Genomic and functional characterization of Pseudomonas aeruginosa-targeting bacteriophages isolated from hospital wastewater

Pseudomonas aeruginosa infections can be difficult to treat and new therapeutic approaches are needed. Bacteriophage therapy is a promising alternative to traditional antibiotics, but large numbers of isolated and characterized phages are lacking. We collected 23 genetically and phenotypically diverse P. aeruginosa isolates from people with cystic fibrosis (CF) and clinical infections, and characterized their genetic, phenotypic, and prophage diversity. We then used these isolates to screen and isolate 14 new P. aeruginosa-targeting phages from hospital wastewater. Phages were characterized with genome sequencing, comparative genomics, and lytic activity screening against all 23 bacterial host isolates. For four different phages, we evolved bacterial mutants that were resistant to phage infection. We then used genome sequencing and functional analysis of the resistant mutants to study their mechanisms of phage resistance as well as changes in virulence factor production and antibiotic resistance, which differed from corresponding parent bacterial isolates. Finally, we tested two phages for their ability to kill P. aeruginosa grown in biofilms in vitro, and observed that both phages reduced viable bacteria in biofilms by least one order of magnitude. One of these phages also showed activity against P. aeruginosa biofilms grown on CF airway epithelial cells. Overall, this study demonstrates how systematic genomic and phenotypic characterization can be deployed to develop bacteriophages as precision antibiotics.

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Calpurnia aurea (Aiton) Benth Extracts Reduce Quorum Sensing Controlled Virulence Factors in Pseudomonas aeruginosa

Molecules ◽

10.3390/molecules25102283 ◽

2020 ◽

Vol 25 (10) ◽

pp. 2283

Author(s):

Sekelwa Cosa ◽

Jostina R. Rakoma ◽

Abdullahi A. Yusuf ◽

Thilivhali E. Tshikalange

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Docking ◽

Quorum Sensing ◽

Virulence Factors ◽

Laser Scanning ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Promising Alternative ◽

The Individual

Pseudomonas aeruginosa is the causative agent of several life-threatening human infections. Like many other pathogens, P. aeruginosa exhibits quorum sensing (QS) controlled virulence factors such as biofilm during disease progression, complicating treatment with conventional antibiotics. Thus, impeding the pathogen’s QS circuit appears as a promising alternative strategy to overcome pseudomonas infections. In the present study, Calpurnia aurea were evaluated for their antibacterial (minimum inhibitory concentrations (MIC)), anti-quorum sensing/antivirulence (AQS), and antibiofilm potential against P. aeruginosa. AQS and antivirulence (biofilm formation, swimming, and swarming motility) activities of plant extracts were evaluated against Chromobacterium violaceum and P. aeruginosa, respectively. The in vitro AQS potential of the individual compounds were validated using in silico molecular docking. Acetone and ethanolic extracts of C. aurea showed MIC at 1.56 mg/mL. The quantitative violacein inhibition (AQS) assay showed ethyl acetate extracts as the most potent at a concentration of 1 mg/mL. GCMS analysis of C. aurea revealed 17 compounds; four (pentadecanol, dimethyl terephthalate, terephthalic acid, and methyl mannose) showed potential AQS through molecular docking against the CviR protein of C. violaceum. Biofilm of P. aeruginosa was significantly inhibited by ≥60% using 1-mg/mL extract of C. aurea. Confocal laser scanning microscopy correlated the findings of crystal violet assay with the extracts significantly altering the swimming motility. C. aurea extracts reduced the virulence of pseudomonas, albeit in a strain- and extract-specific manner, showing their suitability for the identification of lead compounds with QS inhibitory potential for the control of P. aeruginosa infections.

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Selection of Bacteriophages to Control In Vitro 24 h Old Biofilm of Pseudomonas aeruginosa Isolated from Drinking and Thermal Water

Viruses ◽

10.3390/v11080749 ◽

2019 ◽

Vol 11 (8) ◽

pp. 749 ◽

Cited By ~ 1

Author(s):

Vanessa Magin ◽

Nathalie Garrec ◽

Yves Andrés

Keyword(s):

Pseudomonas Aeruginosa ◽

Opportunistic Pathogen ◽

Planktonic Cell ◽

Test Method ◽

Public Healthcare ◽

Bacterial Strains ◽

Promising Alternative ◽

Standard Culture ◽

Stainless Steel Coupon

Pseudomonas aeruginosa is an opportunistic pathogen that causes public healthcare issues. In moist environments, this Gram-negative bacterium persists through biofilm-associated contamination on surfaces. Bacteriophages are seen as a promising alternative strategy to chemical biocides. This study evaluates the potential of nine lytic bacteriophages as biocontrol treatments against nine environmental P. aerginosa isolates. The spot test method is preliminarily used to define the host range of each virus and to identify their minimum infectious titer, depending on the strain. Based on these results, newly isolated bacteriophages 14.1, LUZ7, and B1 are selected and assessed on a planktonic cell culture of the most susceptible isolates (strains MLM, D1, ST395E, and PAO1). All liquid infection assays are achieved in a mineral minimum medium that is much more representative of real moist environments than standard culture medium. Phages 14.1 and LUZ7 eliminate up to 90% of the PAO1 and D1 bacterial strains. Hence, their effectiveness is evaluated on the 24 h old biofilms of these strains, established on a stainless steel coupon that is characteristic of materials found in thermal and industrial environments. The results of quantitative PCR viability show a maximum reduction of 1.7 equivalent Log CFU/cm2 in the coupon between treated and untreated surfaces and shed light on the importance of considering the entire virus/host/environment system for optimizing the treatment.

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Functional characterization of macrophage receptors for in vitro phagocytosis of unopsonized Pseudomonas aeruginosa.

Journal of Clinical Investigation ◽

10.1172/jci113692 ◽

1988 ◽

Vol 82 (3) ◽

pp. 872-879 ◽

Cited By ~ 51

Author(s):

D P Speert ◽

S D Wright ◽

S C Silverstein ◽

B Mah

Keyword(s):

Pseudomonas Aeruginosa ◽

Functional Characterization ◽

Macrophage Receptors

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ID2008 Aerosol-based cell delivery as an innovative treatment for lung diseases

Biomedical Research and Therapy ◽

10.15419/bmrat.v4is.251 ◽

2017 ◽

Vol 4 (S) ◽

pp. 41 ◽

Cited By ~ 1

Author(s):

Badrul Hisham Yahaya

Keyword(s):

Epithelial Cells ◽

Cell Therapy ◽

Airway Epithelial Cells ◽

Cell Delivery ◽

Airway Epithelial ◽

Promising Alternative ◽

Airway Injury ◽

Lung Injuries ◽

And Function

Endogenous repair mechanism of airway epithelial cells often fails to achieve sufficient cellular turnover and diminish with age, thus leading to permanent alterations in the structure and function of the airway epithelium. The therapeutic use of stem cells and progenitor cells represents a promising alternative for clinical strategy in treating acute and chronic lung disorders. Aerosol-based cell therapy is a novel therapeutic strategy in enhancing reparative process following both acute and chronic lung injuries. In such background, this study was aimed to determine the effect of aerosol-based cell delivery using MicroSprayer® Aerosolizer in the setting of acute lung injury (ALI) and ovalbumin-induced airway injury in the rabbit. In vitro evaluation revealed that the aerosol technique didn’t cause a significant effect on cell morphology, viability and proliferation capability over the course of cell culture period. Aerosol delivery of airway epithelial cells (AEC) and mesenchymal stem cell (MSC) resulted in uniform distribution in the distal airway and lung interstitial region. Short term assessment showed that cells delivered to the lungs via aerosol was found to be safe for transplantation with no signs of cell rejection and histopathological alterations in the liver and spleen of all treated animals. Histological evidences also demonstrated that administration of AEC and MSC via aerosolization into the respiratory airway prevented lung inflammation as well as resulted in improvement of both alveolar damage and permeability. To our knowledge, this is the first report of aerosol cell delivery via a Microsprayer® Aerosolizer device to the lungs of rabbits to treat airway injuries. Our findings provide a promising evidence that aerosol-based cell therapy may provide a basis for the development of an innovative approach for the treatment lung injuries.

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Inhaled bacteriophage therapy in a porcine model of ventilator-associated pneumonia caused by pseudomonas aeruginosa.

10.22541/au.161113708.81157558/v1 ◽

2021 ◽

Author(s):

Antoine Guillon ◽

Jeoffrey Pardessus ◽

Guillaume L’Hostis ◽

Cindy Fevre ◽

Celine Barc ◽

...

Keyword(s):

Mechanical Ventilation ◽

Pseudomonas Aeruginosa ◽

Phage Therapy ◽

Antimicrobial Efficacy ◽

Resistant Bacteria ◽

Ventilator Associated Pneumonia ◽

Bacteriophage Therapy ◽

Log Reduction

Background and Purpose. Pseudomonas aeruginosa is a main cause of ventilator-associated pneumonia (VAP) with drug-resistant bacteria. Bacteriophage therapy has experienced resurgence to compensate for the limited development of novel antibiotics. However, phage therapy is limited to a compassionate use so far, resulting from lack of adequate studies in relevant pharmacological models. We used a pig model of VAP caused by P. aeruginosa that recapitulates essential features of human disease to study the antimicrobial efficacy of nebulized-phage therapy. Experimental Approach. (i) Lysis kinetic assays were performed to evaluate in vitro phage antibacterial efficacy against P. aeruginosa and select relevant combinations of lytic phages. (ii) The efficacy of the phage combinations was investigated in vivo (murine model of P. aeruginosa lung infection). (iii) We determined the optimal conditions to ensure efficient phage delivery by aerosol during mechanical ventilation. (iv) Lung antimicrobial efficacy of inhaled-phage therapy was evaluated in pigs, which were anesthetized, mechanically ventilated and infected with P. aeruginosa. Key Results. By selecting an active phage cocktail and optimizing aerosol delivery conditions, we were able to deliver high phage concentrations in the lungs, which resulted in a rapid and marked reduction in P. aeruginosa density (1.5 Log reduction, p<0.001). No phage was detected in the sera and urines throughout the experiment. Conclusion and Implications. Our findings demonstrated: (i) the feasibility of delivering large amounts of active phages by nebulization during mechanical ventilation, (ii) rapid control of in situ infection by inhaled bacteriophage in an experimental model of VAP with high translational value.

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Single-Cell Transcriptomic Attributes and Unbiased Computational Modeling for the Prediction of Immunomodulatory Potency of Mesenchymal Stromal Cells

10.1101/2020.09.12.294850 ◽

2020 ◽

Author(s):

Pallab Pradhan ◽

Paramita Chatterjee ◽

Hazel Y. Stevens ◽

Chad Glen ◽

Camila Medrano-Trochez ◽

...

Keyword(s):

Single Cell ◽

Mesenchymal Stromal Cells ◽

Stromal Cells ◽

Computational Models ◽

Functional Characterization ◽

Symbolic Regression ◽

Phenotypic Characterization ◽

Cell Therapies

ABSTRACTMesenchymal stromal cells (MSCs) are currently being tested in numerous clinical trials as potential cell therapies for the treatment of various diseases and due to their potential immunomodulatory, pro-angiogenic, and regenerative properties. However, variabilities in tissue sources, donors, and manufacturing processes and the lack of defined critical quality attributes (CQAs) and clinically relevant mechanism of action (MoA) pose significant challenges to identify MSC cell therapy products with a predictable therapeutic outcome. This also hinders regulatory considerations and broad clinical translation of MSCs. MSC products are often administered to the patient immediately after thawing from cryopreserved vials (out-of-thaw). However, the qualifying quality-control assays are either performed before cryopreservation, or after culturing the post-thaw cells for 24-48 hours (culture-rescued), none of which represent the out-of-thaw product administered to patients. In this study, we performed a broad functional characterization of out-of-thaw and culture-rescue MSCs from bone marrow (BM-MSCs) and cord tissue (CT-MSCs) using macrophage activation and T cell proliferation-based in vitro potency assays and deep phenotypic characterization using single-cell RNA-sequencing. Using this data, we developed unbiased computational models, specifically symbolic regression (SR) and canonical correlation analysis (CCA) models to predict the immunomodulatory potency of MSCs. Overall, our results suggest that manufacturing conditions (OOT vs. CR) have a strong effect on MSC-function on MSC interactions with macrophages and T cells. Furthermore, single-cell RNA-seq analyses of out-of-thaw BM and CT-MSCs indicate a tissue of origin-dependent variability and heterogeneity in the transcriptome profile. Using symbolic regression modeling we identified specific single-cell transcriptomic attributes of MSCs that predict their immunomodulatory potency. In addition, CCA modeling predicted MSC donors with high or low immunomodulatory potency from their transcriptome profiles. Taken together, our results provide a broad framework for identifying predictive CQAs of MSCs that could ultimately help in better understanding of their MOAs and improved reproducibility and manufacturing control of MSCs.

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A novel in silico antimicrobial peptide DP7 combats MDR Pseudomonas aeruginosa and related biofilm infections

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa308 ◽

2020 ◽

Vol 75 (11) ◽

pp. 3248-3259 ◽

Cited By ~ 1

Author(s):

Qi Yin ◽

Siwen Wu ◽

Lei Wu ◽

Zhenling Wang ◽

Yandong Mu ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Peptide ◽

Genome Sequencing ◽

Mode Of Action ◽

In Silico ◽

Bacterial Colonization ◽

Outer Membrane Proteins ◽

Antibiofilm Activity ◽

Promising Alternative ◽

Biofilm Infection

Abstract Background Antimicrobial peptides are promising alternative antimicrobial agents to combat MDR. DP7, an antimicrobial peptide designed in silico, possesses broad-spectrum antimicrobial activities and immunomodulatory effects. However, the effects of DP7 against Pseudomonas aeruginosa and biofilm infection remain largely unexplored. Objectives To assess (i) the antimicrobial activity of DP7 against MDR P. aeruginosa; and (ii) the antibiofilm activity against biofilm infection. Also, to preliminarily investigate the possible antimicrobial mode of action. Methods The MICs of DP7 for 104 clinical P. aeruginosa strains (including 57 MDR strains) and the antibiofilm activity were determined. RNA-Seq, genome sequencing and cell morphology were conducted. Both acute and chronic biofilm infection mouse models were established. Two mutants, resulting from point mutations associated with LPS and biofilms, were constructed to investigate the potential mode of action. Results DP7, at 8–32 mg/L, inhibited the growth of clinical P. aeruginosa strains and, at 64 mg/L, reduced biofilm formation by 43% to 68% in vitro. In acute lung infection, 0.5 mg/kg DP7 exhibited a 70% protection rate and reduced bacterial colonization by 50% in chronic infection. DP7 mainly suppressed gene expression involving LPS and outer membrane proteins and disrupted cell wall structure. Genome sequencing of the DP7-resistant strain DP7R revealed four SNPs controlling LPS and biofilm production. gshA44 and wbpJ139 mutants displayed LPS reduction and motility deficiency, conferring the reduction of LPS and biofilm biomass of strain DP7R and indicating that LPS was a potential target of DP7. Conclusions These results demonstrate that DP7 may hold potential as an effective antimicrobial agent against MDR P. aeruginosa and related infections.

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Bacteriophage therapy against Pseudomonas aeruginosa biofilms: a review

Annals of Clinical Microbiology and Antimicrobials ◽

10.1186/s12941-020-00389-5 ◽

2020 ◽

Vol 19 (1) ◽

Author(s):

Zahra Chegini ◽

Amin Khoshbayan ◽

Majid Taati Moghadam ◽

Iman Farahani ◽

Parham Jazireian ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Phage Therapy ◽

Bacteriophage Therapy ◽

Antibiotic Resistant ◽

Combined Use ◽

Biofilm Destruction ◽

Limited Host Range ◽

The One

Abstract Multi-Drug Resistant (MDR) Pseudomonas aeruginosa is one of the most important bacterial pathogens that causes infection with a high mortality rate due to resistance to different antibiotics. This bacterium prompts extensive tissue damage with varying factors of virulence, and its biofilm production causes chronic and antibiotic-resistant infections. Therefore, due to the non-applicability of antibiotics for the destruction of P. aeruginosa biofilm, alternative approaches have been considered by researchers, and phage therapy is one of these new therapeutic solutions. Bacteriophages can be used to eradicate P. aeruginosa biofilm by destroying the extracellular matrix, increasing the permeability of antibiotics into the inner layer of biofilm, and inhibiting its formation by stopping the quorum-sensing activity. Furthermore, the combined use of bacteriophages and other compounds with anti-biofilm properties such as nanoparticles, enzymes, and natural products can be of more interest because they invade the biofilm by various mechanisms and can be more effective than the one used alone. On the other hand, the use of bacteriophages for biofilm destruction has some limitations such as limited host range, high-density biofilm, sub-populate phage resistance in biofilm, and inhibition of phage infection via quorum sensing in biofilm. Therefore, in this review, we specifically discuss the use of phage therapy for inhibition of P. aeruginosa biofilm in clinical and in vitro studies to identify different aspects of this treatment for broader use.

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