Effect of biofilm physical characteristics on their susceptibility to antibiotics: impacts of low-frequency ultrasound.

Biofilms are highly resistant to antimicrobials, often causing chronic infections. Combining antimicrobials with low-frequency ultrasound (LFU) enhances antimicrobial efficiency, but little is known about the underlying mechanisms. Biofilm physical characteristics, which depend on factors such as growth conditions and age, can have significant effects on inactivation efficiency. In this study, we investigated the susceptibility of Pseudomonas aeruginosa biofilms to tobramycin, with and without LFU treatment. The biofilms were grown under low and high fluid shear to provide different characteristics. Low-shear biofilms exhibited greater thickness, roughness, and porosity and lower density, compared to high-shear biofilms. The biofilm matrix of the high-shear biofilms had a three times higher protein-to-polysaccharide ratio, suggesting greater biofilm stiffness. This was supported by microrheology measurements of biofilm creep compliance. For the low-shear biofilms without LFU, the viability of the biofilms in their inner regions was largely unaffected by the antibiotic after a 2-hour treatment. However, when tobramycin was combined with LFU, the inactivation for the entire biofilm increased to 80% after 2 h. For the high-shear biofilms without LFU, higher LFU intensities were needed to achieve similar inactivation results. Microrheology measurements revealed that changes in biofilm inactivation profiles were closely related to changes in biofilm mechanical properties. Modeling suggests that LFU changes antibiotic diffusivity within the biofilm, probably due to a "decohesion" effect. Overall, this research suggests that biofilm physical characteristics (e.g., compliance, morphology) are linked to antimicrobial efficiency. LFU weakens the biofilm while increasing its diffusivity for antibiotics.

Prevalence and mechanisms of aminoglycoside resistance among drug-resistant Pseudomonas aeruginosa clinical isolates in Iran.

BACKGROUND: Aminoglycosides have been a cornerstone of the treatment of nosocomial infections caused by Pseudomonas aeruginosa for over 80 years. However, escalating emergence of resistance poses a significant challenge. Therefore, this study aimed to investigate the prevailing patterns of aminoglycoside resistance among clinical isolates of P. aeruginosa in Iran; as well as the underlying resistance mechanisms observed in patients referred to Ardabil hospitals. METHODS: A total of 200 isolates from five hospitals were evaluated. The resistance profiles of P. aeruginosa isolates to tobramycin, amikacin, and netilmicin were determined using the disk diffusion method. The capacity of aminoglycoside-resistant isolates to form biofilms was assessed through a phenotypic assay, and the results were confirmed using the gene amplification technique. The presence of genes associated with aminoglycoside resistance was detected using polymerase chain reaction (PCR). Quantitative reverse transcription PCR (qRT-PCR) was performed to measure the expression levels of genes encoding the MexXY-OprM efflux pump and PhoPQ two-component system (TCS). RESULTS: The prevalence of aminoglycoside-resistant P. aeruginosa isolates was 48%, with 94.7% demonstrating multidrug resistance (MDR). All aminoglycoside-resistant P. aeruginosa strains exhibited biofilm-forming capabilities and harbored all the genes associated with biofilm production. Among the nine genes encoding 16S rRNA methylase and aminoglycoside-modifying enzymes, three genes were detected in these isolates: aac(6')-Ib (85.4%), ant(2'')-Ia (18.7%), and aph(3')-VI (3.1%). Additionally, all aminoglycoside-resistant P. aeruginosa isolates carried mexY and phoP genes, although the expression levels of mexY and phoP were 75% and 87.5%, respectively. CONCLUSION: Given the considerably high prevalence of aminoglycoside-resistant P. aeruginosa strains, urgent measures are warranted to transition towards the use of novel aminoglycosides and to uphold vigilant surveillance of resistance patterns.

Anti-Persisters Activity of Lacticaseibacillus rhamnosus Culture Filtrates against Pseudomonas aeruginosa in Artificial Sputum Medium.

Persisters are antibiotic-tolerant bacteria, playing a role in the recalcitrance and relapse of many bacterial infections, including P. aeruginosa pulmonary infections in Cystic Fibrosis (CF) patients. Among novel antimicrobial strategies, the use of probiotics and their products is emerging as a particularly promising approach. The aim of this study was to evaluate the anti-persisters activity of culture filtrate supernatants of Lacticaseibacillus rhamnosus (LRM-CFS) against P. aeruginosa in artificial sputum medium (ASM), which resembles the CF lung environment. Planktonic persisters of two clinical strains of P. aeruginosa (PaCF1 and PaCF4) were obtained following two different procedures: (i) exposing stationary-phase cultures to cyanide m-chlorophenylhydrazone (CCCP) in LB medium; (ii) incubating stationary-phase cultures with high doses of tobramycin (128-fold MIC) in ASM. In addition, persisters from biofilm were obtained by exposing 48 h old biofilm of P. aeruginosa to 128 x MIC of ciprofloxacin. LRM-CFS at dilutions of 1:6 and 1:4 resulted in being bactericidal in ASM against both PaCF1 and PaCF4 persisters obtained after CCCP or tobramycin treatment. Moreover, LRM-CFS at dilution 1:4 caused a reduction of antibiotic-tolerant bacteria in the biofilm of both P. aeruginosa strains. Overall, LRM-CFS represents a promising adjuvant therapeutic strategy against P. aeruginosa recalcitrant infections in CF patients.

Dual tobramycin and docosahexaenoic acid loaded nanoemulsions combating Pseudomonas aeruginosa-induced pulmonary infection.

Pseudomonas aeruginosa (P. aeruginosa) typically forms biofilms in vivo, which exhibit high resistance and complicate eradication efforts. Additionally, persistent inflammation and excessive oxidative stress can lead to severe lung dysfunction, facilitating bacterial colonization and infection. Herein, we prepared oil-in-water (O/W) nanoemulsions (TD-αT NEs) by using PEG5k-block-PCL5k and α-tocopherol to encapsulate tobramycin (TOB). To enhance TOB's drug load, a hydrophobic ion pair (TDIP) composed of TOB and docosahexaenoic acid (DHA) was pre-prepared. TD-αT NEs was not only easily prepared and aerosolized, but stable in both physics and chemistry. The negatively charged TD-αT NEs facilitated penetration through mucus, reaching infection sites. Subsequently, TD-αT NEs permeated biofilms due to their small size and released drugs via lipase-triggered carrier dissociation, aiding in eradicating internal bacteria within biofilms (with a 16-fold reduction in CFU vs. free TOB group). TD-αT NEs simultaneously exerted superior anti-inflammatory effects, reducing levels of pro-inflammatory cytokines (NO, IL-6, IL-8, and TNF-α) while increasing the level of anti-inflammatory cytokine (IL-10). It was achieved through the upregulation of PPAR-γ and downregulation of NF-κB signaling, thus mitigating the lung damage. In addition, TD-αT NEs demonstrated strong antioxidant activity, alleviating the oxidative stress induced by P. aeruginosa. Notably, when administered via inhalation, TD-αT NEs significantly reduced the lung bacterial burden, lung inflammation, and oxidative stress in vivo compared to TOB solution. TD-αT NEs could prove beneficial in treating chronic pulmonary infections induced by P. aeruginosa through a comprehensive strategy, specifically enhancing biofilm eradication, reducing inflammation, and alleviating oxidative stress.

The uncharacterized PA3040-3042 operon is part of the cell envelope stress response and a tobramycin resistance determinant in a clinical isolate of Pseudomonas aeruginosa.

Bacteriophages (hereafter "phages") are ubiquitous predators of bacteria in the natural world, but interest is growing in their development into antibacterial therapy as complement or replacement for antibiotics. However, bacteria have evolved a huge variety of antiphage defense systems allowing them to resist phage lysis to a greater or lesser extent. In addition to dedicated phage defense systems, some aspects of the general stress response also impact phage susceptibility, but the details of this are not well known. In order to elucidate these factors in the opportunistic pathogen Pseudomonas aeruginosa, we used the laboratory-conditioned strain PAO1 as host for phage infection experiments as it is naturally poor in dedicated phage defense systems. Screening by transposon insertion sequencing indicated that the uncharacterized operon PA3040-PA3042 was potentially associated with resistance to lytic phages. However, we found that its primary role appeared to be in regulating biofilm formation, particularly in a clinical isolate of P. aeruginosa in which it also altered tobramycin resistance. Its expression was highly growth-phase dependent and responsive to phage infection and cell envelope stress. Our results suggest that this operon may be a cryptic but important locus for P. aeruginosa stress tolerance. IMPORTANCE: An important category of bacterial stress response systems is bacteriophage defense, where systems are triggered by bacteriophage infection and activate a response which may either destroy the phage genome or destroy the infected cell so that the rest of the population survives. In some bacteria, the cell envelope stress response is activated by bacteriophage infection, but it is unknown whether this contributes to the survival of the infection. We have found that a conserved uncharacterized operon (PA3040-PA3042) of the cell envelope stress regulon in Pseudomonas aeruginosa, which has very few dedicated phage defense systems, responds to phage infection and stationary phase as well as envelope stress and is important for growth and biofilm formation in a clinical isolate of P. aeruginosa, even in the absence of phages. As homologs of these genes are found in other bacteria, they may be a novel component of the general stress response.

Effect of L-arginine on cystic fibrosis Pseudomonas aeruginosa biofilms.

Cystic fibrosis (CF) airways are L-arginine deficient which may affect susceptibility to infection with certain pathogens. The potential impact of L-arginine supplementation on Pseudomonas aeruginosa, a common CF airway pathogen, is unclear. This study investigated the effects of L-arginine on P. aeruginosa biofilm cultures, using the laboratory strain PAO1 and multi-drug resistant CF clinical isolates. P. aeruginosa biofilms were grown in a chambered cover-glass slide model for 24 h, then exposed to either L-arginine alone or combined with tobramycin for an additional 24 h. Biofilms were visualized using confocal microscopy, and viable cells were measured via plating for colony-forming units. Increasing concentrations of L-arginine in bacterial culture medium reduced the biovolume of P. aeruginosa in a dose-dependent manner. Notably, L-arginine concentrations within the physiological range (50 mM and 100 mM) in combination with tobramycin promoted biofilm growth, while higher concentrations (600 mM and 1200 mM) inhibited growth. These findings demonstrate the potential of L-arginine as an adjuvant therapy to inhaled tobramycin in treating P. aeruginosa infections in people with CF.

Targeted treatment for biofilm-based infections using PEGylated tobramycin.

Chronic infections often involve biofilm-based bacteria, in which the biofilm results in significant resistance against antimicrobial agents and prevents eradication of the infection. The physicochemical barrier presented by the biofilm matrix is a major impediment to the delivery of many antibiotics. Previously, PEGylation has been shown to improve antibiotic penetration into biofilms in vitro. In these studies, PEGylating tobramycin was investigated both in vitro and in vivo. Two distinct PEGylated tobramycin molecules were synthesized (mPEG-SA-Tob and mPEG-AA-Tob). Then, in a P. aeruginosa biofilm in vitro model, we found that mPEG-SA-Tob can operate as a prodrug and showed 7 times more effectiveness than tobramycin (MIC80: 14 µM vs.100 µM). This improved biofilm eradication is attributable to the fact that mPEG-SA-Tob can aid tobramycin to penetrate through the biofilm and overcome the alginate-mediated antibiotic resistance. Finally, we used an in vivo biofilm-based chronic pulmonary infection rat model to confirm the therapeutic impact of mPEG-SA-Tob on biofilm-based chronic lung infection. mPEG-SA-Tob has a better therapeutic impact than tobramycin in that it cannot only stop P. aeruginosa from multiplying in the lungs but can also reduce inflammation caused by infections and prevent a recurrence infection. Overall, our findings show that PEGylated tobramycin is an effective treatment for biofilm-based chronic lung infections.

Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene.

BACKGROUND: The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. OBJECTIVES: To identify sources for the aac(6')-Im fragment found in A. baumannii. METHODS: MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. RESULTS: Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different. CONCLUSIONS: The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

Development of a tobramycin-loaded calcium alginate microsphere/chitosan composite sponge with antibacterial effects as a wound dressing.

Traditional dressings exhibit several disadvantages, as they frequently lead to bacterial infections, cause severe tissue adhesion and perform a relatively single function. Therefore, in this study, a composite sponge dressing with antibacterial properties and excellent physicochemical properties was developed. Six groups of tobramycin-loaded calcium alginate microspheres were prepared by changing the amount of tobramycin added, and the optimal group was selected. Then, seven groups of tobramycin-loaded calcium alginate microsphere/chitosan composite sponges were fabricated via a solvent blending process and a freeze-drying method. The surface morphology, physicochemical properties,in vitrodegradation properties,in vitrodrug release properties, antibacterial properties and cytotoxicity of the composite sponges were examined. Group 3.0 contained the best microspheres with the largest drug loading capacity, good swelling performance and cumulative drug release rate, obvious and sustained antibacterial activity, and good cytocompatibility. The tobramycin-loaded calcium alginate microsphere/chitosan composite sponges exhibited three-dimensional porous structures, and their porosity, swelling rate, water absorption and water retention rates and water vapor transmission rate met the standards needed for an ideal dressing. The comprehensive performance of the sponge was best when 20 mg of drug-loaded microspheres was added (i.e. group 20). The cumulative drug release rate of the sponge was 29.67 ± 4.14% at 7 d, the diameters of the inhibition zones against the three bacteria were greater than 15 mm, and L929 cell proliferation was promoted. These results demonstrated that the tobramycin-loaded calcium alginate microsphere/chitosan composite sponge with 20 mg of tobramycin-loaded microspheres shows promise as a dressing for infected wounds.

Tobramycin-resistant small colony variant mutant of Salmonella enterica serovar Typhimurium shows collateral sensitivity to nitrofurantoin.

The increasing antibiotic resistance poses a significant global health challenge, threatening our ability to combat infectious diseases. The phenomenon of collateral sensitivity, whereby resistance to one antibiotic is accompanied by increased sensitivity to another, offers potential avenues for novel therapeutic interventions against infections unresponsive to classical treatments. In this study, we elucidate the emergence of tobramycin (TOB)-resistant small colony variants (SCVs) due to mutations in the hemL gene, which render S. Typhimurium more susceptible to nitrofurantoin (NIT). Mechanistic studies demonstrate that the collateral sensitivity in TOB-resistant S. Typhimurium SCVs primarily stems from disruptions in haem biosynthesis. This leads to dysfunction in the electron transport chain (ETC) and redox imbalance, ultimately inducing lethal accumulation of reactive oxygen species (ROS). Additionally, the upregulation of nfsA/B expressions facilitates the conversion of NIT prodrug into its active form, promoting ROS-mediated bacterial killing and contributing to this collateral sensitivity pattern. Importantly, alternative NIT therapy demonstrates a significant reduction of bacterial load by more than 2.24-log10 cfu/g in the murine thigh infection and colitis models. Our findings corroborate the collateral sensitivity of S. Typhimurium to nitrofurans as a consequence of evolving resistance to aminoglycosides. This provides a promising approach for treating infections due to aminoglycoside-resistant strains.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

Impact of pH modification of the empirically used tobramycin ophthalmic solution on MIC90 concentration in tears and aqueous humor of donkeys (Equus asinus).

BACKGROUND: Commercial tobramycin ophthalmic solution is frequently used empirically to treat ocular disorders in equines, despite being primarily formulated for use in humans. It has been noted that tobramycin MIC90 concentration (minimal inhibitory concentration to 90% of microbial growth) rapidly declined following topical administration. It is hypothesized that adjustment of the pH of the empirically used tobramycin ophthalmic solution -prepared for human use- with the pH of the tears of donkeys, could increase the bioavailability of the drug and subsequently improve its penetration to the aqueous humor. Therefore, this study aimed to evaluate the impact of pH adjustment of the empirically used tobramycin ophthalmic solution on MIC90 concentration in tears and aqueous humor of donkeys (Equus asinus). The study was conducted on six (n = 6) clinically healthy donkeys. In each donkey, one eye was randomly selected to receive 210 µg tobramycin of the commercial tobramycin (CT) and used as a positive control (C group, n = 6). The other eye (treated eye) received 210 µg of the modified tobramycin ophthalmic solution (MT) (T group, n = 6). Tears and aqueous humor samples were collected 5-, 10-, 15-, 30- min, and 1-, 2-, 4-, and 6 h post-instillation. RESULTS: Modifying the pH of the empirically used commercial tobramycin ophthalmic solution in donkeys at a pH of 8.26 enhanced the drug's bioavailability. The MIC90 of the most hazardous bacteria isolated from equines' eyes such as Pseudomonas aeruginosa (MIC90 = 128 µg/ml) and Staphylococcus aureus (MIC90 = 256 µg/ml) was covered early (5 min post-instillation) and over a longer period in donkey tears (239-342 min) and aqueous humor (238-330 min) with the modified tobramycin solution. CONCLUSIONS: Adjustment of the pH of the commercial tobramycin ophthalmic solution, empirically used by veterinarians to treat donkeys' ophthalmic infections at a pH of 8.26, isotonic with the donkeys' tears pH, resulting in higher concentrations of tobramycin in tears and aqueous humor for a longer time.

Synergistic effects of inhaled aztreonam plus tobramycin on hypermutable cystic fibrosis Pseudomonas aeruginosa isolates in a dynamic biofilm model evaluated by mechanism-based modelling and whole genome sequencing.

OBJECTIVE: Hypermutable Pseudomonas aeruginosa strains are highly prevalent in chronic lung infections of patients with cystic fibrosis (CF). Acute exacerbations of these infections have limited treatment options. This study aimed to investigate inhaled aztreonam and tobramycin against clinical hypermutable P. aeruginosa strains using the CDC dynamic in vitro biofilm reactor (CBR), mechanism-based mathematical modelling (MBM) and genomic studies. METHODS: Two CF multidrug-resistant strains were investigated in a 168 h CBR (n = 2 biological replicates). Regimens were inhaled aztreonam (75 mg 8-hourly) and tobramycin (300 mg 12-hourly) in monotherapies and combination. The simulated pharmacokinetic profiles of aztreonam and tobramycin (t1/2 = 3 h) were based on published lung fluid concentrations in patients with CF. Total viable and resistant counts were determined for planktonic and biofilm bacteria. MBM of total and resistant bacterial counts and whole genome sequencing were completed. RESULTS: Both isolates showed reproducible bacterial regrowth and resistance amplification for the monotherapies by 168 h. The combination performed synergistically, with minimal resistant subpopulations compared to the respective monotherapies at 168 h. Mechanistic synergy appropriately described the antibacterial effects of the combination regimen in the MBM. Genomic analysis of colonies recovered from monotherapy regimens indicated noncanonical resistance mechanisms were likely responsible for treatment failure. CONCLUSION: The combination of aztreonam and tobramycin was required to suppress the regrowth and resistance of planktonic and biofilm bacteria in all biological replicates of both hypermutable multidrug-resistant P. aeruginosa CF isolates. The developed MBM could be utilised for future investigations of this promising inhaled combination.

Dual action of benzaldehydes: Inhibiting quorum sensing and enhancing antibiotic efficacy for controlling Pseudomonas aeruginosa biofilms.

Quorum sensing (QS) has a central role in biofilm lifestyle and antimicrobial resistance, and disrupting these signaling pathways is a promising strategy to control bacterial pathogenicity and virulence. In this study, the efficacy of three structurally related benzaldehydes (4-hydroxybenzaldehyde, 4-hydroxy-3-methoxybenzaldehyde (vanillin) and 4-hydroxy-3,5-dimethoxybenzaldehyde (syringaldehyde)) in disrupting the las and pqs systems of Pseudomonas aeruginosa was investigated using bioreporter strains and computational simulations. Additionally, these benzaldehydes were combined with tobramycin and ciprofloxacin antibiotics to evaluate their ability to increase antibiotic efficacy in preventing and eradicating P. aeruginosa biofilms. To this end, the total biomass, metabolic activity and culturability of the biofilm cells were determined. In vitro assays results indicated that the aromatic aldehydes have potential to inhibit the las and pqs systems by > 80 %. Molecular docking studies supported these findings, revealing the aldehydes binding in the same pocket as the natural ligands or receptor proteins (LasR, PQSA, PQSE, PQSR). Benzaldehydes were shown to act as virulence factor attenuators, with vanillin achieving a 48 % reduction in pyocyanin production. The benzaldehyde-tobramycin combination led not only to a 60 % reduction in biomass production but also to a 90 % reduction in the metabolic activity of established biofilms. A similar result was observed when benzaldehydes were combined with ciprofloxacin. 4-Hydroxybenzaldehyde demonstrated relevant action in increasing biofilm susceptibility to ciprofloxacin, resulting in a 65 % reduction in biomass. This study discloses, for the first time, that the benzaldehydes studied are potent QS inhibitors and also enhancers of antibiotics antibiofilm activity against P. aeruginosa.

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.

An Antibiotic Nanobomb Constructed from pH-Responsive Chemical Bonds in Metal-Phenolic Network Nanoparticles for Biofilm Eradication and Corneal Ulcer Healing.

In the treatment of refractory corneal ulcers caused by Pseudomonas aeruginosa, antibacterial drugs delivery faces the drawbacks of low permeability and short ocular surface retention time. Hence, novel positively-charged modular nanoparticles (NPs) are developed to load tobramycin (TOB) through a one-step self-assembly method based on metal-phenolic network and Schiff base reaction using 3,4,5-trihydroxybenzaldehyde (THBA), ε-poly-ʟ-lysine (EPL), and Cu2+ as matrix components. In vitro antibacterial test demonstrates that THBA-Cu-TOB NPs exhibit efficient instantaneous sterilization owing to the rapid pH responsiveness to bacterial infections. Notably, only 2.6 µg mL-1 TOP is needed to eradicate P. aeruginosa biofilm in the nano-formed THBA-Cu-TOB owing to the greatly enhanced penetration, which is only 1.6% the concentration of free TOB (160 µg mL-1). In animal experiments, THBA-Cu-TOB NPs show significant advantages in ocular surface retention, corneal permeability, rapid sterilization, and inflammation elimination. Based on molecular biology analysis, the toll-like receptor 4 and nuclear factor kappa B signaling pathways are greatly downregulated as well as the reduction of inflammatory cytokines secretions. Such a simple and modular strategy in constructing nano-drug delivery platform offers a new idea for toxicity reduction, physiological barrier penetration, and intelligent drug delivery.

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.

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.

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.

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.