Enhancing bactericidal activities of ciprofloxacin by targeting the trans-translation system that is involved in stress responses in Klebsiella pneumoniae.

Although the trans-translation system is a promising target for antcibiotic development, its antibacterial mechanism in Klebsiella pneumoniae (KP) is unclear. Considering that tmRNA was the core component of trans-translation, this study firstly investigated phenotypic changes caused by various environmental stresses in KP lacking trans-translation activities (tmRNA-deleted), and then aimed to evaluate antibacterial activities of the trans-translation-targeting antibiotic combination (tobramycin/ciprofloxacin) in clinical KP isolates based on inhibition activities of aminoglycosides against trans-translation. We found that the tmRNA-deleted strain P4325/ΔssrA was significantly more susceptible than the wild-type KP strain P4325 under environments with hypertonicity (0.5 and 1 M NaCl), hydrogen peroxide (40 mM), and UV irradiation. No significant differences in biofilm formation and survivals under human serum were observed between P4325/ΔssrA and P4325. tmRNA deletion caused twofold lower MIC values for aminoglycosides. As for the membrane permeability, tmRNA deletion increased ethidium bromide (EtBr) uptake of KP in the presence or absence of verapamil and carbonyl cyanide-m-chlorophenylhydrazone (CCCP), decreased EtBr uptake in presence of reserpine in P4325/ΔssrA, and reduced EtBr efflux in P4325/ΔssrA in the presence of CCCP. The time-kill curve and in vitro experiments revealed significant bactericidal activities of the tmRNA-targeting aminoglycoside-based antibiotic combination (tobramycin/ciprofloxacin). Thus, the corresponding tmRNA-targeting antibiotic combinations (aminoglycoside-based) might be effective and promising treatment options against multi-drug resistant KP.

Zein nanoparticles containing ceftazidime and tobramycin: antibacterial activity against Gram-negative bacteria.

Aims: This work describes the encapsulation of ceftazidime and tobramycin in zein nanoparticles (ZNPs) and the characterization of their antibacterial and antibiofilm activities against Gram-negative bacteria. Materials & methods: ZNPs were synthesized by nanoprecipitation. Cytotoxicity was assessed by MTT assay and antibacterial and antibiofilm assays were performed by broth microdilution and violet crystal techniques. Results: ZNPs containing ceftazidime (CAZ-ZNPs) and tobramycin (TOB-ZNPs) showed drug encapsulation and thermal stability. Encapsulation of the drugs reduced their cytotoxicity 9-25-fold. Antibacterial activity, inhibition and eradication of biofilm by CAZ-ZNPs and TOB-ZNPs were observed. There was potentiation when CAZ-ZNPs and TOB-ZNPs were combined. Conclusion: CAZ-ZNPs and TOB-ZNPs present ideal physical characteristics for in vivo studies of antibacterial and antibiofilm activities.

A nanotechnology product was developed to treat diseases caused by bacteria. This prototype showed the ideal characteristics and could be administered by ingestion through the mouth, aspiration through the nose or injection into the veins. The prototype did not harm or kill human cells. It killed the bacteria and prevented the formation of a type of protection against antibiotics that bacteria can produce, called a biofilm. Nanotechnology products are a promising alternative for the treatment of bacterial infections.

Murepavadin promotes the killing efficacies of aminoglycoside antibiotics against Pseudomonas aeruginosa by enhancing membrane potential.

Murepavadin is a peptidomimetic that specifically targets the lipopolysaccharide transport protein LptD of Pseudomonas aeruginosa. Here, we found that murepavadin enhances the bactericidal efficacies of tobramycin and amikacin. We further demonstrated that murepavadin enhances bacterial respiration activity and subsequent membrane potential, which promotes intracellular uptake of aminoglycoside antibiotics. In addition, the murepavadin-amikacin combination displayed a synergistic bactericidal effect in a murine pneumonia model.

Mutations in the efflux pump regulator MexZ shift tissue colonization by Pseudomonas aeruginosa to a state of antibiotic tolerance.

Mutations in mexZ, encoding a negative regulator of the expression of the mexXY efflux pump genes, are frequently acquired by Pseudomonas aeruginosa at early stages of lung infection. Although traditionally related to resistance to the first-line drug tobramycin, mexZ mutations are associated with low-level aminoglycoside resistance when determined in the laboratory, suggesting that their selection during infection may not be necessarily, or only, related to tobramycin therapy. Here, we show that mexZ-mutated bacteria tend to accumulate inside the epithelial barrier of a human airway infection model, thus colonising the epithelium while being protected against diverse antibiotics. This phenotype is mediated by overexpression of lecA, a quorum sensing-controlled gene, encoding a lectin involved in P. aeruginosa tissue invasiveness. We find that lecA overexpression is caused by a disrupted equilibrium between the overproduced MexXY and another efflux pump, MexAB, which extrudes quorum sensing signals. Our results indicate that mexZ mutations affect the expression of quorum sensing-regulated pathways, thus promoting tissue invasiveness and protecting bacteria from the action of antibiotics within patients, something unnoticeable using standard laboratory tests.

Optimization of an in vitro Pseudomonas aeruginosa Biofilm Model to Examine Antibiotic Pharmacodynamics at the Air-Liquid Interface.

Pseudomonas aeruginosa is an important cause of lower respiratory tract infections, such as ventilator-associated bacterial pneumonia (VABP). Using inhaled antibiotics to treat VABP can achieve high drug concentrations at the infection site while minimizing systemic toxicities. Despite the theoretical advantages, clinical trials have failed to show a benefit for inhaled antibiotic therapy in treating VABP. A potential reason for this discordance is the presence of biofilm-embedded bacteria in lower respiratory tract infections. Drug selection and dosing are often based on data from bacteria grown planktonically. In the present study, an in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model was optimized to evaluate the activity of simulated epithelial lining fluid exposures of inhaled and intravenous doses of polymyxin B and tobramycin against two P. aeruginosa strains. Antibiotic activity was also determined against the P. aeruginosa strains grown planktonically. Our study revealed that inhaled antibiotic exposures were more active than their intravenous counterparts across biofilm and planktonic populations. Inhaled exposures of polymyxin B and tobramycin exhibited comparable activity against planktonic P. aeruginosa. Although inhaled polymyxin B exposures were initially more active against P. aeruginosa biofilms (through 6 h), tobramycin was more active by the end of the experiment (48 h). Together, these data slightly favor the use of inhaled tobramycin for VABP caused by biofilm-forming P. aeruginosa that are not resistant to either antibiotic. The optimized in vitro air-liquid interface pharmacokinetic/pharmacodynamic biofilm model may be beneficial for the development of novel anti-biofilm agents or to optimize antibiotic dosing for infections such as VABP.

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.

P. aeruginosa tRNA-fMet halves secreted in outer membrane vesicles suppress lung inflammation in cystic fibrosis.

Although tobramycin increases lung function in people with cystic fibrosis (pwCF), the density of Pseudomonas aeruginosa (P. aeruginosa) in the lungs is only modestly reduced by tobramycin; hence, the mechanism whereby tobramycin improves lung function is not completely understood. Here, we demonstrate that tobramycin increases 5' tRNA-fMet halves in outer membrane vesicles (OMVs) secreted by laboratory and CF clinical isolates of P. aeruginosa. The 5' tRNA-fMet halves are transferred from OMVs into primary CF human bronchial epithelial cells (CF-HBEC), decreasing OMV-induced IL-8 and IP-10 secretion. In mouse lungs, increased expression of the 5' tRNA-fMet halves in OMVs attenuated KC (murine homolog of IL-8) secretion and neutrophil recruitment. Furthermore, there was less IL-8 and neutrophils in bronchoalveolar lavage fluid isolated from pwCF during the period of exposure to tobramycin versus the period off tobramycin. In conclusion, we have shown in mice and in vitro studies on CF-HBEC that tobramycin reduces inflammation by increasing 5' tRNA-fMet halves in OMVs that are delivered to CF-HBEC and reduce IL-8 and neutrophilic airway inflammation. This effect is predicted to improve lung function in pwCF receiving tobramycin for P. aeruginosa infection.NEW & NOTEWORTHY The experiments in this report identify a novel mechanism, whereby tobramycin reduces inflammation in two models of CF. Tobramycin increased the secretion of tRNA-fMet halves in OMVs secreted by P. aeruginosa, which reduced the OMV-LPS-induced inflammatory response in primary cultures of CF-HBEC and in mouse lung, an effect predicted to reduce lung damage in pwCF.

Membrane fluidity homeostasis is required for tobramycin-enhanced biofilm in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

Tobramycin and Vancomycin in an In Vitro Model of Anterior Cruciate Ligament Allograft Decontamination.

BACKGROUND: Approximately 100,000 anterior cruciate ligament (ACL) reconstructions (ACLRs) occur annually in the United States, and postoperative surgical-site infection is a relatively rare but devastating complication, often leading to graft failure or septic arthritis of the knee, necessitating repeat surgery. Wrapping allografts in vancomycin-soaked gauze has been adopted as a common sterilization technique in the operating room to reduce surgical-site infection; however, identifying effective alternatives to vancomycin has not been extensively pursued. HYPOTHESIS: Tobramycin would be as effective as vancomycin in reducing the concentrations of Staphylococcus epidermidis bacteria on tendon allografts. STUDY DESIGN: Controlled laboratory study. METHODS: S. epidermidis strain ATCC 12228 was inoculated onto the human cadaveric gracilis tendon. The tendons were wrapped in sterile gauze saturated with tobramycin or vancomycin at various experimental concentrations. Bacteria remaining on the tendon were dislodged, serially diluted, and plated for colony counting. Statistical analysis was performed utilizing 2-way analysis of variance testing. Results were considered statistically significant when P < .05. RESULTS: Vancomycin (P = .0001) and tobramycin (P < .0001) reduced bacterial concentration. Tobramycin was found to produce a statistically significant reduction in bacterial concentration at concentrations as low as 0.1 mg/mL (P < .0001 and P = .01 at 10 and 20 minutes), while vancomycin produced a statistically significant reduction at a concentration as low as 2.5 mg/mL (P < .0001 at both 10 and 20 minutes). CONCLUSION: This study demonstrates that tobramycin is as effective as vancomycin in bacterial concentration reduction but can achieve this reduction level at lower doses. Further studies clarifying the biomechanical and cytotoxic effects of tobramycin on tendon tissue are indicated to solidify its use as a clinical alternative to vancomycin in ACLR. CLINICAL RELEVANCE: These results will begin establishing tobramycin as an alternative to vancomycin in ACL graft decontamination. Because of relatively frequent shortages of vancomycin, establishing tobramycin as an alternative agent is a useful option for the orthopaedic surgeon.

Combination treatment to improve mucociliary transport of Pseudomonas aeruginosa biofilms.

People with muco-obstructive pulmonary diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) often have acute or chronic respiratory infections that are difficult to treat due in part to the accumulation of hyperconcentrated mucus within the airway. Mucus accumulation and obstruction promote chronic inflammation and infection and reduce therapeutic efficacy. Bacterial aggregates in the form of biofilms exhibit increased resistance to mechanical stressors from the immune response (e.g., phagocytosis) and chemical treatments including antibiotics. Herein, combination treatments designed to disrupt the mechanical properties of biofilms and potentiate antibiotic efficacy are investigated against mucus-grown Pseudomonas aeruginosa biofilms and optimized to 1) alter biofilm viscoelastic properties, 2) increase mucociliary transport rates, and 3) reduce bacterial viability. A disulfide bond reducing agent (tris(2-carboxyethyl)phosphine, TCEP), a surfactant (NP40), a biopolymer (hyaluronic acid, HA), a DNA degradation enzyme (DNase), and an antibiotic (tobramycin) are tested in various combinations to maximize biofilm disruption. The viscoelastic properties of biofilms are quantified with particle tracking microrheology and transport rates are quantified in a mucociliary transport device comprised of fully differentiated primary human bronchial epithelial cells. The combination of the NP40 with hyaluronic acid and tobramycin was the most effective at increasing mucociliary transport rates, decreasing the viscoelastic properties of mucus, and reducing bacterial viability. Multimechanistic targeting of biofilm infections may ultimately result in improved clinical outcomes, and the results of this study may be translated into future in vivo infection models.

Mutations in genes lpxL1, bamA, and pmrB impair the susceptibility of cystic fibrosis strains of Pseudomonas aeruginosa to murepavadin.

Murepavadin is a peptidomimetic exhibiting specific inhibitory activity against Pseudomonas species. In the present study, its in vitro activity was assessed on 230 cystic fibrosis (CF) strains of Pseudomonas aeruginosa isolated from 12 French hospitals, in comparison with 12 other antipseudomonal antibiotics. Although murepavadin is still in preclinical stage of development, 9.1% (n = 21) of strains had a minimum inhibitory concentration (MIC) >4 mg/L, a level at least 128-fold higher than the modal MIC value of the whole collection (≤0.06 mg/L). Whole-genome sequencing of these 21 strains along with more susceptible isogenic counterparts coexisting in the same patients revealed diverse mutations in genes involved in the synthesis (lpxL1 and lpxL2) or transport of lipopolysaccharides (bamA, lptD, and msbA), or encoding histidine kinases of two-component systems (pmrB and cbrA). Allelic replacement experiments with wild-type reference strain PAO1 confirmed that alteration of genes lpxL1, bamA, and/or pmrB can decrease the murepavadin susceptibility from 8- to 32-fold. Furthermore, we found that specific amino acid substitutions in histidine kinase PmrB (G188D, Q105P, and D45E) reduce the susceptibility of P. aeruginosa to murepavadin, colistin, and tobramycin, three antibiotics used or intended to be used (murepavadin) in aerosols to treat colonized CF patients. Whether colistin or tobramycin may select mutants resistant to murepavadin or the opposite needs to be addressed by clinical studies.

Multifunctional Cationic Hyperbranched Polyaminoglycosides that Target Multiple Mediators for Severe Abdominal Trauma Management.

Trauma and its associated complications, including dysregulated inflammatory responses, severe infection, and disseminated intravascular coagulation (DIC), continue to pose lethal threats worldwide. Following injury, cell-free nucleic acids (cfNAs), categorized as damage-associated molecular patterns (DAMPs), are released from dying or dead cells, triggering local and systemic inflammatory responses and coagulation abnormalities that worsen disease progression. Harnessing cfNA scavenging strategies with biomaterials has emerged as a promising approach for treating posttrauma systemic inflammation. In this study, the effectiveness of cationic hyperbranched polyaminoglycosides derived from tobramycin (HPT) and disulfide-included HPT (ss-HPT) in scavenging cfNAs to mitigate posttrauma inflammation and hypercoagulation is investigated. Both cationic polymers demonstrate the ability to suppress DAMP-induced toll-like receptor (TLR) activation, inflammatory cytokine secretion, and hypercoagulation by efficiently scavenging cfNAs. Additionally, HPT and ss-HPT exhibit potent antibacterial efficacy attributed to the presence of tobramycin in their chemical composition. Furthermore, HPT and ss-HPT exhibit favorable modulatory effects on inflammation and therapeutic outcomes in a cecal ligation puncture (CLP) mouse abdominal trauma model. Notably, in vivo studies reveal that ss-HPT displayed high accumulation and retention in injured organs of traumatized mice while maintaining a higher biodegradation rate in healthy mice, contrasting with findings for HPT. Thus, functionalized ss-HPT, a bioreducible polyaminoglycoside, holds promise as an effective option to enhance therapeutic outcomes for trauma patients by alleviating posttrauma inflammation and coagulation complications.

Amphiphilic tribasic galactosamines potentiate rifampicin in Gram-negative bacteria at low Mg++/Ca++concentrations.

Many antibiotics specific to Gram-positive bacteria like rifampicin (RIF) are inactive in Gram-negative bacteria because of outer membrane (OM) impermeability. Enhancing the OM permeability of these antibiotics with the help of OM perturbants is a promising strategy to develop new agents against Gram-negative bacteria. Here we report the synthesis and biological properties of amphiphilic tribasic galactosamines as potential RIF potentiators. Our results demonstrate that tribasic galactose-based amphiphiles potentiate RIF in multidrug-resistant Acinetobacter baumannii and Escherichia coli but not Pseudomonas aeruginosa in low salt-containing media. Under these conditions, lead compounds 20, 22 and 35 lowered the minimum inhibitory concentration of RIF by 64- to 256-fold against Gram-negative bacteria. However, the RIF-potentiating effect was reduced when bivalent Mg++ or Ca++ ions were added in the media at physiological concentrations. Overall, our results indicate that amphiphilic tribasic galactosamine-based compounds show reduced RIF-potentiating effects when compared to amphiphilic tobramycin antibiotics at physiological salt concentrations.

Thermostable Lactonases Inhibit Pseudomonas aeruginosa Biofilm: Effect In Vitro and in Drosophila melanogaster Model of Chronic Infection.

Pseudomonas aeruginosa is one of the six antimicrobial-resistant pathogens known as "ESKAPE" that represent a global threat to human health and are considered priority targets for the development of novel antimicrobials and alternative therapeutics. The virulence of P. aeruginosa is regulated by a four-chemicals communication system termed quorum sensing (QS), and one main class of QS signals is termed acylhomoserine lactones (acyl-HSLs), which includes 3-Oxo-dodecanoil homoserine lactone (3-Oxo-C12-HSL), which regulates the expression of genes implicated in virulence and biofilm formation. Lactonases, like Paraoxonase 2 (PON2) from humans and the phosphotriesterase-like lactonases (PLLs) from thermostable microorganisms, are able to hydrolyze acyl-HSLs. In this work, we explored in vitro and in an animal model the effect of some lactonases on the production of Pseudomonas virulence factors. This study presents a model of chronic infection in which bacteria were administered by feeding, and Drosophila adults were treated with enzymes and the antibiotic tobramycin, alone or in combination. In vitro, we observed significant effects of lactonases on biofilm formation as well as effects on bacterial motility and the expression of virulence factors. The treatment in vivo by feeding with the lactonase SacPox allowed us to significantly increase the biocidal effect of tobramycin in chronic infection.

The Inactivation of the Putative Two-Component System Sensor PA14_27940 Increases the Susceptibility to Several Antibiotics and Reduces the Motility of Pseudomonas aeruginosa.

The identification of targets whose inactivation increases the activity of antibiotics helps to fight antibiotic resistance. Previous work showed that a transposon-insertion mutant in the gene PA14_27940 increases Pseudomonas aeruginosa susceptibility to aminoglycosides. Since polar effects may affect the phenotype, in the present work, we generated an in-frame PA14_27940 deletion mutant. A PA14_27940 deletion increased the susceptibility to aminoglycosides, tetracycline, tigecycline, erythromycin and fosfomycin. Excepting fosfomycin, the other antibiotics are inducers of the MexXY efflux pump. MexXY induction is required for P. aeruginosa resistance to these antibiotics, which is post-transcriptionally regulated by the anti-repressor ArmZ. Although mexXY is inducible by tobramycin in ΔPA14_27940, the induction level is lower than in the parental PA14 strain. Additionally, armZ is induced by tobramycin in PA14 and not in ΔPA14_27940, supporting that ΔPA14_27940 presents an ArmZ-mediated defect in mexXY induction. For its part, hypersusceptibility to fosfomycin may be due to a reduced expression of nagZ and agmK, which encode enzymes of the peptidoglycan recycling pathway. ΔPA14_27940 also presents defects in motility, an element with relevance in P. aeruginosa's virulence. Overall, our results support that PA14_27940 is a good target for the search of adjuvants that will increase the activity of antibiotics and reduce the virulence of P. aeruginosa.

Development and validation of a rabbit model of Pseudomonas aeruginosa non-ventilated pneumonia for preclinical drug development.

Background: New drugs targeting antimicrobial resistant pathogens, including Pseudomonas aeruginosa, have been challenging to evaluate in clinical trials, particularly for the non-ventilated hospital-acquired pneumonia and ventilator-associated pneumonia indications. Development of new antibacterial drugs is facilitated by preclinical animal models that could predict clinical efficacy in patients with these infections. Methods: We report here an FDA-funded study to develop a rabbit model of non-ventilated pneumonia with Pseudomonas aeruginosa by determining the extent to which the natural history of animal disease reproduced human pathophysiology and conducting validation studies to evaluate whether humanized dosing regimens of two antibiotics, meropenem and tobramycin, can halt or reverse disease progression. Results: In a rabbit model of non-ventilated pneumonia, endobronchial challenge with live P. aeruginosa strain 6206, but not with UV-killed Pa6206, caused acute respiratory distress syndrome, as evidenced by acute lung inflammation, pulmonary edema, hemorrhage, severe hypoxemia, hyperlactatemia, neutropenia, thrombocytopenia, and hypoglycemia, which preceded respiratory failure and death. Pa6206 increased >100-fold in the lungs and then disseminated from there to infect distal organs, including spleen and kidneys. At 5 h post-infection, 67% of Pa6206-challenged rabbits had PaO2 <60 mmHg, corresponding to a clinical cut-off when oxygen therapy would be required. When administered at 5 h post-infection, humanized dosing regimens of tobramycin and meropenem reduced mortality to 17-33%, compared to 100% for saline-treated rabbits (P<0.001 by log-rank tests). For meropenem which exhibits time-dependent bactericidal activity, rabbits treated with a humanized meropenem dosing regimen of 80 mg/kg q2h for 24 h achieved 100% T>MIC, resulting in 75% microbiological clearance rate of Pa6206 from the lungs. For tobramycin which exhibits concentration-dependent killing, rabbits treated with a humanized tobramycin dosing regimen of 8 mg/kg q8h for 24 h achieved Cmax/MIC of 9.8 ± 1.4 at 60 min post-dose, resulting in 50% lung microbiological clearance rate. In contrast, rabbits treated with a single tobramycin dose of 2.5 mg/kg had Cmax/MIC of 7.8 ± 0.8 and 8% (1/12) microbiological clearance rate, indicating that this rabbit model can detect dose-response effects. Conclusion: The rabbit model may be used to help predict clinical efficacy of new antibacterial drugs for the treatment of non-ventilated P. aeruginosa pneumonia.

Rapid AMR prediction in Pseudomonas aeruginosa combining MALDI-TOF MS with DNN model.

BACKGROUND: Pseudomonas aeruginosa is a significant clinical pathogen that poses a substantial threat due to its extensive drug resistance. The rapid and precise identification of this resistance is crucial for effective clinical treatment. Although matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been used for antibiotic susceptibility differentiation of some bacteria in recent years, the genetic diversity of P. aeruginosa complicates population analysis. Rapid identification of antimicrobial resistance (AMR) in P. aeruginosa based on a large amount of MALDI-TOF-MS data has not yet been reported. In this study, we employed publicly available datasets for P. aeruginosa, which contain data on bacterial resistance and MALDI-TOF-MS spectra. We introduced a deep neural network model, synergized with a strategic sampling approach (SMOTEENN) to construct a predictive framework for AMR of three widely used antibiotics. RESULTS: The framework achieved area under the curve values of 90%, 85%, and 77% for Tobramycin, Cefepime, and Meropenem, respectively, surpassing conventional classifiers. Notably, random forest algorithm was used to assess the significance of features and post-hoc analysis was conducted on the top 10 features using Cohen's d. This analysis revealed moderate effect sizes (d = 0.5-0.8) in Tobramycin and Cefepime models. Finally, putative AMR biomarkers were identified in this study. CONCLUSIONS: This work presented an AMR prediction tool specifically designed for P. aeruginosa, which offers a hopeful pathway for clinical decision-making.

Development of a novel tobramycin dependent riboswitch.

We herein report the selection and characterization of a new riboswitch dependent on the aminoglycoside tobramycin. Its dynamic range rivals even the tetracycline dependent riboswitch to be the current best performing, synthetic riboswitch that controls translation initiation. The riboswitch was selected with RNA Capture-SELEX, a method that not only selects for binding but also for structural changes in aptamers on binding. This study demonstrates how this method can fundamentally reduce the labour required for the de novo identification of synthetic riboswitches. The initially selected riboswitch candidate harbours two distinct tobramycin binding sites with KDs of 1.1 nM and 2.4 µM, respectively, and can distinguish between tobramycin and the closely related compounds kanamycin A and B. Using detailed genetic and biochemical analyses and 1H NMR spectroscopy, the proposed secondary structure of the riboswitch was verified and the tobramycin binding sites were characterized. The two binding sites were found to be essentially non-overlapping, allowing for a separate investigation of their contribution to the activity of the riboswitch. We thereby found that only the high-affinity binding site was responsible for regulatory activity, which allowed us to engineer a riboswitch from only this site with a minimal sequence size of 33 nt and outstanding performance.

Limited effects of azithromycin on the oropharyngeal microbiome in children with CF and early pseudomonas infection.

BACKGROUND: Tobramycin inhalation solution (TIS) and chronic azithromycin (AZ) have known clinical benefits for children with CF, likely due to antimicrobial and anti-inflammatory activity. The effects of chronic AZ in combination with TIS on the airway microbiome have not been extensively investigated. Oropharyngeal swab samples were collected in the OPTIMIZE multicenter, randomized, placebo-controlled trial examining the addition of AZ to TIS in 198 children with CF and early P. aeruginosa infection. Bacterial small subunit rRNA gene community profiles were determined. The effects of TIS and AZ were assessed on oropharyngeal microbial diversity and composition to uncover whether effects on the bacterial community may be a mechanism of action related to the observed changes in clinical outcomes. RESULTS: Substantial changes in bacterial communities (total bacterial load, diversity and relative abundance of specific taxa) were observed by week 3 of TIS treatment for both the AZ and placebo groups. On average, these shifts were due to changes in non-traditional CF taxa that were not sustained at the later study visits (weeks 13 and 26). Bacterial community measures did not differ between the AZ and placebo groups. CONCLUSIONS: This study provides further evidence that the mechanism for AZ's effect on clinical outcomes is not due solely to action on airway microbial composition.

Efficacy of antibiotic combinations in an experimental sepsis model with Pseudomonas aeruginosa.

This study aimed to compare the efficacy of fosfomycin, colistin, tobramycin and their dual combinations in an experimental sepsis model. After sepsis was established with a Pseudomonas aeruginosa isolate (P1), antibiotic-administered rats were divided into six groups: Fosfomycin, tobramycin, colistin and their dual combinations were administered by the intravenous or intraperitoneal route to the groups. The brain, heart, lung, liver, spleen and kidney tissues of rats were cultured to investigate bacterial translocation caused by P1. Given the antibiotics and their combinations, bacterial colony counts in liver tissues were decreased in colistin alone and colistin plus tobramycin groups compared with control group, but there were no significant differences. In addition, a non-statistical decrease was found in the spleen tissues of rats in the colistin plus tobramycin group. There was a > 2 log10 CFU/ml decrease in the number of bacterial colonies in the kidney tissues of the rats in the fosfomycin group alone, but the decrease was not statistically significant. However, there was an increase in the number of bacterial colonies in the spleen and kidney samples in the group treated with colistin as monotherapy compared to the control group. The number of bacterial colonies in the spleen samples in fosfomycin plus tobramycin groups increased compared to the control group. Bacterial colony numbers in all tissue samples in the fosfomycin plus colistin group were found to be close to those in the control group. Colistin plus tobramycin combinations are effective against P. aeruginosa in experimental sepsis, and clinical success may be achieved. New in vivo studies demonstrating the ability of P. aeruginosa to biofilm formation in tissues other than the lung are warranted in future.