red bar highlighted the successful combination treatment that potentiated the efficacy of tobramycin. Each group contained 5 mice, and 3 technical replicates were performed for CFU counting. Error bars indicate standard deviation, *** p < 0.001 (one-way ANOVA, with post-hoc Dunnett’s tests for multiple comparisons between 2 groups).

Article Snippet: The Minimum inhibitory concentration (MIC) values of tobramycin (MedChemExpress), ciprofloxacin hydrochloride monohydrate (MedChemExpress), ceftazidime (MedChemExpress) and meropenem (Fresenius Kabi, Belgium) were determined in Mueller-Hinton broth (MHB, Neogen, UK) using EUCAST microdilution method.

Techniques: Infection, Lactate Dehydrogenase Assay, Standard Deviation, Transmission Assay

Journal: mBio

Article Title: Parallel Evolution of Tobramycin Resistance across Species and Environments

doi: 10.1128/mBio.00932-20

Figure Lengend Snippet: Parallel evolution of tobramycin resistance level across species and environments. Populations of A. baumannii and P. aeruginosa were propagated in minimal medium with either increasing concentrations of tobramycin or no drug and in either a planktonic or a biofilm lifestyle. Five replicate populations were propagated per treatment. (A) Populations were either propagated for 12 days in no antibiotic or inoculated into half the MIC of tobramycin, with the concentrations being doubled every 72 h. Samples of each population were archived for later phenotypic analysis and sequencing periodically throughout the experiment (red arrows). (B) Populations were propagated with selection for either planktonic growth through a daily 1:100 dilution or biofilm growth through a daily bead transfer, which forces cells to undergo the entire biofilm life cycle of attachment, growth, dispersion, and reattachment every 24 h, as described in previous work . (C) Tobramycin resistance level relative to that for the ancestral clone for three randomly chosen populations per treatment after 12 days of evolution. MICs were determined by microdilution in Mueller-Hinton broth according to CLSI guidelines. The fold change in the MIC for three replicates per population is shown, with the median fold change and range indicated. The A. baumannii ancestral clone MIC was 1.0 mg/liter and the P. aeruginosa ancestral clone MIC was 0.5 mg/liter in Mueller-Hinton broth. Populations had to acquire resistance to TOB at a concentration 4× the MIC for the ancestral strain in order to survive the experiment (gray dashed line).

Article Snippet: Lineages propagated with antibiotic selection were treated with tobramycin sulfate (Alfa Aesar, Wardhill, MA) starting at 0.5× MIC for the ancestral strain in the experimental minimal medium (0.5 mg/liter for A. baumannii and 2.0 mg/liter for P. aeruginosa ), with doubling of the concentration every 72 h. The experiment was performed for 12 days, with samples collected on days 3, 4, 6, 7, 9, 10, and 12 and frozen at −80°C in either 25% glycerol for P. aeruginosa or 9% dimethyl sulfoxide for A. baumannii .

Techniques: Sequencing, Selection, Dispersion, Concentration Assay

Journal: mBio

Article Title: Parallel Evolution of Tobramycin Resistance across Species and Environments

doi: 10.1128/mBio.00932-20

Figure Lengend Snippet: Population sequencing reveals interspecies parallelism and the lifestyle-associated molecular targets of evolution. Mutations identified by whole-population genome sequencing of tobramycin-treated populations of A. baumannii and P. aeruginosa . Five populations per treatment were sequenced after 12 days of experimental evolution. Shading indicates the total frequency of all mutations in each gene within a population at day 12.

Article Snippet: Lineages propagated with antibiotic selection were treated with tobramycin sulfate (Alfa Aesar, Wardhill, MA) starting at 0.5× MIC for the ancestral strain in the experimental minimal medium (0.5 mg/liter for A. baumannii and 2.0 mg/liter for P. aeruginosa ), with doubling of the concentration every 72 h. The experiment was performed for 12 days, with samples collected on days 3, 4, 6, 7, 9, 10, and 12 and frozen at −80°C in either 25% glycerol for P. aeruginosa or 9% dimethyl sulfoxide for A. baumannii .

Techniques: Sequencing

Journal: mBio

Article Title: Parallel Evolution of Tobramycin Resistance across Species and Environments

doi: 10.1128/mBio.00932-20

Figure Lengend Snippet: Evolutionary dynamics of bacterial populations in increasing concentrations of tobramycin. Muller plot diagrams display the genotype frequencies as a proportion of the population throughout 12 days of evolution for three populations per treatment. Genotypes are shaded by the putative driver loci that are mutated. Different lineages of the same color represent mutations at different positions within the same locus that coexist within the population. The frequency of genotypes at every time point is represented by the height of the graph that it spans at that time point. In situations in which a first mutation arises in the background of the ancestral genotype, the color representing that genotype can be seen beginning from the white background, whereas in situations in which a mutation arises in the background of another mutation, thus generating a new genotype, the new color arises in the middle of the existing genotype. Mutations occurring in the background of putative driver mutations are not shown but may be viewed in linear allele frequency plots of each population in Fig. S4 in the supplemental material.

Article Snippet: Lineages propagated with antibiotic selection were treated with tobramycin sulfate (Alfa Aesar, Wardhill, MA) starting at 0.5× MIC for the ancestral strain in the experimental minimal medium (0.5 mg/liter for A. baumannii and 2.0 mg/liter for P. aeruginosa ), with doubling of the concentration every 72 h. The experiment was performed for 12 days, with samples collected on days 3, 4, 6, 7, 9, 10, and 12 and frozen at −80°C in either 25% glycerol for P. aeruginosa or 9% dimethyl sulfoxide for A. baumannii .

Techniques: Mutagenesis