An Amino Acid Substitution in Elongation Factor EF-G1A Alters the Antibiotic Susceptibility of Pseudomonas aeruginosa LasR-Null Mutants.

The opportunistic bacterium Pseudomonas aeruginosa uses the LasR-I quorum-sensing system to increase resistance to the aminoglycoside antibiotic tobramycin. Paradoxically, lasR-null mutants are commonly isolated from chronic human infections treated with tobramycin, suggesting there may be a mechanism that permits the emergence of lasR-null mutants under tobramycin selection. We hypothesized that some other genetic mutations that emerge in these isolates might modulate the effects of lasR-null mutations on antibiotic resistance. To test this hypothesis, we inactivated lasR in several highly tobramycin-resistant isolates from long-term evolution experiments. In some of these isolates, inactivating lasR further increased resistance, compared with decreasing resistance of the wild-type ancestor. These strain-dependent effects were due to a G61A nucleotide polymorphism in the fusA1 gene encoding amino acid substitution A21T in the translation elongation factor EF-G1A. The EF-G1A mutational effects required the MexXY efflux pump and the MexXY regulator ArmZ. The fusA1 mutation also modulated ΔlasR mutant resistance to two other antibiotics, ciprofloxacin and ceftazidime. Our results identify a gene mutation that can reverse the direction of the antibiotic selection of lasR mutants, a phenomenon known as sign epistasis, and provide a possible explanation for the emergence of lasR-null mutants in clinical isolates. IMPORTANCE One of the most common mutations in Pseudomonas aeruginosa clinical isolates is in the quorum sensing lasR gene. In laboratory strains, lasR disruption decreases resistance to the clinical antibiotic tobramycin. To understand how lasR mutations emerge in tobramycin-treated patients, we mutated lasR in highly tobramycin-resistant laboratory strains and determined the effects on resistance. Disrupting lasR enhanced the resistance of some strains. These strains had a single amino acid substitution in the translation factor EF-G1A. The EF-G1A mutation reversed the selective effects of tobramycin on lasR mutants. These results illustrate how adaptive mutations can lead to the emergence of new traits in a population and are relevant to understanding how genetic diversity contributes to the progression of disease during chronic infections.

Adaptation of Pseudomonas aeruginosa biofilms to tobramycin and the quorum sensing inhibitor C-30 during experimental evolution requires multiple genotypic and phenotypic changes.

In the present study we evaluated the fitness, antimicrobial susceptibility, metabolic activity, gene expression, in vitro production of virulence factors and in vivo virulence of experimentally evolved Pseudomonas aeruginosa PAO1. These strains were previously evolved in the presence of tobramycin and the quorum sensing inhibitor furanone C-30 (C-30) and carried mutations in mexT and fusA1. Compared to the wild-type (WT), the evolved strains show a different growth rate and different metabolic activity, suggesting they have an altered fitness. mexT mutants were less susceptible to C-30 than WT strains; they also show reduced susceptibility to chloramphenicol and ciprofloxacin, two substrates of the MexEF-OprN efflux pump. fusA1 mutants had a decreased susceptibility to aminoglycoside antibiotics, and an increased susceptibility to chloramphenicol. The decreased antimicrobial susceptibility and decreased susceptibility to C-30 was accompanied by a changed metabolic activity profile during treatment. The expression of mexE was significantly increased in mexT mutants and induced by C-30, suggesting that MexEF-OprN exports C-30 out of the bacterial cell. The in vitro production of virulence factors as well as virulence in two in vivo models of the strains evolved in the presence of C-30 was unchanged compared to the virulence of the WT. Finally, the evolved strains were less susceptible towards tobramycin (alone and combined with C-30) in an in vivo mouse model. In conclusion, this study shows that mutations acquired during experimental evolution of P. aeruginosa biofilms in the presence of tobramycin and C-30, are accompanied by an altered fitness, metabolism, mexE expression and in vitro and in vivo antimicrobial susceptibility.

Impact of fluoroquinolones and aminoglycosides on P. aeruginosa virulence factor production and cytotoxicity.

The opportunistic pathogen Pseudomonas aeruginosa is one of leading causes of disability and mortality worldwide and the world health organisation has listed it with the highest priority for the need of new antimicrobial therapies. P. aeruginosa strains responsible for the poorest clinical outcomes express either ExoS or ExoU, which are injected into target host cells via the type III secretion system (T3SS). ExoS is a bifunctional cytotoxin that promotes intracellular survival of invasive P. aeruginosa by preventing targeting of the bacteria to acidified intracellular compartments. ExoU is a phospholipase which causes destruction of host cell plasma membranes, leading to acute tissue damage and bacterial dissemination. Fluoroquinolones are usually employed as a first line of therapy as they have been shown to be more active against P. aeruginosa in vitrothan other antimicrobial classes. Their overuse over the past decade, however, has resulted in the emergence of antibiotic resistance. In certain clinical situations, aminoglycosides have been shown to be more effective then fluoroquinolones, despite their reduced potency towards P. aeruginosa in vitro. In this study, we evaluated the effects of fluoroquinolones (moxifloxacin and ciprofloxacin) and aminoglycosides (tobramycin and gentamycin) on T3SS expression and toxicity, in corneal epithelial cell infection models. We discovered that tobramycin disrupted T3SS expression and reduced both ExoS and ExoU mediated cytotoxicity, protecting infected HCE-t cells at concentrations below the minimal inhibitory concentration (MIC). The fluoroquinolones moxifloxacin and ciprofloxacin, however, up-regulated the T3SS and did not inhibit and may have increased the cytotoxic effects of ExoS and ExoU.

Mucoid Pseudomonas aeruginosa Can Produce Calcium-Gelled Biofilms Independent of the Matrix Components Psl and CdrA.

Biofilms are aggregates of microorganisms embedded in an extracellular matrix comprised largely of exopolysaccharides (EPSs), nucleic acids, and proteins. Pseudomonas aeruginosa is an opportunistic human pathogen that is also a model organism for studying biofilms in the laboratory. Here, we define a novel program of biofilm development used by mucoid (alginate-overproducing) P. aeruginosa in the presence of elevated calcium. Calcium cations cross-link negatively charged alginate polymers, resulting in individual cells being suspended in an alginate gel. The formation of this type of structurally distinct biofilm is not reliant on the canonical biofilm EPS components Psl and Pel or the matrix protein CdrA. We also observed that mucoid P. aeruginosa biofilm cells do not have the typical elevated levels of the secondary messenger cyclic di-GMP (c-di-GMP), as expected of biofilm cells, nor does the overproduction of alginate rely on high c-di-GMP. This contrasts with nonmucoid biofilms in which the production of the matrix components Psl, Pel, and CdrA is positively regulated by elevated c-di-GMP. We further demonstrate that calcium-gelled alginate biofilms impede the penetration of the antibiotic tobramycin, thus protecting the biofilm community from antibiotic-mediated killing. Finally, we show that bacterial aggregates with a dispersed cell arrangement like laboratory-grown calcium-alginate biofilm structures are present in explanted cystic fibrosis (CF) lung samples. Our findings illustrate the diverse nature of biofilm formation and structure in P. aeruginosa. IMPORTANCE The opportunistic pathogen Pseudomonas aeruginosa produces a complex biofilm matrix comprised of exopolysaccharides (EPSs), nucleic acids, and proteins. P. aeruginosa biofilm formation canonically depends on a variable combination of the exopolysaccharides Psl and Pel and the matrix protein CdrA. We demonstrate that mucoid P. aeruginosa, which overproduces the EPS alginate, possesses an entirely alternate and calcium-dependent method of biofilm formation. These mucoid biofilm structures do not require Psl, Pel, or CdrA, and they display a unique organization of individually suspended cells similar to bacterial aggregates observed in cystic fibrosis airways. Furthermore, calcium-gelled mucoid biofilms impede the penetration and killing action of the antibiotic tobramycin, illustrating their potential clinical significance. Our findings highlight the compositional and structural variety of P. aeruginosa biofilm aggregates.

Nitric oxide inhibits alginate biosynthesis in Pseudomonas aeruginosa and increases its sensitivity to tobramycin by downregulating algU gene expression.

In the process of chronic cystic fibrosis (CF) infection, Pseudomonas aeruginosa (PA) is converted into a mucoid phenotype characterized by an overproduction of exopolysaccharide alginate. The alginate forms a thick mucus that causes difficulty in patient's breathing, drug resistance and contributes to both the morbidity and mortality of the patient. AlgU of PA, an extracytoplasmic function sigma factor, is responsible for the alginate overproduction and leads to mucoidy and chronic infection of CF patients. In this report, we found that endogenous and exogenous nitric oxide (NO) can significantly reduce algU expression, leading to down-regulation of a series of alginate synthesis-related genes (algD, alg8, algX, and algK), eventually down-regulated alginate synthesis. A fluorescent reporter strain was constructed to clarify the inhibitory effect of alginate synthesis through real-time monitoring in different conditions. The results showed that NO presented inhibitory effect on alginate synthesis in nine clinical PA isolates as in the PA reference strain, and the reduction of alginate was more significant in three mucoid strains (by about 51%, 70% and 61%, respectively, while 47% for the reference strain). In the co-culture system, effect of NO on PA fluorescence intensity is similar to that in monocultures, with the best effect at 10 µM NO donor sodium nitroprusside (SNP). Finally, we examined the changes in the antibiotic susceptibility of PA under NO-inhibited alginate conditions. In the presence of 10 µM SNP, the number of planktonic cells increased, and both adherent and planktonic PA cells showed increased susceptibility to tobramycin. We thus suggest that NO can potentially be employed as a therapeutic strategy to prevent cystic fibrosis lungs from PA infection.

Microtiter plate with built-in oxygen sensors: a novel approach to investigate the dynamics of Pseudomonas aeruginosa growth suppression in the presence of divalent cations and antibiotics.

The depletion of dissolved oxygen in a defined synthetic medium can be measured in real time, using a micro-well plate format, associated with a fluorescent plate reader. This technology is appropriate for investigating the effect of antibiotics on cell kinetics because there is a direct correlation between the latter and the amount of dissolved oxygen in the medium of an assay. In this study, the metabolic activity of the opportunistic human pathogen Pseudomonas aeruginosa PA01 was investigated using the OxoPlate OP96U optical sensor technology. The response of P. aeruginosa to aminoglycoside antibiotics when Ca2+and Mg2+ ions are present in the Evans defined synthetic medium was measured. The results revealed that the effect of antibiotics on P. aeruginosa is influenced by the concentration of divalent cations present in the test medium, although the efficiency of Ca2+ in supressing antibiotic activity was found to be greater than that of Mg2+. By comparison to tobramycin, the effect of amikacin is largely inhibited by the Ca2+and Mg2+concentrations. The study results underscore that the reliability of the observation of growth inhibitors is enhanced by the oxygen consumption measurements. Thus, the OxoPlate OP96U system is proven to be an accurate method to test the effectiveness of antibiotic treatments against P. aeruginosa.

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.

Kidney Injury Monitoring in Tobramycin-Treated Rhesus Monkeys: Supplementing Urinary Kidney Biomarkers With Kidney Biopsy Gene Expression Profiling.

Kidney biopsies are used sparingly to diagnose kidney injury in the clinic. Here we have conducted a small exploratory study to directly compare the low-grade kidney injury monitoring performance of serum safety biomarkers, novel urine safety biomarkers, microscopic histopathology and targeted gene expression alterations in kidney biopsy specimens in rhesus monkeys treated with tobramycin. Targeted gene expression increases were observed in the kidney biopsy samples and whole kidney sections for kidney injury molecule 1 (KIM-1), clusterin (CLU), osteopontin (OPN) messenger RNA transcripts. In addition, increases of the urinary kidney safety protein biomarkers including KIM-1, CLU, OPN were also observed. These increases in gene expression and urinary protein end point were in concordance with the eventual low-grade kidney lesions seen in terminal tissue sections. In contrast, conventional serum biomarkers blood urea nitrogen and serum creatinine were not as sensitive in monitoring kidney injury. Although these data do not support routinely adding kidney biopsies to regular toxicology studies, they provide evidence on the value and limitations of incorporating gene expression profiling on kidney biopsy specimens, further underscore the value of urinary kidney safety biomarkers for improved low-grade kidney injury monitoring, and open the door for future definitive studies.

Characterization and utilization of methyltransferase for apramycin production in Streptoalloteichus tenebrarius.

A structurally unique aminoglycoside produced in Streptoalloteichus tenebrarius, Apramycin is used in veterinary medicine or the treatment of Salmonella, Escherichia coli, and Pasteurella multocida infections. Although apramycin was discovered nearly 50 years ago, many biosynthetic steps of apramycin remain unknown. In this study, we identified a HemK family methyltransferase, AprI, to be the 7'-N-methyltransferase in apramycin biosynthetic pathway. Biochemical experiments showed that AprI converted demethyl-aprosamine to aprosamine. Through gene disruption of aprI, we identified a new aminoglycoside antibiotic demethyl-apramycin as the main product in aprI disruption strain. The demethyl-apramycin is an impurity in apramycin product. In addition to demethyl-apramycin, carbamyltobramycin is another major impurity. However, unlike demethyl-apramycin, tobramycin is biosynthesized by an independent biosynthetic pathway in S. tenebrarius. The titer and rate of apramycin were improved by overexpression of the aprI and disruption of the tobM2, which is a crucial gene for tobramycin biosynthesis. The titer of apramycin increased from 2227 ± 320 mg/L to 2331 ± 210 mg/L, while the titer of product impurity demethyl-apramycin decreased from 196 ± 36 mg/L to 51 ± 9 mg/L. Moreover, the carbamyltobramycin titer of the wild-type strain was 607 ± 111 mg/L and that of the engineering strain was null. The rate of apramycin increased from 68% to 87% and that of demethyl-apramycin decreased from 1.17% to 0.34%.

Thermodynamics of an aminoglycoside modifying enzyme with low substrate promiscuity: The aminoglycoside N3 acetyltransferase-VIa.

Kinetic, thermodynamic, and structural properties of the aminoglycoside N3-acetyltransferase-VIa (AAC-VIa) are determined. Among the aminoglycoside N3-acetyltransferases, AAC-VIa has one of the most limited substrate profiles. Kinetic studies showed that only five aminoglycosides are substrates for this enzyme with a range of fourfold difference in kcat values. Larger differences in KM (∼40-fold) resulted in ∼30-fold variation in kcat /KM . Binding of aminoglycosides to AAC-VIa was enthalpically favored and entropically disfavored with a net result of favorable Gibbs energy (ΔG < 0). A net deprotonation of the enzyme, ligand, or both accompanied the formation of binary and ternary complexes. This is opposite of what was observed with several other aminoglycoside N3-acetyltransferases, where ligand binding causes more protonation. The change in heat capacity (ΔCp) was different in H2 O and D2 O for the binary enzyme-sisomicin complex but remained the same in both solvents for the ternary enzyme-CoASH-sisomicin complex. Unlike, most other aminoglycoside-modifying enzymes, the values of ΔCp were within the expected range of protein-carbohydrate interactions. Solution behavior of AAC-VIa was also different from the more promiscuous aminoglycoside N3-acetyltransferases and showed a monomer-dimer equilibrium as detected by analytical ultracentrifugation (AUC). Binding of ligands shifted the enzyme to monomeric state. Data also showed that polar interactions were the most dominant factor in dimer formation. Overall, thermodynamics of ligand-protein interactions and differences in protein behavior in solution provide few clues on the limited substrate profile of this enzyme despite its >55% sequence similarity to the highly promiscuous aminoglycoside N3-acetyltransferase. Proteins 2017; 85:1258-1265. © 2017 Wiley Periodicals, Inc.

Association between the Presence of Aminoglycoside-Modifying Enzymes and In Vitro Activity of Gentamicin, Tobramycin, Amikacin, and Plazomicin against Klebsiella pneumoniae Carbapenemase- and Extended-Spectrum-ß-Lactamase-Producing Enterobacter Species.

We compared the in vitro activities of gentamicin (GEN), tobramycin (TOB), amikacin (AMK), and plazomicin (PLZ) against 13 Enterobacter isolates possessing both Klebsiella pneumoniae carbapenemase and extended-spectrum ß-lactamase (KPC+/ESBL+) with activity against 8 KPC+/ESBL-, 6 KPC-/ESBL+, and 38 KPC-/ESBL- isolates. The rates of resistance to GEN and TOB were higher for KPC+/ESBL+ (100% for both) than for KPC+/ESBL- (25% and 38%, respectively), KPC-/ESBL+ (50% and 17%, respectively), and KPC-/ESBL- (0% and 3%, respectively) isolates. KPC+/ESBL+ isolates were more likely than others to possess an aminoglycoside-modifying enzyme (AME) (100% versus 38%, 67%, and 5%; P = 0.007, 0.06, and <0.0001, respectively) or multiple AMEs (100% versus 13%, 33%, and 0%, respectively; P < 0.01 for all). KPC+/ESBL+ isolates also had a greater number of AMEs (mean of 4.6 versus 1.5, 0.9, and 0.05, respectively; P < 0.01 for all). GEN and TOB MICs were higher against isolates with >1 AME than with ≤1 AME. The presence of at least 2/3 of KPC, SHV, and TEM predicted the presence of AMEs. PLZ MICs against all isolates were ≤4 µg/ml, regardless of KPC/ESBL pattern or the presence of AMEs. In conclusion, GEN and TOB are limited as treatment options against KPC+ and ESBL+ Enterobacter PLZ may represent a valuable addition to the antimicrobial armamentarium. A full understanding of AMEs and other aminoglycoside resistance mechanisms will allow clinicians to incorporate PLZ rationally into treatment regimens. The development of molecular assays that accurately and rapidly predict antimicrobial responses among KPC- and ESBL-producing Enterobacter spp. should be a top research priority.

Two mechanisms of killing of Pseudomonas aeruginosa by tobramycin assessed at multiple inocula via mechanism-based modeling.

Bacterial resistance is among the most serious threats to human health globally, and many bacterial isolates have emerged that are resistant to all antibiotics in monotherapy. Aminoglycosides are often used in combination therapies against severe infections by multidrug-resistant bacteria. However, models quantifying different antibacterial effects of aminoglycosides are lacking. While the mode of aminoglycoside action on protein synthesis has often been studied, their disruptive action on the outer membrane of Gram-negative bacteria remains poorly characterized. Here, we developed a novel quantitative model for these two mechanisms of aminoglycoside action, phenotypic tolerance at high bacterial densities, and adaptive bacterial resistance in response to an aminoglycoside (tobramycin) against three Pseudomonas aeruginosa strains. At low-intermediate tobramycin concentrations (<4 mg/liter), bacterial killing due to the effect on protein synthesis was most important, whereas disruption of the outer membrane was the predominant killing mechanism at higher tobramycin concentrations (≥8 mg/liter). The extent of killing was comparable across all inocula; however, the rate of bacterial killing and growth was substantially lower at the 10(8.9) CFU/ml inoculum than that at the lower inocula. At 1 to 4 mg/liter tobramycin for strain PAO1-RH, there was a 0.5- to 6-h lag time of killing that was modeled via the time to synthesize hypothetical lethal protein(s). Disruption of the outer bacterial membrane by tobramycin may be critical to enhance the target site penetration of antibiotics used in synergistic combinations with aminoglycosides and thereby combat multidrug-resistant bacteria. The two mechanisms of aminoglycoside action and the new quantitative model hold great promise to rationally design novel, synergistic aminoglycoside combination dosage regimens.

Mucin Binding Reduces Colistin Antimicrobial Activity.

Colistin has found increasing use in treating drug-resistant bacterial lung infections, but potential interactions with pulmonary biomolecules have not been investigated. We postulated that colistin, like aminoglycoside antibiotics, may bind to secretory mucin in sputum or epithelial mucin that lines airways, reducing free drug levels. To test this hypothesis, we measured binding of colistin and other antibiotics to porcine mucin, a family of densely glycosylated proteins used as a surrogate for human sputum and airway mucin. Antibiotics were incubated in dialysis tubing with or without mucin, and concentrations of unbound antibiotics able to penetrate the dialysis tubing were measured over time using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The percentage of antibiotic measured in the dialysate after 4 h in the presence of mucin, relative to the amount without mucin, was 15% for colistin, 16% for polymyxin B, 19% for tobramycin, 52% for ciprofloxacin, and 78% for daptomycin. Antibiotics with the strongest mucin binding had an overall polybasic positive charge, whereas those with comparatively little binding were less basic. When comparing MICs measured with or without added mucin, colistin and polymyxin B showed >100-fold increases in MICs for multiple Gram-negative bacteria. Preclinical evaluation of mucin binding should become a standard procedure when considering the potential pulmonary use of new or existing antibiotics, particularly those with a polybasic overall charge. In the airways, mucin binding may reduce the antibacterial efficacy of inhaled or intravenously administered colistin, and the presence of sub-MIC effective antibiotic concentrations could result in the development of antibiotic resistance.

Tobramycin Variants with Enhanced Ribosome-Targeting Activity.

With the increased evolution of aminoglycoside (AG)-resistant bacterial strains, the need to develop AGs with 1) enhanced antimicrobial activity, 2) the ability to evade resistance mechanisms, and 3) the capability of targeting the ribosome with higher efficiency is more and more pressing. The chemical derivatization of the naturally occurring tobramycin (TOB) by attachment of 37 different thioether groups at the 6''-position led to the identification of generally poorer substrates of TOB-targeting AG-modifying enzymes (AMEs). Thirteen of these displayed better antibacterial activity than the parent TOB while retaining ribosome-targeting specificity. Analysis of these compounds in vitro shed light on the mechanism by which they act and revealed three with clearly enhanced ribosome-targeting activity.

Combination of hypothiocyanite and lactoferrin (ALX-109) enhances the ability of tobramycin and aztreonam to eliminate Pseudomonas aeruginosa biofilms growing on cystic fibrosis airway epithelial cells.

OBJECTIVES: Chelating iron may be a promising new therapy to eliminate Pseudomonas aeruginosa biofilms in the lungs of cystic fibrosis (CF) patients. Here, we investigate whether ALX-109 [a defined combination of an investigational drug containing lactoferrin (an iron-binding glycoprotein) and hypothiocyanite (a bactericidal agent)], alone and in combination with tobramycin or aztreonam, reduces P. aeruginosa biofilms grown on human CF airway epithelial cells. METHODS: P. aeruginosa (PAO1 and six clinical isolates of Pseudomonas) biofilms grown at the apical surface of confluent monolayers of CF airway epithelial cells were treated with ALX-109, either alone or in combination with tobramycin or aztreonam. Bacterial cfu remaining after treatment were determined by plate counting. RESULTS: ALX-109 alone reduced PAO1 biofilm formation, but had no effect on established biofilms. ALX-109 enhanced the ability of tobramycin and aztreonam to inhibit PAO1 biofilm formation and to reduce established PAO1 biofilms. ALX-109 and tobramycin were additive in disrupting established biofilms formed by six clinical isolates of P. aeruginosa obtained from the sputum of CF patients. Mucoid P. aeruginosa isolates were most susceptible to the combination of ALX-109 and tobramycin. In addition, ALX-109 also enhanced the ability of aztreonam to reduce established PAO1 biofilms. CONCLUSIONS: Inhalation therapy combining hypothiocyanite and lactoferrin with TOBI(®) (tobramycin) or Cayston(®) (aztreonam) may be beneficial to CF patients by decreasing the airway bacterial burden of P. aeruginosa.

Targeted release of tobramycin from a pH-responsive grafted bilayer challenged with S. aureus.

A stimuli-responsive, controlled release bilayer for the prevention of bacterial infection on biomaterials is presented. Drug release is locally controlled by the pH-responsiveness of the bilayer, comprised of an inner poly(acrylic acid) (PAA) monolayer grafted to a biomaterial and cross-linked with an outer chitosan (CH) brush. Tobramycin (TOB) is loaded in the inner PAA in part to minimize bacteria resistance. Because biofilm formation causes a decrease in local pH, TOB is released from PAA and permeates through the CH, which is in contact with the biofilm. Antibiotic capacity is controlled by the PAA thickness, which depends on PAA brush length and the extent of cross-linking between CH and PAA at the bilayer interface. This TOB-loaded, pH-responsive bilayer exhibits significantly enhanced antibacterial activity relative to controls.

Assessment of aminoglycoside dosing and estimated glomerular filtration rate in determining gentamicin and tobramycin area under the curve and clearance.

BACKGROUND: Aminoglycoside clearance depends on kidney function, but the Australian Therapeutic Guidelines for antibiotics (version 14, 2010) recommend initial dosing based on weight without consideration of kidney function. Other guidelines that modify dosing based on kidney function estimates often use the Cockroft-Gault equation, but the role of the estimated glomerular filtration rate equations for this purpose is unclear. AIM: To determine the performance of current guideline dosing in achieving target area-under-the-curve and examine the relative precision of the estimated glomerular filtration rate equations compared with traditional Cockroft-Gault creatinine clearance in predicting aminoglycoside clearance. METHODS: We analysed 496 aminoglycoside treatment episodes involving 1377 infusions in adult patients. Conformity with antibiotic guideline dosing was achieved if the discrepancy between prescribed and recommended dose was less than 15%. Aminoglycoside clearance was determined from linear regression using a one compartment model with the Aminoglycoside Levels and Daily Dose Indicator programme. We assessed the precision of the Cockroft-Gault, Modification of Diet in renal Disease Study and Chronic Kidney Disease-Epidemiology Collaboration (CKD-EPI) equations in predicting aminoglycoside clearance by correlation and linear regression. RESULTS: Conformity with guideline dosing was not associated with achieving target area-under-the-curve. The CKD-EPI estimated glomerular filtration rate adjusted for body surface area showed the highest correlation (gentamicin, r = 0.66; tobramycin, r = 0.82) and best predictive model for aminoglycoside clearance. CONCLUSION: Current guideline dosing may be suboptimal for achieving target area-under-the-curve. The CKD-EPI equation adjusted for patient body surface area best predicts aminoglycoside clearance, and could be evaluated as a covariate in determining initial aminoglycoside dosing.

Development and characterization of a ricinoleic acid poloxamer gel system for transdermal eyelid delivery.

OBJECTIVE: This study deals with the preparation and evaluation of a pluronic lecithin organogel (PLO gel) containing ricinoleic acid for the transdermal eyelid delivery of dexamethasone and tobramycin. METHODS: Five different PLO gel formulations (F1, F2, F3, F4 and F5) containing tobramycin (0.3%) and dexamethasone (0.1%) were prepared and compared to a conventional PLO gel (light mineral oil PLO gel, F6) with respect to physical appearance and viscosity. The optimized ricinoleic acid PLO gel formulation (F2) was further characterized for pH, gelation temperature, morphology and drug content. Ex vivo permeability of dexamethasone and bactericidal activity of tobramycin from formulation F2 was tested, and values were compared to the marketed Tobradex® eye ointment. RESULTS: No apparent changes in the physical appearance and consistency were observed when ricinoleic acid was used as the oil phase. The pH of the optimized ricinoleic acid PLO gel (formulation F2) was found to be 6.54 with a gelation temperature of 31 °C. The drug content of tobramycin and dexamethasone were found to be 102.8% and 100.14%, respectively. The penetration profile of dexamethasone from formulation F2 was found to be much higher than the marketed Tobradex® eye ointment. F2 showed a better antimicrobial activity and higher zones of inhibition when compared to the marketed Tobradex® eye ointment. CONCLUSION: The findings of this investigation indicate that the ricinoleic acid PLO gel has the potential for use as a transdermal eyelid delivery system.

[ThE COMPARATIVE CHARACTERISTIC OF PHENOTYPIC AND GENOTYPIC RESISTANCE TO AMINOGLYCOSIDES OF STRAINS STAPHYLOCOCCUS AUREUS ISOLATED IN TRAUMATOLOGICAI ORTHOPEDIC HOSPITAL].

The clinical isolates of Staphylococcus aureus (n = 102) were analyzed on sensitivity and to gentamicin, tobramicin, netimicin and amikacin. The disc diffusing technique was applied. The technique ofpolymerase chain reaction was applied to analyze all strains establishing presence in their genomes genes aac (6'-Ie/aph(2"), ant1, aac, ant(6)-Ia, aph (3')-IIIa and ant(4')-Ia coding amino-glycoside-modifying enzymes. The strains sensitive to amino-glycosides had no the given genes in genome. The genome of all strains resistant to amino-glycosides included no less than two of enumerated genes. The 100% correlation was established between phenotypic resistance of analyzed strains to amino-glycosides and availability in them of gene aac(6')-Ie/aph(2").

Systemic absorption of nasally administered tobramycin and colistin in patients with cystic fibrosis.

OBJECTIVES: In cystic fibrosis (CF) patients the paranasal sinuses can constitute a niche for bacteria, which can migrate to the lungs. Nasal administration of antibiotics may be effective, but safety of this treatment has to be established first. The objective of this study was to investigate the systemic absorption of nasally administered tobramycin, colistin (administered as colistin sulfomethate sodium; CMS) and a combination of both drugs using systemic absorption as surrogate for safety. In addition, tolerability of the nasal irrigations was examined. METHODS: Ten adult CF patients performed three different nasal irrigations: 300 mg of tobramycin; 160 mg of CMS; and 300 mg of tobramycin combined with 160 mg of CMS. Serum concentrations of tobramycin and colistin A and B (the main components of colistin) were analysed. Tolerability was measured using a visual analogue scale. Dutch Trial Register: NTR 4008. RESULTS: Following the tobramycin and the combined irrigation, only two patients had detectable tobramycin serum levels, with the highest being 0.054 mg/L. Serum levels of colistin A and B were not detectable. All three nasal irrigation solutions were well tolerated with a higher tolerability for CMS compared with tobramycin. CONCLUSIONS: Nasal irrigations with tobramycin, CMS and a combination of tobramycin and CMS resulted in safe serum levels and were well tolerated.