Antimicrobial and antibiotic-potentiating effect of calcium peroxide nanoparticles on oral bacterial biofilms.

Bacterial biofilms represent a prominent biological barrier against physical and chemical attacks. Disturbing the anaerobic microenvironment within biofilms by co-delivery of oxygen appears as a promising strategy to enhance the activity of an antibiotic. Here, we report the effect of oxygen-producing calcium peroxide nanoparticles (CaO2 NP) in combination with tobramycin sulfate (Tob). On Pseudomonas aeruginosa PAO1 biofilms in vitro, the additive effect of CaO2 NP towards Tob activity enhanced biofilm eradication by 2 log compared to Tob alone. For natural biofilms grown in the oral cavity of human volunteers in situ, treatment by CaO2 NP alone slightly increased the fraction of dead bacteria from 44% in various controls, including Tob alone, to 57%. However, the combination of CaO2 NP with Tob further increased the fraction of dead bacteria to 69%. These data confirm the intrinsic antimicrobial and antibiotic-potentiating effect of CaO2 NP also in a clinically relevant setting.

A biomimetic human disease model of bacterial keratitis using a cornea-on-a-chip system.

Bacterial keratitis is a common form of inflammation caused by the bacterial invasion of the corneal stroma after trauma. In extreme cases, it can lead to severe visual impairment or even blindness; therefore, timely medical intervention is imperative. Unfortunately, widespread misuse of antibiotics has led to the development of drug resistance. In recent years, organ-on-chips that integrate multiple cell co-cultures have extensive applications in fundamental research and drug screening. In this study, immortalized human corneal epithelial cells and primary human corneal fibroblasts were co-cultured on a porous polydimethylsiloxane membrane to create a cornea-on-a-chip model. The developed multilayer epithelium closely mimicked clinical conditions, demonstrating high structural resemblance and repeatability. By introducing a consistently defective epithelium and bacterial infection using the space-occupying method, we successfully established an in vitro model of bacterial keratitis using S. aureus. We validate this model by evaluating the efficacy of antibiotics, such as levofloxacin, tobramycin, and chloramphenicol, through simultaneously observing the reactions of bacteria and the two cell types to these antibiotics. Our study has revealed the barrier function of epithelium of the model and differentiated efficacy of three drugs in terms of bactericidal activity, reducing cellular apoptosis, and mitigating scar formation. Altogether, the cornea on chip enables the assessment of ocular antibiotics, distinguishing the impact on corneal cells and structural integrity. This study introduced a biomimetic in vitro disease model to evaluate drug efficacy and provided significant insights into the extensive effects of antibiotics on diverse cell populations within the cornea.

Hybrids of 3-Hydroxypyridin-4(1H)-ones and Long-Chain 4-Aminoquinolines as Potent Biofilm Inhibitors of Pseudomonas aeruginosa Potentiate Tobramycin and Polymyxin B Activity.

The biofilm formation of Pseudomonas aeruginosa involves multiple complex regulatory pathways; thus, blocking a single pathway is unlikely to achieve the desired antibiofilm efficacy. Herein, a series of hybrids of 3-hydroxypyridin-4(1H)-ones and long-chain 4-aminoquinolines were synthesized as biofilm inhibitors against P. aeruginosa based on a multipathway antibiofilm strategy. Comprehensive structure-activity relationship studies identified compound 30b as the most valuable antagonist, which significantly inhibited P. aeruginosa biofilm formation (IC50 = 5.8 µM) and various virulence phenotypes. Mechanistic studies revealed that 30b not only targets the three quorum sensing systems but also strongly induces iron deficiency signals in P. aeruginosa. Furthermore, 30b demonstrated a favorable in vitro and in vivo safety profile. Moreover, 30b specifically enhanced the antibacterial activity of tobramycin and polymyxin B in in vitro and in vivo combination therapy. Overall, these results highlight the potential of 30b as a novel anti-infective candidate for treating P. aeruginosa infections.

Design and application of an ultrasensitive and selective tobromycin electrochemiluminescence aptasensor using MXene /Ni/Sm-LDH-based nanocomposite.

Using a chemiluminescence reaction between luminol and H2O2 in basic solution, an ultrasensitive electrochemiluminescence (ECL) aptasensor was developed for the determination of tobramycin (TOB), as an aminoglycoside antibiotic. Ti3C2/Ni/Sm-LDH-based nanocomposite effectively catalyzes the oxidation of luminol and decomposition of H2O2, leading to the formation of different reactive oxygen species (ROSs), thus amplifying the ECL signal intensity of luminol, which can be used for the determination of TOB concentration. To evaluate the performance of the electrochemiluminescence aptasensor and synthesized nanocomposite, different methods such as cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) analyses were performed. The considerable specific area, large number of active sites, and enhanced electron transfer reaction on this nanocomposite led to the development of an ECL aptasensor with high sensitivity and electrocatalytic activity. After optimizing the preparation method and analysis conditions, the aptasensor revealed a wide linear response ranging from 1.0 pM to 1.0 µM with a detection limit of 18 pM, displaying outstanding accuracy, specificity, and response stability. The developed ECL sensor was found to be applicable to the determination of TOB in human serum samples and is anticipated to possess excellent clinical potentials for detecting other antibiotics, as well.

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.

Electrochemical aptamer sensor based on AgNPs@PDANSs and "sandwich" structure guidance for the detection of tobramycin in water samples.

In this study, an ultrasensitive detection platform for tobramycin (TOB) was developed, featuring a "sandwich" structure guided by AgNCs@PDANSs and Thi-AuNCs@ZnONSs. To address the issue of large background current peak signals in tagless sensors, Thi-AuNCs@ZnONSs composites were synthesized as signal tags. Zinc oxide nanosheets (ZnONSs) served as the loading agent, and AuNCs with the electroactive molecule Thi acted as carriers. Furthermore, AgNPs@PDANSs nanocomposites, possessing excellent electrical conductivity and large specific surface areas, were prepared as substrate materials for the modified electrodes. A "sandwich" structure strategy was also introduced to enhance the accuracy of the electrochemical aptasensor. This strategy, utilizing a dual sequence for target labeling and capture, yielded higher sensitivity and simplified the sensor construction compared to methods employing a single sequence. Under optimal conditions, the detection limit for TOB was established at 1.41 pM, with a detection range of 0.05-5000 nM. The aptasensor was effectively applied in the detection of TOB in tap and lake water, demonstrating outstanding reproducibility, selectivity, and stability. These results may serve as a reference for environmental TOB detection.

A sensitive tobramycin electrochemical aptasensor based on multiple signal amplification cascades.

The residue of tobramycin, a broad spectrum antibiotic commonly used in animal husbandry, has evitable impact on human health, which may cause kidney damage, respiratory paralysis, neuromuscular blockade and cross-allergy in humans. Sensitive monitoring of tobramycin in animal-derived food products is therefore of great importance. Herein, a new aptamer electrochemical biosensor for sensing tobramycin with high sensitivity is demonstrated via exonuclease III (Exo III) and metal ion-dependent DNAzyme recycling and hybridization chain reaction (HCR) signal amplification cascades. Tobramycin analyte binds aptamer-containing hairpin probe to switch its conformation to expose the toehold sequence, which triggers Exo III-based catalytic digestion of the secondary hairpin to release many DNAzyme strands. The substrate hairpins immobilized on the Au electrode (AuE) are then cyclically cleaved by the DNAzymes to form ssDNAs, which further initiate HCR formation of lots of long methylene blue (MB)-tagged dsDNA polymers on the AuE. Subsequently electro-oxidation of these MB labels thus exhibit highly enhanced currents for sensing tobramycin within the 5-1000 nM concentration range with an impressive detection limit of 3.51 nM. Furthermore, this strategy has high selectivity for detecting tobramycin in milk and shows promising potential for detect other antibiotics for food safety monitoring.

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.

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.

In Vivo Intra-Articular Antibiotic Concentrations at 24 Hours After TKA Fall Below the Minimum Inhibitory Concentration for Most Bacteria: A Randomized Study of Commercially Available Bone Cement.

BACKGROUND: The use of antibiotic-loaded bone cement (ALBC) to help reduce the risk of infection after primary total knee arthroplasty (TKA) is controversial. There is a paucity of in vivo data on the elution characteristics of ALBC. We aimed to determine whether the antibiotic concentrations of 2 commercially available ALBCs met the minimum inhibitory concentration (MIC) and minimum biofilm eradication concentration (MBEC) for common infecting organisms. METHODS: Forty-five patients undergoing TKA were randomized to receive 1 of the following: bone cement without antibiotic (the negative control; n = 5), a commercially available formulation containing 1 g of tobramycin (n = 20), or a commercially available formulation containing 0.5 g of gentamicin (n = 20). Intra-articular drains were placed, and fluid was collected at 4 and 24 hours postoperatively. An automated immunoassay measuring antibiotic concentration was performed, and the results were compared against published MIC and MBEC thresholds. RESULTS: The ALBC treatment groups were predominantly of White (65%) or Black (32.5%) race and were 57.5% female and 42.4% male. The mean age (and standard deviation) was 72.6 ± 7.2 years in the gentamicin group and 67.6 ± 7.4 years in the tobramycin group. The mean antibiotic concentration in the tobramycin group was 55.1 ± 37.7 µg/mL at 4 hours and 19.5 ± 13.0 µg/mL at 24 hours, and the mean concentration in the gentamicin group was 38.4 ± 25.4 µg/mL at 4 hours and 17.7 ± 15.4 µg/mL at 24 hours. Time and antibiotic concentration had a negative linear correlation coefficient (r = -0.501). Most of the reference MIC levels were reached at 4 hours. However, at 24 hours, a considerable percentage of patients had concentrations below the MIC for many common pathogens, including Staphylococcus epidermidis (gentamicin: 65% to 100% of patients; tobramycin: 50% to 85%), methicillin-sensitive Staphylococcus aureus (gentamicin: 5% to 90%; tobramycin: 5% to 50%), methicillin-resistant S . aureus (gentamicin: 5% to 65%; tobramycin: 50%), Streptococcus species (gentamicin: 10% to 100%), and Cutibacterium acnes (gentamicin: 10% to 65%; tobramycin: 100%). The aforementioned ranges reflect variation in the MIC among different strains of each organism. Gentamicin concentrations reached MBEC threshold values at 4 hours only for the least virulent strains of S . aureus and Escherichia coli. Tobramycin concentrations did not reach the MBEC threshold for any of the bacteria at either time point. CONCLUSIONS: The elution of antibiotics from commercially available ALBC decreased rapidly following TKA, and only at 4 hours postoperatively did the mean antibiotic concentrations exceed the MIC for most of the pathogens. Use of commercially available ALBC may not provide substantial antimicrobial coverage following TKA. LEVEL OF EVIDENCE: Therapeutic Level I . See Instructions for Authors for a complete description of levels of evidence.

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.

Inhalable tobramycin EEG powder formulation for treating Pseudomonas aeruginosa-induced lung infection.

Pulmonary delivery of antibiotics is an effective strategy in treating bacterial lung infection for cystic fibrosis patients, by achieving high local drug concentrations and reducing overall systemic exposure compared to systemic administration. However, the inherent anatomical lung defense mechanisms, formulation characteristics, and drug-device combination determine the treatment efficacy of the aerosol delivery approach. In this study, we prepared a new tobramycin (Tobi) dry powder aerosol using excipient enhanced growth (EEG) technology and evaluated the in vitro and in vivo aerosol performance. We further established a Pseudomonas aeruginosa-induced lung infection rat model using an in-house designed novel liquid aerosolizer device. Notably, novel liquid aerosolizer yields comparable lung infection profiles despite administering 3-times lower P. aeruginosa CFU per rat in comparison to the conventional intratracheal administration. Dry powder insufflator (e.g. Penn-Century DP-4) to administer small powder masses to experimental animals is no longer commercially available. To address this gap, we developed a novel rat air-jet dry powder insufflator (Rat AJ DPI) that can emit 68-70 % of the loaded mass for 2 mg and 5 mg of Tobi-EEG powder formulations, achieving a high rat lung deposition efficiency of 79 % and 86 %, respectively. Rat AJ DPI can achieve homogenous distribution of Tobi EEG powder formulations at both loaded mass (2 mg and 5 mg) over all five lung lobes in rats. We then demonstrated that Tobi EEG formulation delivered by Rat AJ DPI can significantly decrease CFU counts in both trachea and lung lobes at 2 mg (p < 0.05) and 5 mg (p < 0.001) loaded mass compared to the untreated P. aeruginosa-infected group. Tobi EEG powder formulation delivered by the novel Rat AJ DPI showed excellent efficiencies in substantially reducing the P. aeruginosa-induced lung infection in rats.

Mutations Affecting Cellular Levels of Cobalamin (Vitamin B12) Confer Tolerance to Bactericidal Antibiotics in Burkholderia cenocepacia.

The Burkholderia cepacia complex (Bcc) consists of opportunistic pathogens known to cause pneumonia in immunocompromised individuals, especially those with cystic fibrosis. Treating Bcc pneumonia is challenging due to the pathogens' high multidrug resistance. Therefore, inhalation therapy with tobramycin powder, which can achieve high antibiotic concentrations in the lungs, is a promising treatment option. In this study, we investigated potential mechanisms that could compromise the effectiveness of tobramycin therapy. By selecting for B. cenocepacia survivors against tobramycin, we identified three spontaneous mutations that disrupt a gene encoding a key enzyme in the biosynthesis of cobalamin (Vitamin B12). This disruption may affect the production of succinyl-CoA by methylmalonyl-CoA mutase, which requires adenosylcobalamin as a cofactor. The depletion of cellular succinyl-CoA may impact the tricarboxylic acid (TCA) cycle, which becomes metabolically overloaded upon exposure to tobramycin. Consequently, the mutants exhibited significantly reduced reactive oxygen species (ROS) production. Both the wild-type and mutants showed tolerance to tobramycin and various other bactericidal antibiotics under microaerobic conditions. This suggests that compromised ROS-mediated killing, due to the impacted TCA cycle, underlies the mutants' tolerance to bactericidal antibiotics. The importance of ROS-mediated killing and the potential emergence of mutants that evade it through the depletion of cobalamin (Vitamin B12) provide valuable insights for developing strategies to enhance antibiotic treatments of Bcc pneumonia.

Does Local Aqueous Tobramycin Injection Reduce Open Fracture-Related Infection Rates?

OBJECTIVES: To examine the effect of local aqueous tobramycin injection adjunct to perioperative intravenous (IV) antibiotic prophylaxis in reducing fracture-related infections (FRIs) following reduction and internal fixation of open fractures. DESIGN: Retrospective cohort study. SETTING: Single academic Level I trauma center. PATIENTS SELECTION CRITERIA: Patients with open extremity fractures treated with reduction and internal fixation with (intervention group) or without (control group) 80 mg of local aqueous (2 mg/mL) tobramycin injected during closure at the time of definitive fixation were identified from December 2018 to August 2021 based on population-matched demographic and injury characteristics. OUTCOME MEASURES AND COMPARISONS: The primary outcome was FRI within 6 months of definitive fixation. Secondary outcomes consisted of fracture nonunion and bacterial speciation. Differences in outcomes between the 2 groups were assessed and logistic regression models were created to assess the difference in infection rates between groups, with and without controlling for potential confounding variables, such as sex, fracture location, and Gustilo-Anderson classification. RESULTS: An analysis of 157 patients was performed with 78 patients in the intervention group and 79 patients in the control group. In the intervention group, 30 (38.5%) patients were women with a mean age of 47.1 years. In the control group, 42 (53.2%) patients were women with a mean age of 46.4 years. The FRI rate was 11.5% in the intervention group compared with 25.3% in the control group ( P = 0.026). After controlling for sex, Gustilo-Anderson classification, and fracture location, the difference in FRI rates between groups remained significantly different ( P = 0.014). CONCLUSIONS: Local aqueous tobramycin injection at the time of definitive internal fixation of open extremity fractures was associated with a significant reduction in FRI rates when administered as an adjunct to intravenous antibiotics, even after controlling for potential confounding variables. LEVEL OF EVIDENCE: Therapeutic Level III. See Instructions for Authors for a complete description of levels of evidence.

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.

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.

Escherichia coli exopolysaccharides disrupt Pseudomonas aeruginosa biofilm and increase its antibiotic susceptibility.

Pseudomonas aeruginosa (P. aeruginosa) is a major pathogen that causes infectious diseases. It has high tendency to form biofilms, resulting in the failure of traditional antibiotic therapies. Inspired by the phenomenon that co-culture of Escherichia coli (E. coli) and P. aeruginosa leads to a biofilm reduction, we reveal that E. coli exopolysaccharides (EPS) can disrupt P. aeruginosa biofilm and increase its antibiotic susceptibility. The results show that E. coli EPS effectively inhibit biofilm formation and disrupt mature biofilms in P. aeruginosa, Staphylococcus aureus, and E. coli itself. The maximal inhibition and disruption rates against P. aeruginosa biofilm are 40 % and 47 %, respectively. Based on the biofilm-disrupting ability of E. coli EPS, we develop an E. coli EPS/antibiotic combining strategy for the treatment of P. aeruginosa biofilms. The combination with E. coli EPS increases the antibacterial efficiency of tobramycin against P. aeruginosa biofilms in vitro and in vivo. This study provides a promising strategy for treating biofilm infections. STATEMENT OF SIGNIFICANCE: Biofilm formation is a leading cause of chronic infections. It blocks antibiotics, increases antibiotic-tolerance, and aids in immune evasion, thus representing a great challenge in clinic. This study proposes a promising approach to combat pathogenic Pseudomonas aeruginosa (P. aeruginosa) biofilms by combining Escherichia coli exopolysaccharides with antibiotics. This strategy shows high efficiency in different P. aeruginosa stains, including two laboratory strains, PAO1 and ATCC 10145, as well as a clinically acquired carbapenem-resistant strain. In addition, in vivo experiments have shown that this approach is effective against implanted P. aeruginosa biofilms and can prevent systemic inflammation in mice. This strategy offers new possibilities to address the clinical failure of conventional antibiotic therapies for microbial biofilms.

Dual-sensitized heterojunction Ag2S/ZnS/NiS composites with entire visible-light region absorption for ultrasensitive photoelectrochemical detection of tobramycin.

In this study, an ultrasensitive photoelectrochemical (PEC) aptasensor based on dual-sensitized heterojunction Ag2S/ZnS/NiS composites as a signal probe was proposed for the detection of tobramycin (TOB) by combining a cascaded quadratic signal amplification strategy. Specifically, compared to the limited visible light-harvesting capability of single sensitized composites, Ag2S/ZnS/NiS composites with p-n and n-n heterojunction could greatly improve the light energy utilization to tremendously strengthen the optical absorption in the entire visible-light region. Moreover, dual-sensitized heterojunction could effectively hinder the rapid recombination of photoelectrons and holes (carriers) to obtain a good photocurrent for improving the sensitivity of the aptasensor. Furthermore, a cascaded quadratic signal amplification strategy was applied to convert trace target TOB into plentiful gold nanoclusters (Au NCs) labelled double-stranded DNA for the construction of PEC aptasensor, with a broad linear detection range from 0.01 to 100 ng mL-1 and a low detection limit of 3.38 pg mL-1. Importantly, this study provided a versatile and sensitive PEC biosensing platform for TOB analysis, and demonstrated its successful application for TOB detection in milk samples. This protocol provides a novel dual-sensitized heterojunction composites to develop a highly efficient and harmfulless PEC aptasensor, which is expected to be used in food safety, environmental monitoring and other areas.

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.