A new model of endotracheal tube biofilm identifies combinations of matrix-degrading enzymes and antimicrobials able to eradicate biofilms of pathogens that cause ventilator-associated pneumonia.

Ventilator-associated pneumonia is defined as pneumonia that develops in a patient who has been on mechanical ventilation for more than 48 hours through an endotracheal tube. It is caused by biofilm formation on the indwelling tube, which introduces pathogenic microbes such as Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans into the patient's lower airways. Currently, there is a lack of accurate in vitro models of ventilator-associated pneumonia development. This greatly limits our understanding of how the in-host environment alters pathogen physiology and the efficacy of ventilator-associated pneumonia prevention or treatment strategies. Here, we showcase a reproducible model that simulates the biofilm formation of these pathogens in a host-mimicking environment and demonstrate that the biofilm matrix produced differs from that observed in standard laboratory growth medium. In our model, pathogens are grown on endotracheal tube segments in the presence of a novel synthetic ventilated airway mucus medium that simulates the in-host environment. Matrix-degrading enzymes and cryo-scanning electron microscopy were employed to characterize the system in terms of biofilm matrix composition and structure, as compared to standard laboratory growth medium. As seen in patients, the biofilms of ventilator-associated pneumonia pathogens in our model either required very high concentrations of antimicrobials for eradication or could not be eradicated. However, combining matrix-degrading enzymes with antimicrobials greatly improved the biofilm eradication of all pathogens. Our in vitro endotracheal tube model informs on fundamental microbiology in the ventilator-associated pneumonia context and has broad applicability as a screening platform for antibiofilm measures including the use of matrix-degrading enzymes as antimicrobial adjuvants.

Screening and isolation of quorum sensing inhibitors of Pseudomonas aeruginosa from Phellodendron amurense extracts using bio-affinity chromatography.

Drug-resistant bacterial infections pose a significant challenge in the field of bacterial disease treatment. Finding new antibacterial pathways and targets to combat drug-resistant bacteria is crucial. The bacterial quorum sensing (QS) system regulates the expression of bacterial virulence factors. Inhibiting bacterial QS and reducing bacterial virulence can achieve antibacterial therapeutic effects, making QS inhibition an effective strategy to control bacterial pathogenicity. This article mainly focused on the PqsA protein in the QS system of Pseudomonas aeruginosa. An affinity chromatography medium was developed using the SpyTag/SpyCatcher heteropeptide bond system. Berberine, which can interact with the PqsA target, was screened from Phellodendron amurense by affinity chromatography. We characterized its structure, verified its inhibitory activity on P. aeruginosa, and preliminarily analyzed its mechanism using molecular docking technology. This method can also be widely applied to the immobilization of various protein targets and the effective screening of active substances.

An in-vitro model for bacteria-related catheter encrustations.

PURPOSE: About 50% of individuals with long-term indwelling catheters are affected by catheter encrustations and bladder stone formation. Therefore, prophylaxis of catheter encrustations is important. Currently, however, neither an established prophylaxis nor a standardized in-vitro model to test different measures exist. We have therefore developed and qualitatively evaluated an in-vitro model of catheter encrustation. METHODS: Size 14 French suprapubic catheters were incubated under sterile conditions at 37 degrees Celsius in five different media: (1) sterile artificial urine (n = 16), (2) artificial urine with E. coli (n = 8), (3) with Pseudomonas aeruginosa (n = 8), (4) with Proteus mirabilis (n = 8), and (5) with a mix of these three strains (n = 8). Catheter balloons were inflated either a glycerine or a bactericidal solution. After 6 weeks, the catheters were removed from the solution, dried, and weighed, and a photometric determination of the retrieved encrustations was performed. RESULTS: Most frequently and pronounced encrustations were detected in the Pseudomonas group. The median weight of these encrustations (50% struvite and brushite) was 84.4 mg (47.7 mg / 127.3 mg). Even on catheters stored in sterile urine, encrustations (69.2% struvite) were found. Bacterial growth was not affected by the medium used for catheter blockage. CONCLUSION: Although in-vitro models appear to be limited because they lack "the human factor", they are valuable for systematically assessing physico-chemical factors affecting encrustations. Therefore, our model, being reliable and cost-effective, may foster further research despite its limitations.

Antibiotic Nanoparticles-Loaded Wound Dressings Against Pseudomonas aeruginosa's Skin Infection: A Systematic Review.

Pseudomonas aeruginosa (P. aeruginosa) is a common nosocomial pathogen that can cause severe infections in critically ill patients. Due to its resistance to multiple drugs, it is challenging to treat, which can result in serious illness and death. Conventional treatments for infected wounds often involve the topical or systemic application of antibiotics, which can lead to systemic toxicity and the development of drug resistance. The combination of wound dressings that promote wound healing with nanoparticles (NPs) represents a revolutionary strategy for optimizing the safety and efficacy of antibiotics. This review assesses a systematic search to identify the latest approaches where the evaluation of wound dressings loaded with antibiotic NPs is conducted. The properties of NPs, the features of wound dressings, the antimicrobial activity and biocompatibility of the different strategies are analyzed. The results indicate that most research in this field is focused on dressings loaded with silver NPs (57.1%) or other inorganic materials (22.4%). Wound dressings loaded with polymeric NPs and carbon-based NPs represent 14.3% and 6.1% of the evaluated studies, respectively. Nevertheless, there are no clinical trials that have evaluated the efficacy of NPs-loaded wound dressings in patients. Further research is required to ensure the safety of these treatments and to translate the findings from the bench to the bedside.

Small RNA GadY in Escherichia coli enhances conjugation system of IncP-1 by targeting SdiA.

Plasmid-mediated conjugation is a common mechanism for most bacteria to transfer antibiotic resistance genes (ARGs). The conjugative transfer of ARGs is emerging as a major threat to human beings. Although several transfer-related factors are known to regulate this process, small RNAs (sRNAs)-based regulatory roles remain to be clarified. Here, the Hfq-binding sRNA GadY in donor strain Escherichia coli (E. coli) SM10λπ was identified as a new regulator for bacterial conjugation. Two conjugation models established in our previous studies were used, which SM10λπ carrying a chromosomally integrated IncP-1α plasmid RP4 and a mobilizable plasmid pUCP24T served as donor cells, and P. aeruginosa PAO1 or E. coli EC600 as the recipients. GadY was found to promote SM10λπ-PAO1 conjugation by base-pairing with its target mRNA SdiA, an orphan LuxR-type receptor that responds to exogenous N-acylated homoserine lactones (AHLs). However, SM10λπ-EC600 conjugation was not affected due to EC600 lacking AHLs synthase. It indicates that the effects of GadY on conjugation depended on AHLs-SdiA signalling. Further study found GadY bound SdiA to negatively regulate the global RP4 repressors KorA and KorB. When under ciprofloxacin or levofloxacin treatment, GadY expression in donor strain was enhanced, and it positively regulated quinolone-induced SM10λπ-PAO1 conjugation. Thus, our study provides a novel role for sRNA GadY in regulating plasmid-mediated conjugation, which helps us better understand bacterial conjugation to counter antibiotic resistance.

Low-dose zinc oxide nanoparticles trigger the growth and biofilm formation of Pseudomonas aeruginosa: a hormetic response.

INTRODUCTION: Hormesis describes an inverse dose-response relationship, whereby a high dose of a toxic compound is inhibitory, and a low dose is stimulatory. This study explores the hormetic response of low concentrations of zinc oxide nanoparticles (ZnO NPs) toward Pseudomonas aeruginosa. METHOD: Samples of P. aeruginosa, i.e. the reference strain, ATCC 27,853, together with six strains recovered from patients with cystic fibrosis, were exposed to ten decreasing ZnO NPs doses (0.78-400 µg/mL). The ZnO NPs were manufactured from Peganum harmala using a chemical green synthesis approach, and their properties were verified utilizing X-ray diffraction and scanning electron microscopy. A microtiter plate technique was employed to investigate the impact of ZnO NPs on the growth, biofilm formation and metabolic activity of P. aeruginosa. Real-time polymerase chain reactions were performed to determine the effect of ZnO NPs on the expression of seven biofilm-encoding genes. RESULT: The ZnO NPs demonstrated concentration-dependent bactericidal and antibiofilm efficiency at concentrations of 100-400 µg/mL. However, growth was significantly stimulated at ZnO NPs concentration of 25 µg/mL (ATCC 27853, Pa 3 and Pa 4) and at 12.5 µg/mL and 6.25 µg/mL (ATCC 27853, Pa 2, Pa 4 and Pa 5). No significant positive growth was detected at dilutions < 6.25 µg/mL. similarly, biofilm formation was stimulated at concentration of 12.5 µg/mL (ATCC 27853 and Pa 1) and at 6.25 µg/mL (Pa 4). At concentration of 12.5 µg/mL, ZnO NPs upregulated the expression of LasB ( ATCC 27853, Pa 1 and Pa 4) and LasR and LasI (ATCC 27853 and Pa 1) as well as RhII expression (ATCC 27853, Pa 2 and Pa 4). CONCLUSION: When exposed to low ZnO NPs concentrations, P. aeruginosa behaves in a hormetic manner, undergoing positive growth and biofilm formation. These results highlight the importance of understanding the response of P. aeruginosa following exposure to low ZnO NPs concentrations.

Phages produce persisters.

Arguably, the greatest threat to bacteria is phages. It is often assumed that those bacteria that escape phage infection have mutated or utilized phage-defence systems; however, another possibility is that a subpopulation forms the dormant persister state in a manner similar to that demonstrated for bacterial cells undergoing nutritive, oxidative, and antibiotic stress. Persister cells do not undergo mutation and survive lethal conditions by ceasing growth transiently. Slower growth and dormancy play a key physiological role as they allow host phage defence systems more time to clear the phage infection. Here, we investigated how bacteria survive lytic phage infection by isolating surviving cells from the plaques of T2, T4, and lambda (cI mutant) virulent phages and sequencing their genomes. We found that bacteria in plaques can escape phage attack both by mutation (i.e. become resistant) and without mutation (i.e. become persistent). Specifically, whereas T4-resistant and lambda-resistant bacteria with over a 100,000-fold less sensitivity were isolated from plaques with obvious genetic mutations (e.g. causing mucoidy), cells were also found after T2 infection that undergo no significant mutation, retain wild-type phage sensitivity, and survive lethal doses of antibiotics. Corroborating this, adding T2 phage to persister cells resulted in 137,000-fold more survival compared to that of addition to exponentially growing cells. Furthermore, our results seem general in that phage treatments with Klebsiella pneumonia and Pseudomonas aeruginosa also generated persister cells. Hence, along with resistant strains, bacteria also form persister cells during phage infection.

Exploring the Effects of Lignin Nanoparticles in Different Zebrafish Inflammatory Models.

Purpose: Lignin is the most abundant source of aromatic biopolymers and has gained interest in industrial and biomedical applications due to the reported biocompatibility and defense provided against bacterial and fungal pathogens, besides antioxidant and UV-blocking properties. Especially in the form of nanoparticles (NPs), lignin may display also antioxidant and anti-inflammatory activities. Methods: To evaluate these characteristics, sonochemically nano-formulated pristine lignin (LigNPs) and enzymatically-phenolated one (PheLigNPs) were used to expose zebrafish embryos, without chorion, at different concentrations. Furthermore, two different zebrafish inflammation models were generated, by injecting Pseudomonas aeruginosa lipopolysaccharide (LPS) and by provoking a wound injury in the embryo caudal fin. The inflammatory process was investigated in both models by qPCR, analyzing the level of genes as il8, il6, il1ß, tnfα, nfkbiaa, nfk2, and ccl34a.4, and by the evaluation of neutrophils recruitment, taking advantage of the Sudan Black staining, in the presence or not of LigNPs and PheLigNPs. Finally, the Wnt/ß-catenin pathway, related to tissue regeneration, was investigated at the molecular level in embryos wounded and exposed to NPs. Results: The data obtained demonstrated that the lignin-based NPs showed the capacity to induce a positive response during an inflammatory event, increasing the recruitment of cytokines to accelerate their chemotactic function. Moreover, the LigNPs and PheLigNPs have a role in the resolution of wounds, favoring the regeneration process. Conclusion: In this paper, we used zebrafish embryos within 5 days post fertilization (hpf). Despite being an early-stage exemplary, the zebrafish embryos have proven their potential as predicting models. Further long-term experiments in adults will be needed to explore completely the biomedical capabilities of lignin NPs. The results underlined the safety of both NPs tested paved the way for further evaluations to exploit the anti-inflammatory and pro-healing properties of the lignin nanoparticles examined.

Capsid structure of bacteriophage ΦKZ provides insights into assembly and stabilization of jumbo phages.

Jumbo phages are a group of tailed bacteriophages with large genomes and capsids. As a prototype of jumbo phage, ΦKZ infects Pseudomonas aeruginosa, a multi-drug-resistant (MDR) opportunistic pathogen leading to acute or chronic infection in immunocompromised individuals. It holds potential to be used as an antimicrobial agent and as a model for uncovering basic phage biology. Although previous low-resolution structural studies have indicated that jumbo phages may have more complicated capsid structures than smaller phages such as HK97, the detailed structures and the assembly mechanism of their capsids remain largely unknown. Here, we report a 3.5-Å-resolution cryo-EM structure of the ΦKZ capsid. The structure unveiled ten minor capsid proteins, with some decorating the outer surface of the capsid and the others forming a complex network attached to the capsid's inner surface. This network seems to play roles in driving capsid assembly and capsid stabilization. Similar mechanisms of capsid assembly and stabilization are probably employed by many other jumbo viruses.

From Proteome to Potential Drugs: Integration of Subtractive Proteomics and Ensemble Docking for Drug Repurposing against Pseudomonas aeruginosa RND Superfamily Proteins.

Pseudomonas aeruginosa (P. aeruginosa) poses a significant threat as a nosocomial pathogen due to its robust resistance mechanisms and virulence factors. This study integrates subtractive proteomics and ensemble docking to identify and characterize essential proteins in P. aeruginosa, aiming to discover therapeutic targets and repurpose commercial existing drugs. Using subtractive proteomics, we refined the dataset to discard redundant proteins and minimize potential cross-interactions with human proteins and the microbiome proteins. We identified 12 key proteins, including a histidine kinase and members of the RND efflux pump family, known for their roles in antibiotic resistance, virulence, and antigenicity. Predictive modeling of the three-dimensional structures of these RND proteins and subsequent molecular ensemble-docking simulations led to the identification of MK-3207, R-428, and Suramin as promising inhibitor candidates. These compounds demonstrated high binding affinities and effective inhibition across multiple metrics. Further refinement using non-covalent interaction index methods provided deeper insights into the electronic effects in protein-ligand interactions, with Suramin exhibiting superior binding energies, suggesting its broad-spectrum inhibitory potential. Our findings confirm the critical role of RND efflux pumps in antibiotic resistance and suggest that MK-3207, R-428, and Suramin could be effectively repurposed to target these proteins. This approach highlights the potential of drug repurposing as a viable strategy to combat P. aeruginosa infections.

Biosynthesis and Characterization of Aeonium arboreum-Derived Silver Nanoparticles: Antimicrobial Activity, Biofilm Inhibition, Antihemolytic Activity, and In Silico Studies.

Environmentally friendly biosynthesis of silver nanoparticles (AgNPs) from Aeonium arboreum (L.) Webb & Berthel is reported for the first time. The synthesized AgNPs were characterized using UV-Vis, FTIR, TEM, Zeta potential, and XRD analysis, revealing high stability (-29.1 mV), spherical shape, and an average size of 100 nm. The antimicrobial activity levels of both A. arboreum extract and biosynthesized AgNPs were evaluated against five uropathogens (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans). Both the extract and the AgNPs exhibited significant efficacy, particularly against E. coli, with inhibition zones of 27 mm and 30 mm, respectively. LC-MS analysis tentatively identified 11 secondary metabolites in the extract, including quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, myricetin 3-glucoside, and daphneresinol. In silico docking studies revealed promising binding affinities of these metabolites in relation to key enzymes involved in bacterial folate synthesis (dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS)) and DNA replication (DNA gyrase). These findings demonstrate the potential of A. arboreum-based AgNPs and their associated metabolites as a novel therapeutic approach for combating urinary tract infections. Their antimicrobial, antihemolytic, and antibiofilm properties warrant further investigation.

An unnatural amino acid dependent, conditional Pseudomonas vaccine prevents bacterial infection.

Live vaccines are ideal for inducing immunity but suffer from the need to attenuate their pathogenicity or replication to preclude the possibility of escape. Unnatural amino acids (UAAs) provide a strategy to engineer stringent auxotrophies, yielding conditionally replication incompetent live bacteria with excellent safety profiles. Here, we engineer Pseudomonas aeruginosa to maintain auxotrophy for the UAA p-benzoyl-L-phenylalanine (BzF) through its incorporation into the essential protein DnaN. In vivo evolution using an Escherichia coli-based two-hybrid selection system enabled engineering of a mutant DnaN homodimeric interface completely dependent on a BzF-specific interaction. This engineered strain, Pa Vaccine, exhibits undetectable escape frequency (<10-11) and shows excellent safety in naïve mice. Animals vaccinated via intranasal or intraperitoneal routes are protected from lethal challenge with pathogenic P. aeruginosa PA14. These results establish UAA-auxotrophic bacteria as promising candidates for bacterial vaccine therapy and outline a platform for expanding this technology to diverse bacterial pathogens.

Resistance of Pseudomonas aeruginosa and Staphylococcus aureus to the airway epithelium oxidative response assessed by a cell-free in vitro assay.

The antibacterial oxidative response, which relies on the production of hydrogen peroxide (H2O2) and hypothiocyanite (OSCN-), is a major line of defense protecting the human airway epithelium (HAE) from lesions when infected. The in vitro studies of the oxidative responses are performed mainly by one-shot H2O2 exposure that does not recapitulate the complex H2O2/LPO/SCN- system releasing the reactive oxygen species in airway secretions. A cell-free in vitro assay mimicking this system has been described but was not fully characterized. Here, we comprehensively characterized the hourly H2O2/OSCN- concentrations produced within this in vitro assay and assessed the resistance of Pseudomonas aeruginosa and Staphylococcus aureus clinical strains to the HAE oxidative response. We found that H2O2/OSCN- were steadily produced from 7h and up to 25h, but OSCN- was detoxified in 15 minutes by bacteria upon exposure. Preliminary tests on PA14 showed survival rates at 1-hour post-exposure (hpe) to H2O2 of roughly 50% for 105 and 107 colony-forming unit (CFU)/mL inocula, while 102 and 104 CFU/mL inocula were cleared after one hpe. Thirteen clinical strains were then exposed, highlighting that conversely to P. aeruginosa, S. aureus showed resistance to oxidative stress independently of its antibiotic resistance phenotype. Our results demonstrated how this in vitro assay can be helpful in assessing whether pathogens can resist the antibacterial oxidative HAE response. We anticipate these findings as a starting point for more sophisticated in vitro models that could serve as high-throughput screening for molecules targeting the bacterial antioxidant response.

A new protocol for multispecies bacterial infections in zebrafish and their monitoring through automated image analysis.

The zebrafish Danio rerio has become a popular model host to explore disease pathology caused by infectious agents. A main advantage is its transparency at an early age, which enables live imaging of infection dynamics. While multispecies infections are common in patients, the zebrafish model is rarely used to study them, although the model would be ideal for investigating pathogen-pathogen and pathogen-host interactions. This may be due to the absence of an established multispecies infection protocol for a defined organ and the lack of suitable image analysis pipelines for automated image processing. To address these issues, we developed a protocol for establishing and tracking single and multispecies bacterial infections in the inner ear structure (otic vesicle) of the zebrafish by imaging. Subsequently, we generated an image analysis pipeline that involved deep learning for the automated segmentation of the otic vesicle, and scripts for quantifying pathogen frequencies through fluorescence intensity measures. We used Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae, three of the difficult-to-treat ESKAPE pathogens, to show that our infection protocol and image analysis pipeline work both for single pathogens and pairwise pathogen combinations. Thus, our protocols provide a comprehensive toolbox for studying single and multispecies infections in real-time in zebrafish.

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.

Outcomes of inadequate empiric therapy and timing of newer antibacterial therapy in hospitalized adults with culture-positive Enterobacterales and Pseudomonas aeruginosa: a multicenter analysis.

BACKGROUND: Infections caused by multi-drug resistant Gram-negative pathogens are associated with worse clinical outcomes in critically ill patients. We evaluated hospital outcomes based on adequacy of overall and newer antibacterial therapy for Enterobacterales (ENT) and Pseudomonas aeruginosa (PsA) in US patients. METHODS: Hospitalized adults ≥ 18 years old with facility-reported antibiotic susceptibility from 2018-2022 across 161 facilities in the BD Insights Research Database were identified as ENT- or PsA-positive. Generalized linear mixed models were used to evaluate the impact of inadequate empiric therapy (IET) and time to initiate newer antibacterials (ceftazidime-avibactam; ceftolozane-tazobactam; cefiderocol; meropenem-vaborbactam; eravacycline; and imipenem-cilcastatin-relebactam) on hospital mortality and post-culture length of stay (LOS). RESULTS: Among 229,320 ENT and 36,027 PsA susceptibility results, 1.7% and 16.8% were carbapenem non-susceptible (carb-NS), respectively. Median time to first susceptibility result was longer for carb-NS vs. carb susceptible in ENT (64 h vs. 48 h) and PsA (67 h vs. 60 h). For ENT, IET was associated with significantly higher mortality (odds ratio [OR],1.29 [95% CI, 1.16-1.43, P < 0.0001]) and longer hospital LOS (14.8 vs. 13.3, P < 0.0001). Delayed start to newer antibacterial therapy was associated with significantly greater hospital mortality for ENT (P = 0.0182) and PsA (P = 0.0249) and significantly longer post-culture LOS for ENT (P < 0.0001) and PsA (P < 0.0001). CONCLUSIONS: Overall, IET and delayed use of newer antibacterials are associated with significantly worse hospital outcomes. More rapid identification of high-risk patients can facilitate adequate therapy and timely use of newer antibacterials developed for resistant Gram-negative pathogens.

Evaluation of the antibacterial and antifungal properties of oleuropein, olea Europea leaf extract, and thymus vulgaris oil.

BACKGROUND: Although synthetic preservatives and antioxidants may have high antimicrobial and antioxidant activity, they are usually associated with adverse effects on human health. Currently, there is a growing interest in natural antimicrobial and antioxidant agents. This study aimed to evaluate the antimicrobial activity of two medicinal plant extracts and one active compound. Olive leaf extracts (0.2, 0.3, and 0.4% w/v), oleuropein (0.2, 0.4, and 0.6% w/v), thyme oil (0.1%), and oleuropein in combination with thyme oil (0.4% w/v and 0.1% v/v) were used against three bacterial strains (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) and two fungal strains (Candida albicans and Aspergillus niger). RESULTS: The use of oleuropein resulted in complete antimicrobial activity against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. In this context, a reduction of 7 logs was achieved during the storage period (4 weeks). Oleuropein showed no fungal activity at low concentrations (0.2%), but Aspergillus niger was reduced by 2.35 logs at higher concentrations (0.6% w/v). Similar antibacterial and antifungal properties were observed for the olive leaf extracts. Oleuropein at a concentration of 0.4 w/v and a mixture of oleuropein and thyme at concentrations of 0.4 and 0.1 (v/v) showed strong antimicrobial activity against the studied microorganisms. CONCLUSION: Olive leaf extract, thyme oil, and oleuropein have strong antibacterial and weak antifungal properties. There was a good synergistic effect between oleuropein and thymol.

Convergent evolution in toxin detection and resistance provides evidence for conserved bacterial-fungal interactions.

Microbes rarely exist in isolation and instead form complex polymicrobial communities. As a result, microbes have developed intricate offensive and defensive strategies that enhance their fitness in these complex communities. Thus, identifying and understanding the molecular mechanisms controlling polymicrobial interactions is critical for understanding the function of microbial communities. In this study, we show that the gram-negative opportunistic human pathogen Pseudomonas aeruginosa, which frequently causes infection alongside a plethora of other microbes including fungi, encodes a genetic network which can detect and defend against gliotoxin, a potent, disulfide-containing antimicrobial produced by the ubiquitous filamentous fungus Aspergillus fumigatus. We show that gliotoxin exposure disrupts P. aeruginosa zinc homeostasis, leading to transcriptional activation of a gene encoding a previously uncharacterized dithiol oxidase (herein named as DnoP), which detoxifies gliotoxin and structurally related toxins. Despite sharing little homology to the A. fumigatus gliotoxin resistance protein (GliT), the enzymatic mechanism of DnoP from P. aeruginosa appears to be identical that used by A. fumigatus. Thus, DnoP and its transcriptional induction by low zinc represent a rare example of both convergent evolution of toxin defense and environmental cue sensing across kingdoms. Collectively, these data provide compelling evidence that P. aeruginosa has evolved to survive exposure to an A. fumigatus disulfide-containing toxin in the natural environment.

Pseudomonas aeruginosa mucinous phenotypes and algUmucABD operon mutant characteristics obtained from inpatients with bronchiectasis and their correlation with acute aggravation.

Objective: Although the mechanism is unclear, Pseudomonas aeruginosa (PA) infection directly affects the frequency of acute exacerbations in patients with bronchiectasis. The aims of this article are to analyze the genetic mutation characteristics of the algUmucABD operon in PA, isolated from hospitalized patients with bronchiectasis, and to explore independent risk factors for frequent acute exacerbations of bronchiectasis. Methods: Based on the number of acute exacerbations that occurred in the past year, these patients with bronchiectasis were divided into those with frequent acute exacerbations (Group A) and those with non-frequent acute exacerbations (Group B). We identified the distribution of mucoid phenotypes (MPs) and alginate morphotypes (AMs) in PA, and classified them into I-IV categories based on their different AMs; otherwise, the gene mutation types (GMTs) of the algUmucABD operon were tested. Subsequently, the relationship between GMT, MP, and AM and the independent risk factors for frequent acute exacerbations in patients with bronchiectasis were explored. Results: A total of 93 patients and 75 PA strains, from January 2019 to August 2023, were included in this study. The MP and AM distributions of PA were as follows: 64 strains (85.33%) of mucoid (the AMs were 38 strains of type I, 3 strains of type II, and 23 strains of type IV) and 11 strains of non-mucoid (the AM was type III only). Mucoid PA with algU, mucA, mucB, and mucD mutations accounted for 19.61%, 74.51%, 31.37%, and 50.98%, respectively. GMT was divided into the following: mucA mutations only, mucA combined with other gene mutations, other gene mutations without mucA mutations, and without gene mutations. In 91.7% of PA with type I of AM, only mucA mutations occurred, and in both separate MP and AM, the GMT differences were statistically significant. Lastly, the number of lung lobes with bronchiectasis and the number of PA with mucA mutations only were the independent risk factors for frequent acute exacerbations. Conclusion: The mucA mutation was primarily responsible for the mucoid of MP and type I of AM in PA, and it was also an independent risk factor for frequent exacerbations of bronchiectasis.