Antimicrobial Delivery Using Metallophore-Responsive Dynamic Nanocarriers.

The increasing prevalence of multidrug-resistant (MDR) pathogens has promoted the development of innovative approaches, such as drug repurposing, synergy, and efficient delivery, in complement to traditional antibiotics. In this study, we present an approach based on biocompatible nanocarriers containing antimicrobial cations and known antibiotics. The matrices were prepared by coordinating GaIII or InIII to formulations of chitosan/tripolyphosphate or catechol-functionalized chitosan with or without encapsulated antibiotics, yielding particles of 100-200 nm in hydrodynamic diameter. MDR clinical isolates of Pseudomonas aeruginosa were found to be effectively inhibited by the nanocarriers under nutrient-limiting conditions. Fractional inhibitory concentration (FIC) indices revealed that cation- and antibiotic-encapsulated nanomatrices were effective against both Gram-negative and Gram-positive pathogens. Metallophores, such as deferoxamine (DFO), were probed to facilitate the sequestration and transport of the antimicrobial cations GaIII or InIII. Although the antimicrobial activities were less significant with DFO, the eradication of biofilm-associated bacteria showed promising trends against P. aeruginosa and Staphylococcus epidermidis. Interestingly, indium-containing compounds showed enhanced activity on biofilm formation and eradication, neutralizing P. aeruginosa under Fe-limiting conditions. In particular, InIII-cross-linked catechol-modified chitosan matrices were able to inhibit pathogenic growth together with DFO. The nanocarriers showed low cytotoxicity toward A549 cells and improvable CC50 values with NIH/3T3 cells.

Application of rosemary oil nano-emulsion as antimicrobial and antioxidant natural alternative in pasteurized cream and Karish cheese.

Essential oils possess significant antimicrobial and antioxidant properties and are increasingly used as natural substitutes for food preservation. Therefore, this study investigated the potential application of rosemary essential oil (REO) and REO nano-emulsion in the dairy plant. The antimicrobial effects of REO and REO nano-emulsion were determined by an agar well diffusion assay after chemical profiling by Gas Chromatography-Mass Spectrometry (GC-MS). The REO nano-emulsion was characterized by a Transmission Electron Microscope (TEM). The REO chemical profile revealed the presence of 42 chemical compounds, including 1, 8-cineole (9.72 %), and α-pinene (5.46 %) as major active components. REO nano-emulsion demonstrated significant antimicrobial activity compared to REO (P < 0.05) with a MIC value of 0.0001 mg/ml against Listeria monocytogenes and Aspergillus flavus and 0.001 mg/ml against Pseudomonas aeruginosa and Bacillus cereus. REO nano-emulsion enhanced the oxidative stability of pasteurized fresh cream, revealing a non-significant difference compared with that inoculated with butylated hydroxy anisol (BHA; synthetic antioxidant) (P˃ 0.05). Fortified cream and Karish cheese with REO nano-emulsion were evaluated organoleptically, and the results showed higher grades of overall acceptability when compared to control samples with a statistically significant difference (P < 0.05). Viability studies were estimated using the previously mentioned microorganisms in fortified fresh cream and Karish cheese with REO nano-emulsion. Results of the fortified cream showed a complete reduction of L. monocytogenes, A. flavus, and B. cereus on days 5, 7, and 10, respectively, and a 96.93 % reduction of P. aeruginosa by the end of the storage period. Regarding Karish cheese viability studies, C. albicans, A. flavus, and P. aeruginosa exhibited complete reduction on days 10, 10, and 15 of storage, respectively. In conclusion, REO nano-emulsion was recommended as a natural, safe, and effective antimicrobial and antioxidant additive in the dairy industry.

A Label-Free Fluorescent Amplification Strategy for High-Sensitive Detection of Pseudomonas aeruginosa based on Protective-EXPAR (p-EXPAR) and Catalytic Hairpin Assembly.

This study presents a fluorescent mechanism for two-step amplification by combining two widely used techniques, exponential amplification reaction (EXPAR) and catalytic hairpin assembly (CHA). Pseudomonas aeruginosa (P. aeruginosa) engaged in competition with the complementary DNA in order to attach to the aptamer that had been fixed on the magnetic beads. The unbound complementary strand in the liquid above was utilized as a trigger sequence to initiate the protective-EXPAR (p-EXPAR) process, resulting in the generation of a substantial quantity of short single-stranded DNA (ssDNA). The amplified ssDNA can initiate the second CHA amplification process, resulting in the generation of many double-stranded DNA (dsDNA) products. The CHA reaction was initiated by the target/trigger DNA, resulting in the release of G-quadruplex sequences. These sequences have the ability to bond with the fluorescent amyloid dye thioflavin T (ThT), generating fluorescence signals. The method employed in this study demonstrated a detection limit of 16 cfu/mL and exhibited a strong linear correlation within the concentration range of 50 cfu/mL to 105 cfu/mL. This method of signal amplification has been effectively utilized to create a fluorescent sensing platform without the need for labels, enabling the detection of P. aeruginosa with high sensitivity.

Biofilm generation and antibiotic resistant profile of extensive and multidrug resistant Pseudomonas aeruginosa from burn patients in Ahvaz: A cross-sectional study.

Background and Aims: Multidrug and extensive drug-resistant Pseudomonas aeruginosa was extracted from burn patients referring to burn centers in southwest Iran so that biofilm generation and antibiotic resistance could be investigated. Methods: A specific primer was used to confirm all our considered 110 P. aeruginosa culture-positive reports on 345 burn patients. The resistance of P. aeruginosa to seven antibiotics and Colistin with minimum inhibitory concentration (MIC) was assessed. Biofilm formation was assessed by the phenotypic study of specimens under Congo red agar and microtiter plate assays. Results: One hundred and 10 clinical P. aeruginosa isolates taken from burn wound infections were validated. Among P. aeruginosa isolates, Piperacillin, Ceftazidime, Maeropenem, Gentamycin, and Gatifloacin had the highest resistance to antibiotics, while Ticarcillin-Clavulanic acid and Ceftolozane-Tazobactam showed the least resistance. MICs were then evaluated via the E test. Seven isolates were resistant to colistin. Colistin reference MICs for multidrug-resistant P. aeruginosa prevalence was 38%, while it was 22% for extensively drug-resistant (XDR) P. aeruginosa. One P. aeruginosa was pandrug-resistant (PDR). Under Congo red agar test, 66 isolates (67%) formed biofilms and black colonies, whereas 44 isolates (50%) had red colonies. In MTP, 76% formed biofilm. 40%, 32%, 21% of the isolates were strong, moderate, and weak biofilm formers, respectively, while 43% did not form biofilms. Conclusion: The P. aeruginosa resistance to antimicrobial agents has largely challenged the control of the infection. Accordingly, a higher resistance occurred when the isolates were transferred to the patients. Less than 50% P. aeruginosa samples generated strong biofilms. Consequently, hygienic measurements are essential to inhibit P. aeruginosa transmission to hospitalized patients.

Effects of quorum sensing-interfering agents, including macrolides and furanone C-30, and an efflux pump inhibitor on nitrosative stress sensitivity in Pseudomonas aeruginosa.

Long-term administration of certain macrolides is efficacious in patients with persistent pulmonary Pseudomonas aeruginosa infection, despite how limited the clinically achievable concentrations are, being far below their MICs. An increase in the sub-MIC of macrolide exposure-dependent sensitivity to nitrosative stress is a typical characteristic of P. aeruginosa. However, a few P. aeruginosa clinical isolates do not respond to sub-MIC of macrolide treatment. Therefore, we examined the effects of sub-MIC of erythromycin (EM) on the sensitivity to nitrosative stress together with an efflux pump inhibitor (EPI) phenylalanine arginyl ß-naphthylamide (PAßN). The sensitivity to nitrosative stress increased, suggesting that the efflux pump was involved in inhibiting the sub-MIC of macrolide effect. Analysis using efflux pump-mutant P. aeruginosa revealed that MexAB-OprM, MexXY-OprM, and MexCD-OprJ are factors in reducing the sub-MIC of macrolide effect. Since macrolides interfere with quorum sensing (QS), we demonstrated that the QS-interfering agent furanone C-30 (C-30) producing greater sensitivity to nitric oxide (NO) stress than EM. The effect of C-30 was decreased by overproduction of MexAB-OprM. To investigate whether the increase in the QS-interfering agent exposure-dependent sensitivity to nitrosative stress is characteristic of P. aeruginosa clinical isolates, we examined the viability of P. aeruginosa treated with NO. Although treatment with EM could reduce cell viability, a high variability in EM effects was observed. Conversely, C-30 was highly effective at reducing cell viability. Treatment with both C-30 and PAßN was sufficiently effective against the remaining isolates. Therefore, the combination of a QS-interfering agent and an EPI could be effective in treating P. aeruginosa infections.

Saccharomyces cerevisiae ß-glucan improves the response of trained macrophages to severe P. aeruginosa infections.

OBJECTIVE P. AERUGINOSA: (PA), the major pathogen of lung cystic fibrosis (CF), polarizes macrophages into hyperinflammatory tissue damaging phenotype. The main aim of this study was to verify whether training of macrophages with ß-glucan might improve their response to P. aeruginosa infections. METHODS: To perform this task C57BL/6 mice sensitive to infections with P. aeruginosa were used. Peritoneal macrophages were trained with Saccharomyces cerevisiae ß-glucan and exposed to PA57, the strong biofilm-forming bacterial strain isolated from the patient with severe lung CF. The release of cytokines and the expression of macrophage phenotypic markers were measured. A quantitative proteomic approach was used for the characterization of proteome-wide changes in macrophages. The effect of in vivo ß-glucan-trained macrophages in the air pouch model of PA57 infection was investigated. In all experiments the effect of trained and naïve macrophages was compared. RESULTS: Trained macrophages acquired a specific phenotype with mixed pro-inflammatory and pro-resolution characteristics, however they retained anti-bacterial properties. Most importantly, transfer of trained macrophages into infected air pouches markedly ameliorated the course of infection. PA57 bacterial growth and formation of biofilm were significantly suppressed. The level of serum amyloid A (SAA), a systemic inflammation biomarker, was reduced. CONCLUSIONS: Training of murine macrophages with S. cerevisiae ß-glucan improved macrophage defense properties along with inhibition of secretion of some detrimental inflammatory agents. We suggest that training of macrophages with such ß-glucans might be a new therapeutic strategy in P. aeruginosa biofilm infections, including CF, to promote eradication of pathogens and resolution of inflammation.

Repression of resistance mechanisms of Pseudomonas aeruginosa: implications of the combination of antibiotics and phytoconstituents.

Antimicrobial resistance is a prevalent problem witnessed globally and creating an alarming situation for the treatment of infections caused by resistant pathogens. Available armaments such as antibiotics often fail to exhibit the intended action against resistant pathogens, leading to failure in the treatments that are causing mortality. New antibiotics or a new treatment approach is necessary to combat this situation. P. aeruginosa is an opportunistic drug resistant pathogen and is the sixth most common cause of nosocomial infections. P. aeruginosa due to its genome organization and other factors are exhibiting resistance against drugs. Bacterial biofilm formation, low permeability of outer membrane, the production of the beta-lactamase, and the production of several efflux systems limits the antibacterial potential of several classes of antibiotics. Combination of phytoconstituents with antibiotics is a promising strategy to combat multidrug resistant P. aeruginosa. Phytoconstituents such as flavonoids, terpenoids, alkaloids, polypeptides, phenolics, and essential oils are well known antibacterial agents. In this review, the activity of combination of the phytoconstituents and antibiotics, and their corresponding mechanism of action was discussed elaborately. The combination of antibiotics and plant-derived compounds exhibited better efficacy compared to antibiotics alone against the antibiotic resistance P. aeruginosa infections.

Pseudomonas aeruginosa producing FIM-1-metallo-ß-lactamase: emergence and cross-transmission in a long-term acute care rehabilitation hospital.

FIM-1 metallo-ß-lactamase was previously detected in sporadic Pseudomonas aeruginosa clinical isolates. Here, we report on FIM-1-positive P. aeruginosa from two patients who had shared the same ward in a long-term acute care rehabilitation hospital. Whole-genome sequencing analysis revealed close relatedness of these isolates, which belonged to an ST235 sublineage (clade 8/14) different from those previously reported. Results highlighted the occurrence of clonal diversity among FIM-positive strains and the possibility of their cross-transmission in some healthcare settings.

Pilot Evaluation of S-(3-[18F]Fluoropropyl)-D-Homocysteine and O-(2-[18F]Fluoroethyl)-D-Tyrosine as Bacteria-Specific Radiotracers for PET Imaging of Infection.

PURPOSE: There is currently no ideal radiotracer for imaging bacterial infections. Radiolabelled D-amino acids are promising candidates because they are actively incorporated into the peptidoglycan of the bacterial cell wall, a structural feature which is absent in human cells. This work describes fluorine-18 labelled analogues of D-tyrosine and D-methionine, O-(2-[18F]fluoroethyl)-D-tyrosine (D-[18F]FET) and S-(3-[18F]fluoropropyl)-D-homocysteine (D-[18F]FPHCys), and their pilot evaluation studies as potential radiotracers for imaging bacterial infection. PROCEDURES: D-[18F]FET and D-[18F]FPHCys were prepared in classical fluorination-deprotection reactions, and their uptake in Staphylococcus aureus and Pseudomonas aeruginosa was evaluated over 2 h. Heat killed bacteria were used as controls. A clinically-relevant foreign body model of S. aureus infection was established in Balb/c mice, as well as a sterile foreign body to mimic inflammation. The ex vivo biodistribution of D-[18F]FPHCys in the infected and inflamed mice was evaluated after 1 h, by dissection and gamma counting. The uptake was compared to that of [18F]FDG. RESULTS: In vitro uptake of both D-[18F]FET and D-[18F]FPHCys was specific to live bacteria. Uptake was higher in S. aureus than in P. aeruginosa for both radiotracers, and of the two, higher for D-[18F]FPHCys than D-[18F]FET. Blocking experiments with non-radioactive D-[19F]FPHCys confirmed specificity of uptake. In vivo, D-[18F]FPHCys had greater accumulation in S. aureus infection compared with sterile inflammation, which was statistically significant. As anticipated, [18F]FDG showed no significant difference in uptake between infection and inflammation. CONCLUSIONS: D-[18F]FPHCys uptake was higher in infected tissues than inflammation, and represents a fluorine-18 labelled D-AA with potential to detect a S. aureus reference strain (Xen29) in vivo. Additional studies are needed to evaluate uptake of this radiotracer in clinical isolates.

Novel antimicrobial applications of copper oxide nanoparticles after combination with tissue conditioner used in complete prostheses.

BACKGROUND: Tissue conditioners are used for treating and improving the tissues supporting complete dentures. On the other hand, recent advances in nanotechnology have revolutionized various fields of science, including dentistry. The present study aimed to investigate novel antimicrobial applications of copper oxide nanoparticle-based tissue conditioner used in complete prostheses. METHODS: The present experimental study included 126 tissue conditioner samples with different concentrations of copper oxide nanoparticles (20%, 10%, 5%, 2.5%, 1.25%, 0.625%, and 0% w/w). The samples were incubated with Enterococcus faecalis, Pseudomonas aeruginosa, and Candida albicans in 24-well plates for 24 h. Then, samples from the wells were re-incubated for 24 h, and the microorganisms were counted. RESULTS: The culture media containing E. faecalis and P. aeruginosa showed significantly different growth between different nanoparticle concentrations following 24 h (P < 0.001), showing a reduction in bacterial growth with increased nanoparticle concentration. Both bacteria did not show any growth at the 20% concentration. However, C. albicans showed significant differences in growth between different nanoparticle concentrations following 48 h (P < 0.001), showing a reduction in growth with increased nanoparticle concentration. Also, the least growth was observed at the 20% concentration. CONCLUSIONS: In conclusion, the CuO nanoparticles were prepared using a green synthesis methon in the suitable sizes. Moreover, the tissue conditioners containing CuO nanoparticles showed acceptable antimicrobial properties against E. faecalis, P. aeruginosa, and C. albicans.

Characterization of biofilm formation and multi-drug resistance among Pseudomonas aeruginosa isolated from hospital wastewater in Dhaka, Bangladesh.

Hospital wastewater has been identified as a hotspot for the emergence and transmission of multidrug-resistant (MDR) pathogens that present a serious threat to public health. Therefore, we investigated the current status of antibiotic resistance as well as the phenotypic and genotypic basis of biofilm formation in Pseudomonas aeruginosa from hospital wastewater in Dhaka, Bangladesh. The disc diffusion method and the crystal violet assay were performed to characterize antimicrobial resistance and biofilm formation, respectively. Biofilm and integron-associated genes were amplified by the polymerase chain reaction. Isolates exhibited varying degrees of resistance to different antibiotics, in which >80% of isolates showed sensitivity to meropenem, amikacin, and gentamicin. The results indicated that 93.82% of isolates were MDR and 71 out of 76 MDR isolates showed biofilm formation activities. We observed the high prevalence of biofilm-related genes, in which algD+pelF+pslD+ (82.7%) was found to be the prevalent biofilm genotypic pattern. Sixteen isolates (19.75%) possessed class 1 integron (int1) genes. However, statistical analysis revealed no significant association between biofilm formation and multidrug resistance (χ2 = 0.35, P = 0.55). Taken together, hospital wastewater in Dhaka city may act as a reservoir for MDR and biofilm-forming P. aeruginosa, and therefore, the adequate treatment of wastewater is recommended to reduce the occurrence of outbreaks.

Possible impact of revisions in disc diffusion breakpoints for aminoglycosides and piperacillin/tazobactam in the 33rd edition of CLSI M100 document on clinical reporting and use in Indian settings with low susceptibility.

PURPOSE: The study explores the impact of significant interpretative breakpoint changes for aminoglycosides and piperacillin-tazobactam in Enterobacterales and Pseudomonas aeruginosa, considering PK/PD, clinical data, and susceptibility on clinical reporting and use. PROCEDURE: Between January 2021 and June 2023, a total of 189,583 samples were processed for bacterial pathogens and antimicrobial susceptibility testing was performed using disc diffusion method/VITEK® 2 Compact system/broth microdilution. WHONET software was utilised to capture and analyse the changes in the interpretation of disc diffusion method, following updates to CLSI M100 documents in comparison to previous editions. Antimicrobial consumption data was collected and interpreted as DDD/100 bed days using AMC tool software. Here, we present data for 13,615 members of Order Enterobacterales and 1793 Pseudomonas aeruginosa isolates. FINDING: Enterobacterales exhibited a significant susceptibility drop of 14.7% for gentamicin and 21.7% for amikacin. Pseudomonas aeruginosa showed an increase in isolates with intermediate tobramycin susceptibility, from 0.6% to 29.7%, with relatively minor changes in piperacillin-tazobactam interpretation. CONCLUSION: The changes indicate a shift toward increased 'resistance' and 'intermediate susceptibility' for these antibiotics, emphasizing the need for cautious use and leveraging PK/PD knowledge for improved antibiotic utilization, patient outcomes, and antimicrobial stewardship.

Evaluation of fortimicin antibiotic combinations against MDR Pseudomonas aeruginosa and resistome analysis of a whole genome sequenced pan-drug resistant isolate.

BACKGROUND: Multidrug-resistant (MDR) P. aeruginosa is a rising public health concern, challenging the treatment of such a ubiquitous pathogen with monotherapeutic anti-pseudomonal agents. Worryingly, its genome plasticity contributes to the emergence of P. aeruginosa expressing different resistant phenotypes and is now responsible for notable epidemics within hospital settings. Considering this, we aimed to evaluate the synergistic combination of fortimicin with other traditional anti-pseudomonal agents and to analyze the resistome of pan-drug resistant (PDR) isolate. METHODS: Standard methods were used for analyzing the antimicrobial susceptibility tests. The checkerboard technique was used for the in vitro assessment of fortimicin antibiotic combinations against 51 MDR P. aeruginosa and whole genome sequencing was used to determine the resistome of PDR isolate. RESULTS: Out of 51 MDR P. aeruginosa, the highest synergistic effect was recorded for a combination of fortimicin with ß-lactam group as meropenem, ceftazidime, and aztreonam at 71%, 59% and 43%, respectively. Of note, 56.8%, 39.2%, and 37.2% of the tested MDR isolates that had synergistic effects were also resistant to meropenem, ceftazidime, and aztreonam, respectively. The highest additive effects were recorded for combining fortimicin with amikacin (69%) and cefepime (44%) against MDR P. aeruginosa. Resistome analysis of the PDR isolate reflected its association with the antibiotic resistance phenotype. It ensured the presence of a wide variety of antibiotic-resistant genes (ß-lactamases, aminoglycosides modifying enzymes, and efflux pump), rendering the isolate resistant to all clinically relevant anti-pseudomonal agents. CONCLUSION: Fortimicin in combination with classical anti-pseudomonal agents had shown promising synergistic activity against MDR P. aeruginosa. Resistome profiling of PDR P. aeruginosa enhanced the rapid identification of antibiotic resistance genes that are likely linked to the appearance of this resistant phenotype and may pave the way to tackle antimicrobial resistance issues shortly.

Harnessing the power of native biocontrol agents against wilt disease of Pigeonpea incited by Fusarium udum.

Fusarium wilt, caused by (Fusarium udum Butler), is a significant threat to pigeonpea crops worldwide, leading to substantial yield losses. Traditional approaches like fungicides and resistant cultivars are not practical due to the persistent and evolving nature of the pathogen. Therefore, native biocontrol agents are considered to be more sustainable solution, as they adapt well to local soil and climatic conditions. In this study, five isolates of F. udum infecting pigeonpea were isolated from various cultivars and characterized morphologically and molecularly. The isolate from the ICP 8858 cultivar displayed the highest virulence of 90%. Besides, 100 endophytic bacteria, 100 rhizosphere bacteria and three Trichoderma spp. were isolated and tested against F. udum isolated from ICP 8858 under in vitro conditions. Out of the 200 bacteria tested, nine showed highest inhibition, including Rb-4 (Bacillus sp.), Rb-11 (B. subtilis), Rb-14 (B. megaterium), Rb-18 (B. subtilis), Rb-19 (B. velezensis), Eb-8 (Bacillus sp.), Eb-11 (B. subtilis), Eb-13 (P. aeruginosa), and Eb-21 (P. aeruginosa). Similarly, Trichoderma spp. were identified as T. harzianum, T. asperellum and Trichoderma sp. Notably, Rb-18 (B. subtilis) and Eb-21 (P. aeruginosa) exhibited promising characteristics such as the production of hydrogen cyanide (HCN), cellulase, siderophores, ammonia and nutrient solubilization. Furthermore, treating pigeonpea seedlings with these beneficial microorganisms led to increased levels of key enzymes (POD, PPO, and PAL) associated with resistance to Fusarium wilt, compared to untreated controls. In field trials conducted for four seasons, the application of these potential biocontrol agents as seed treatments on the susceptible ICP2376 cultivar led to the lowest disease incidence. Specifically, treatments T2 (33.33) (P. aeruginosa) and T3 (35.41) (T. harzianium) exhibited the lowest disease incidence, followed by T6 (36.5) (Carbendizim), T1 (36.66) (B. subtilis), T4 (52.91) (T. asperellum) and T5 (53.33) (Trichoderma sp.). Results of this study revealed that, P. aeruginosa (Eb-21), B. subtilis (Rb-18) and T. harzianum can be used for plant growth promotion and management of Fusarium wilt of pigeonpea.

Vitamin D and vitamin K1 as novel inhibitors of biofilm in Gram-negative bacteria.

BACKGROUND: The persistent surge in antimicrobial resistance represents a global disaster. The initial attachment and maturation of microbial biofilms are intimately related to antimicrobial resistance, which in turn exacerbates the challenge of eradicating bacterial infections. Consequently, there is a pressing need for novel therapies to be employed either independently or as adjuvants to diminish bacterial virulence and pathogenicity. In this context, we propose a novel approach focusing on vitamin D and vitamin K1 as potential antibiofilm agents that target Gram-negative bacteria which are hazardous to human health. RESULTS: Out of 130 Gram-negative bacterial isolates, 117 were confirmed to be A. baumannii (21 isolates, 17.9%), K. pneumoniae (40 isolates, 34.2%) and P. aeruginosa (56 isolates, 47.9%). The majority of the isolates were obtained from blood and wound specimens (27.4% each). Most of the isolates exhibited high resistance rates to ß-lactams (60.7-100%), ciprofloxacin (62.5-100%), amikacin (53.6-76.2%) and gentamicin (65-71.4%). Approximately 93.2% of the isolates were biofilm producers, with 6.8% categorized as weak, 42.7% as moderate, and 50.4% as strong biofilm producers. The minimum inhibitory concentrations (MICs) of vitamin D and vitamin K1 were 625-1250 µg mL-1 and 2500-5000 µg mL-1, respectively, against A. baumannii (A5, A20 and A21), K. pneumoniae (K25, K27 and K28), and P. aeruginosa (P8, P16, P24 and P27) clinical isolates and standard strains A. baumannii (ATCC 19606 and ATCC 17978), K. pneumoniae (ATCC 51503) and P. aeruginosa PAO1 and PAO14. Both vitamins significantly decreased bacterial attachment and significantly eradicated mature biofilms developed by the selected standard and clinical Gram-negative isolates. The anti-biofilm effects of both supplements were confirmed by a notable decrease in the relative expression of the biofilm-encoding genes cusD, bssS and pelA in A. baumannii A5, K. pneumoniae K28 and P. aeruginosa P16, respectively. CONCLUSION: This study highlights the anti-biofilm activity of vitamins D and K1 against the tested Gram-negative strains, which emphasizes the potential of these vitamins for use as adjuvant therapies to increase the efficacy of treatment for infections caused by multidrug-resistant (MDR) strains and biofilm-forming phenotypes. However, further validation through in vivo studies is needed to confirm these promising results.

Lippia graveolens Essential Oil to Enhance the Effect of Imipenem against Axenic and Co-Cultures of Pseudomonas aeruginosa and Acinetobacter baumannii.

This research focuses on assessing the synergistic effects of Mexican oregano (Lippia graveolens) essential oil or carvacrol when combined with the antibiotic imipenem, aiming to reduce the pathogenic viability and virulence of Acinetobacter baumannii and Pseudomonas aeruginosa. The study highlighted the synergistic effect of combining L. graveolens essential oil or carvacrol with imipenem, significantly reducing the required doses for inhibiting bacterial growth. The combination treatments drastically lowered the necessary imipenem doses, highlighting a potent enhancement in efficacy against A. baumannii and P. aeruginosa. For example, the minimum inhibitory concentrations (MIC) for the essential oil/imipenem combinations were notably low, at 0.03/0.000023 mg/mL for A. baumannii and 0.0073/0.000023 mg/mL for P. aeruginosa. Similarly, the combinations significantly inhibited biofilm formation at lower concentrations than when the components were used individually, demonstrating the strategic advantage of this approach in combating antibiotic resistance. For OXA-51, imipenem showed a relatively stable interaction during 30 ns of dynamic simulation of their interaction, indicating changes (<2 nm) in ligand positioning during this period. Carvacrol exhibited similar fluctuations to imipenem, suggesting its potential inhibition efficacy, while thymol showed significant variability, particularly at >10 ns, suggesting potential instability. With IMP-1, imipenem also displayed very stable interactions during 38 ns and demonstrated notable movement and positioning changes within the active site, indicating a more dynamic interaction. In contrast, carvacrol and thymol maintained their position within the active site only ~20 and ~15 ns, respectively. These results highlight the effectiveness of combining L. graveolens essential oil and carvacrol with imipenem in tackling the difficult-to-treat pathogens A. baumannii and P. aeruginosa.

Advances in Bioreceptor Layer Engineering in Nanomaterial-based Sensing of Pseudomonas Aeruginosa and its Metabolites.

Pseudomonas aeruginosa is an opportunistic pathogen that infects wounds and burns and causes severe infections in humans. The high virulence, the emergence of antibiotic-resistant strains, and the easy transmissibility of P. aeruginosa necessitate its fast detection and control. The gold standard for detecting P. aeruginosa,  the plate culture method, though reliable, takes several days to complete. Therefore, developing accurate, rapid, and easy-to-use diagnostic tools for P. aeruginosa is highly desirable. Nanomaterial-based biosensors are at the forefront of detecting P. aeruginosa and its secondary metabolites. This review summarises the biorecognition elements, biomarkers, immobilisation strategies, and current state-of-the-art biosensors for P. aeruginosa. The review highlights the underlying principles of bioreceptor layer engineering and the design of nanomaterial-based optical, electrochemical, mass-based, and thermal biosensors. The advantages and disadvantages of these biosensors and their future point-of-care applications are also discussed.

Cu-MOF loaded chitosan based freeze-dried highly porous dressings with anti-biofilm and pro-angiogenic activities accelerated Pseudomonas aeruginosa infected wounds healing in rats.

Metal-organic frameworks (MOFs)-based therapy opens a new area for antibiotic-drug free infections treatment. In the present study, chitosan membranes (CS) loaded with two concentrations of copper-MOF 10 mg/20 ml (Cu-MOF10/CS) & 20 mg/20 ml (Cu-MOF20/CS) were prepared by a simple lyophilization procedure. FTIR spectra of Cu-MOF10/CS and Cu-MOF20/CS dressings confirmed absence of any undesirable chemical changes after loading Cu-MOF. The SEM images of the synthesized materials (CS, Cu-MOF10/CS & Cu-MOF20/CS) showed interconnected porous structures. Cytocompatibility of the materials was confirmed by fibroblasts cells culturing and the materials were hemocompatible, with blood clotting index <5 %. Cu-MOF20/CS showed comparatively higher effective antibacterial activity against the tested strains; E. coli (149.2 %), P. aeruginosa (165 %) S. aureus (117.8 %) and MRSA (142 %) as compared to Amikacin, CS and Cu-MOF10/CS membranes. Similarly, Cu-MOF20/CS dressing significantly eradicated the biofilms; P. aeruginosa (37 %) and MRSA (52 %) respectively. In full thickness infected wound rat model, on day 23, Cu-MOF10/CS and Cu-MOF20/CS promoted wound healing up to 87.7 % and 82 % respectively. H&E staining of wounded tissues treated with Cu-MOF10/CS & Cu-MOF20/CS demonstrated enhanced neovascularization and re-epithelization along-with reduced inflammation, while trichrome staining exhibited increased collagen deposition. Overall, this study declares Cu-MOFs loaded chitosan dressings a multifunctional platform for the healing of infected wounds.

The Antimicrobial Activity of Combination of Vitamin D3 and Omega-3 against Pathogenic Microorganisms.

BACKGROUND: Food supplements such as vitamin D3 and omega-3 have a significant role in activating the immune system and impacting the diversity of gut microbiota; thus, controlling the growth of invading pathogens indirectly. OBJECTIVE: This study aims to evaluate the direct antimicrobial activity of vitamin D3 and omega- 3 individually, combined together, and combined with low concentrations of gentamicin or amphotericin B against selected pathogenic microorganisms. In addition, this study hypothesizes the potential antimicrobial mechanism and recommends suitable studies to be conducted. METHOD: Minimum inhibitory concentration of different serial dilutions of vitamin D3 [0.7µg/mL-83.3µg/mL] or omega-3 [0.7mg/mL-100mg/mL] or combined [vitamin D3:1.3µg/mL-83.3µg/mL and omega-3:1.56mg/mL-100mg/mL] with/without antibiotic have been investigated on Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans using check board technique. RESULTS: The highest concentration of vitamin D3 [83.3 µg/mL] demonstrated a complete eradication of the tested microorganisms. Conversely, omega-3 had a lower effect on them. The highest concentration of combining vitamin D3 and omega-3 with/without gentamicin resulted in a complete eradication of the S. aureus, E. coli and P. aeruginosa with a 6.8 to 7 log reduction. On the other hand, C. albicans was inhibited when using vitamin D3 [83.3 µg/mL] or when this concentration is combined with 100mg/mL of omega-3. However, when these two concentrations were added to amphotericin B the log reduction dropped to 0.45 suggesting antagonistic effect. CONCLUSION: These findings suggested that, unlike omega 3, vitamin D3 possesses good antimicrobial effects against pathogenic microorganisms. The combination of the studied food supplement showed enhanced microbial inhibition at high concentration, while they had antagonistic effect when combined with amphotericin B and applied on C. albicans combined. Further studies on the exact antimicrobial mechanism are still required to understand the measured data here.

Mucus polymer concentration and in vivo adaptation converge to define the antibiotic response of Pseudomonas aeruginosa during chronic lung infection.

The airway milieu of individuals with muco-obstructive airway diseases (MADs) is defined by the accumulation of dehydrated mucus due to hyperabsorption of airway surface liquid and defective mucociliary clearance. Pathological mucus becomes progressively more viscous with age and disease severity due to the concentration and overproduction of mucin and accumulation of host-derived extracellular DNA (eDNA). Respiratory mucus of MADs provides a niche for recurrent and persistent colonization by respiratory pathogens, including Pseudomonas aeruginosa, which is responsible for the majority of morbidity and mortality in MADs. Despite high concentration inhaled antibiotic therapies and the absence of antibiotic resistance, antipseudomonal treatment failure in MADs remains a significant clinical challenge. Understanding the drivers of antibiotic tolerance is essential for developing more effective treatments that eradicate persistent infections. The complex and dynamic environment of diseased airways makes it difficult to model antibiotic efficacy in vitro. We aimed to understand how mucin and eDNA concentrations, the two dominant polymers in respiratory mucus, alter the antibiotic tolerance of P. aeruginosa. Our results demonstrate that polymer concentration and molecular weight affect P. aeruginosa survival post antibiotic challenge. Polymer-driven antibiotic tolerance was not explicitly associated with reduced antibiotic diffusion. Lastly, we established a robust and standardized in vitro model for recapitulating the ex vivo antibiotic tolerance of P. aeruginosa observed in expectorated sputum across age, underlying MAD etiology, and disease severity, which revealed the inherent variability in intrinsic antibiotic tolerance of host-evolved P. aeruginosa populations. IMPORTANCE: Antibiotic treatment failure in Pseudomonas aeruginosa chronic lung infections is associated with increased morbidity and mortality, illustrating the clinical challenge of bacterial infection control. Understanding the underlying infection environment, as well as the host and bacterial factors driving antibiotic tolerance and the ability to accurately recapitulate these factors in vitro, is crucial for improving antibiotic treatment outcomes. Here, we demonstrate that increasing concentration and molecular weight of mucin and host eDNA drive increased antibiotic tolerance to tobramycin. Through systematic testing and modeling, we identified a biologically relevant in vitro condition that recapitulates antibiotic tolerance observed in ex vivo treated sputum. Ultimately, this study revealed a dominant effect of in vivo evolved bacterial populations in defining inter-subject ex vivo antibiotic tolerance and establishes a robust and translatable in vitro model for therapeutic development.