scholarly journals Serial Passage of Cryptococcus neoformans in Galleria mellonella Results in Increased Capsule and Intracellular Replication in Hemocytes, but Not Increased Resistance to Hydrogen Peroxide

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 732 ◽  
Author(s):  
Muhammad Fariz Ali ◽  
Stephen M. Tansie ◽  
John R. Shahan ◽  
Rebecca L. Seipelt-Thiemann ◽  
Erin E. McClelland
Keyword(s):  
Hydrogen Peroxide ◽  
Cryptococcus Neoformans ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Histopathological Examination ◽  
Serial Passage ◽  
Infection Model ◽  
Hydrogen Peroxide Production ◽  
Species Response

To gain insight into how pathogens adapt to new hosts, Cryptococcus neoformans (H99W) was serially passaged in Galleria mellonella. The phenotypic characteristics of the passaged strain (P15) and H99W were evaluated. P15 grew faster in hemolymph than H99W, in vitro and in vivo, suggesting that adaptation had occurred. However, P15 was more susceptible to hydrogen peroxide in vitro, killed fewer mouse macrophages, and had less fungal burden in human ex vivo macrophages than H99W. Analysis of gene expression changes during Galleria infection showed only a few different genes involved in the reactive oxygen species response. As P15 sheds more GXM than H99W, P15 may have adapted by downregulating hemocyte hydrogen peroxide production, possibly through increased capsular glucuronoxylomannan (GXM) shedding. Hemocytes infected with P15 produced less hydrogen peroxide, and hydrogen peroxide production in response to GXM-shedding mutants was correlated with shed GXM. Histopathological examination of infected larvae showed increased numbers and sizes of immune nodules for P15 compared to H99W, suggesting an enhanced, but functionally defective, response to P15. These results could explain why this infection model does not always correlate with murine models. Overall, C. neoformans’ serial passage in G. mellonella resulted in a better understanding of how this yeast evolves under selection.

In Vitro, In Silico and Ex Vivo Studies of Dihydroartemisinin Derivatives as Antitubercular Agents

2019 ◽  
Vol 19 (8) ◽  
pp. 633-644 ◽  
Author(s):  
Komal Kalani ◽  
Sarfaraz Alam ◽  
Vinita Chaturvedi ◽  
Shyam Singh ◽  
Feroz Khan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
In Silico ◽  
Ex Vivo ◽  
Virulent Strain ◽  
Infection Model ◽  
Mouse Bone Marrow ◽  
Significant Activity ◽  
Macrophage Infection ◽  
In Silico Studies

Introduction: As a part of our drug discovery program for anti-tubercular agents, dihydroartemisinin (DHA-1) was screened against Mtb H37Rv, which showed moderate anti-tubercular activity (>25.0 µg/mL). These results prompted us to carry out the chemical transformation of DHA-1 into various derivatives and study their antitubercular potential. Materials and Methods: DHA-1 was semi-synthetically converted into four new acyl derivatives (DHA-1A – DHA-1D) and in-vitro evaluated for their anti-tubercular potential against Mycobacterium tuberculosis H37Rv virulent strain. The derivatives, DHA-1C (12-O-(4-nitro) benzoyl; MIC 12.5 µg/mL) and DHA-1D (12-O-chloro acetyl; MIC 3.12µg/mL) showed significant activity against the pathogen. Results: In silico studies of the most active derivative (DHA-1D) showed interaction with ARG448 inhibiting the mycobacterium enzymes. Additionally, it showed no cytotoxicity towards the Vero C1008 cells and Mouse bone marrow derived macrophages. Conclusion: DHA-1D killed 62% intracellular M. tuberculosis in Mouse bone marrow macrophage infection model. To the best of our knowledge, this is the first-ever report on the antitubercular potential of dihydroartemisinin and its derivatives. Since dihydroartemisinin is widely used as an antimalarial drug; these results may be of great help in anti-tubercular drug development from a very common, inexpensive, and non-toxic natural product.

scholarly journals In Silico Predicted Antifungal Peptides: In Vitro and In Vivo Anti-Candida Activity

2021 ◽  
Vol 7 (6) ◽  
pp. 439
Author(s):  
Tecla Ciociola ◽  
Walter Magliani ◽  
Tiziano De Simone ◽  
Thelma A. Pertinhez ◽  
Stefania Conti ◽  
...  
Keyword(s):  
In Silico ◽  
Mammalian Cells ◽  
Galleria Mellonella ◽  
Ex Vivo ◽  
Consensus Sequence ◽  
In Silico Analysis ◽  
Significant Activity ◽  
Synthetic Antibody

It has been previously demonstrated that synthetic antibody-derived peptides could exert a significant activity in vitro, ex vivo, and/or in vivo against microorganisms and viruses, as well as immunomodulatory effects through the activation of immune cells. Based on the sequence of previously described antibody-derived peptides with recognized antifungal activity, an in silico analysis was conducted to identify novel antifungal candidates. The present study analyzed the candidacidal and structural properties of in silico designed peptides (ISDPs) derived by amino acid substitutions of the parent peptide KKVTMTCSAS. ISDPs proved to be more active in vitro than the parent peptide and all proved to be therapeutic in Galleria mellonella candidal infection, without showing toxic effects on mammalian cells. ISDPs were studied by circular dichroism spectroscopy, demonstrating different structural organization. These results allowed to validate a consensus sequence for the parent peptide KKVTMTCSAS that may be useful in the development of novel antimicrobial molecules.

scholarly journals Designing P. aeruginosa synthetic phages with reduced genomes

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diana P. Pires ◽  
Rodrigo Monteiro ◽  
Dalila Mil-Homens ◽  
Arsénio Fialho ◽  
Timothy K. Lu ◽  
...  
Keyword(s):  
Galleria Mellonella ◽  
Antibacterial Properties ◽  
Genetically Engineered ◽  
In Vivo Studies ◽  
Infection Model ◽  
Promising Therapeutic Approach ◽  
Genes Encoding ◽  
First Time

AbstractIn the era where antibiotic resistance is considered one of the major worldwide concerns, bacteriophages have emerged as a promising therapeutic approach to deal with this problem. Genetically engineered bacteriophages can enable enhanced anti-bacterial functionalities, but require cloning additional genes into the phage genomes, which might be challenging due to the DNA encapsulation capacity of a phage. To tackle this issue, we designed and assembled for the first time synthetic phages with smaller genomes by knocking out up to 48% of the genes encoding hypothetical proteins from the genome of the newly isolated Pseudomonas aeruginosa phage vB_PaeP_PE3. The antibacterial efficacy of the wild-type and the synthetic phages was assessed in vitro as well as in vivo using a Galleria mellonella infection model. Overall, both in vitro and in vivo studies revealed that the knock-outs made in phage genome do not impair the antibacterial properties of the synthetic phages, indicating that this could be a good strategy to clear space from phage genomes in order to enable the introduction of other genes of interest that can potentiate the future treatment of P. aeruginosa infections.

Emergence of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae in vivo

2019 ◽  
Vol 74 (11) ◽  
pp. 3211-3216 ◽  
Author(s):  
Stephan Göttig ◽  
Denia Frank ◽  
Eleonora Mungo ◽  
Anika Nolte ◽  
Michael Hogardt ◽  
...  
Keyword(s):  
Combination Therapy ◽  
Klebsiella Pneumoniae ◽  
Selection Pressure ◽  
Galleria Mellonella ◽  
Phenotypic Characterization ◽  
Infection Model ◽  
Development Of Resistance ◽  
Time Kill

Abstract Objectives The β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is active against KPC-producing Enterobacterales. Herein, we present molecular and phenotypic characterization of ceftazidime/avibactam resistance in KPC-3-producing Klebsiella pneumoniae that emerged in vivo and in vitro. Methods Sequence analysis of blaKPC-3 was performed from clinical and in vitro-generated ceftazidime/avibactam-resistant K. pneumoniae isolates. Time–kill kinetics and the Galleria mellonella infection model were applied to evaluate the activity of ceftazidime/avibactam and imipenem alone and in combination. Results The ceftazidime/avibactam-resistant clinical K. pneumoniae isolate revealed the amino acid change D179Y in KPC-3. Sixteen novel mutational changes in KPC-3 among in vitro-selected ceftazidime/avibactam-resistant isolates were described. Time–kill kinetics showed the emergence of a resistant subpopulation under selection pressure with either imipenem or ceftazidime/avibactam. However, combined selection pressure with imipenem plus ceftazidime/avibactam prevented the development of resistance and resulted in bactericidal activity. Concordantly, the G. mellonella infection model revealed that monotherapy with ceftazidime/avibactam is prone to select for resistance in vivo and that combination therapy with imipenem results in significantly better survival. Conclusions Ceftazidime/avibactam is a valuable antibiotic against MDR and carbapenem-resistant Enterobacterales. Based on time–kill kinetics as well as an in vivo infection model we postulate a combination therapy of ceftazidime/avibactam and imipenem as a strategy to prevent the development of ceftazidime/avibactam resistance in KPC-producing Enterobacterales in vivo.

scholarly journals Activity of Telavancin against Staphylococcus aureus Isolates, Including Those with Decreased Susceptibility to Ceftaroline, from Cystic Fibrosis Patients

2018 ◽  
Vol 62 (9) ◽  
Author(s):  
Melanie Roch ◽  
Maria Celeste Varela ◽  
Agustina Taglialegna ◽  
Warren E. Rose ◽  
Adriana E. Rosato
Keyword(s):  
Cystic Fibrosis ◽  
Staphylococcus Aureus ◽  
Galleria Mellonella ◽  
Therapeutic Option ◽  
The United States ◽  
Infection Model ◽  
Content Type ◽  
Complicated Skin ◽  
Hospital Acquired

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) acquisition in cystic fibrosis (CF) patients confers a clinical outcome worse than that in non-CF patients with an increased rate of declined lung function. Telavancin, an approved lipoglycopeptide used to treat infections due to S. aureus, has a dual mode of action causing inhibition of peptidoglycan synthesis and membrane depolarization. MRSA infections in CF patients remain an important problem with no foreseeable decline in prevalence rates. Although telavancin is currently in clinical use for the treatment of complicated skin infections and hospital-acquired pneumonia, the activity against S. aureus infections in CF patients has not been investigated. In this work, we studied the activity of telavancin against CF patient-derived S. aureus strains collected from geographically diverse CF centers in the United States. We found that the telavancin MIC90 was 0.06 μg/ml, 8-fold lower than the ceftaroline or daptomycin MIC90 and 25-fold lower than the linezolid and vancomycin MIC90. We demonstrate that telavancin at serum free concentrations has rapid bactericidal activity, with a decrease of more than 3 log10 CFU/ml being achieved during the first 4 to 6 h of treatment, performing better in this assay than vancomycin and ceftaroline, including against S. aureus strains resistant to ceftaroline. Telavancin resistance was infrequent (0.3%), although we found that it can occur in vitro in both CF- and non-CF patient-derived S. aureus strains by progressive passages with subinhibitory concentrations. Genetic analysis of telavancin-resistant in vitro mutants showed gene polymorphisms in cell wall and virulence genes and increased survival in a Galleria mellonella infection model. Thus, we conclude that telavancin represents a promising therapeutic option for infections in CF patients with potent in vitro activity and a low resistance development potential.

scholarly journals Treatment with Exogenous Trypsin Expands In Vitro Cellular Tropism of the Avian Coronavirus Infectious Bronchitis Virus

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1102
Author(s):  
Phoebe Stevenson-Leggett ◽  
Sarah Keep ◽  
Erica Bickerton
Keyword(s):  
Cell Culture ◽  
Infectious Bronchitis Virus ◽  
Ex Vivo ◽  
Serial Passage ◽  
In Ovo ◽  
Infectious Bronchitis ◽  
Protease Treatment ◽  
Cellular Tropism ◽  
Passage Cell

The Gammacoronavirus infectious bronchitis virus (IBV) causes a highly contagious and economically important respiratory disease in poultry. In the laboratory, most IBV strains are restricted to replication in ex vivo organ cultures or in ovo and do not replicate in cell culture, making the study of their basic virology difficult. Entry of IBV into cells is facilitated by the large glycoprotein on the surface of the virion, the spike (S) protein, comprised of S1 and S2 subunits. Previous research showed that the S2′ cleavage site is responsible for the extended tropism of the IBV Beaudette strain. This study aims to investigate whether protease treatment can extend the tropism of other IBV strains. Here we demonstrate that the addition of exogenous trypsin during IBV propagation in cell culture results in significantly increased viral titres. Using a panel of IBV strains, exhibiting varied tropisms, the effects of spike cleavage on entry and replication were assessed by serial passage cell culture in the presence of trypsin. Replication could be maintained over serial passages, indicating that the addition of exogenous protease is sufficient to overcome the barrier to infection. Mutations were identified in both S1 and S2 subunits following serial passage in cell culture. This work provides a proof of concept that exogenous proteases can remove the barrier to IBV replication in otherwise non-permissive cells, providing a platform for further study of elusive field strains and enabling sustainable vaccine production in vitro.

scholarly journals Interspecies Inhibition ofPorphyromonas gingivalisby Yogurt-DerivedLactobacillus delbrueckiiRequires Active Pyruvate Oxidase

2019 ◽  
Vol 85 (18) ◽  
Author(s):  
Louis P. Cornacchione ◽  
Brian A. Klein ◽  
Margaret J. Duncan ◽  
Linden T. Hu
Keyword(s):  
Hydrogen Peroxide ◽  
Periodontal Disease ◽  
Microbial Interactions ◽  
Lactobacillus Delbrueckii ◽  
Species Selection ◽  
Probiotic Bacteria ◽  
Pyruvate Oxidase ◽  
Hydrogen Peroxide Production ◽  
Content Type

ABSTRACTDespite a growing interest in using probiotic microorganisms to prevent disease, the mechanisms by which probiotics exert their action require further investigation.Porphyromonas gingivalisis an important pathogen implicated in the development of periodontitis. We isolated several strains ofLactobacillus delbrueckiifrom dairy products and examined their ability to inhibitP. gingivalisgrowthin vitro. We observed strain-specific inhibition ofP. gingivalisgrowthin vitro. Whole-genome sequencing of inhibitory and noninhibitory strains ofL. delbrueckiirevealed significant genetic differences supporting the strain specificity of the interaction. Extracts of theL. delbrueckiiSTYM1 inhibitory strain contain inhibitory activity that is abolished by treatment with heat, proteinase K, catalase, and sodium sulfite. We purified the inhibitory protein(s) fromL. delbrueckiiSTYM1 extracts using ammonium sulfate precipitation, anion-exchange chromatography, and gel filtration chromatography. Pyruvate oxidase was highly enriched in the purified samples. Lastly, we showed that purified, catalytically active, recombinant pyruvate oxidase is sufficient to inhibitP. gingivalisgrowthin vitrowithout the addition of cofactors. Further, using a saturated transposon library, we isolated transposon mutants ofP. gingivalisin thefeoB2(PG_1294) gene that are resistant to killing by inhibitoryL. delbrueckii, consistent with a mechanism of hydrogen peroxide production by pyruvate oxidase. Our results support the current understanding of the importance of strain selection, not simply species selection, in microbial interactions. SpecificL. delbrueckiistrains or their products may be effective in the treatment and prevention ofP. gingivalis-associated periodontal disease.IMPORTANCEP. gingivalisis implicated in the onset and progression of periodontal disease and associated with some systemic diseases. Probiotic bacteria represent an attractive preventative therapy for periodontal disease. However, the efficacy of probiotic bacteria can be variable between studies. Our data support the known importance of selecting particular strains of bacteria for probiotic use, not simply a single species. Specifically, in the context of probiotic intervention of periodontitis, our data suggest that high-level expression of pyruvate oxidase with hydrogen peroxide production inL. delbrueckiicould be an important characteristic for the design of a probiotic supplement or a microbial therapeutic.

scholarly journals Nanocrystals of Fusidic Acid for Dual Enhancement of Dermal Delivery and Antibacterial Activity: In Vitro, Ex Vivo and In Vivo Evaluation

Pharmaceutics ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 199 ◽  
Author(s):  
Iman S. Ahmed ◽  
Osama S. Elnahas ◽  
Nouran H. Assar ◽  
Amany M. Gad ◽  
Rania El Hosary
Keyword(s):  
Fusidic Acid ◽  
Therapeutic Efficacy ◽  
Ex Vivo ◽  
Skin Permeation ◽  
Fold Increase ◽  
Spherical Particles ◽  
Resistant Bacteria ◽  
Infection Model

With the alarming rise in incidence of antibiotic-resistant bacteria and the scarcity of newly developed antibiotics, it is imperative that we design more effective formulations for already marketed antimicrobial agents. Fusidic acid (FA), one of the most widely used antibiotics in the topical treatment of several skin and eye infections, suffers from poor water-solubility, sub-optimal therapeutic efficacy, and a significant rise in FA-resistant Staphylococcus aureus (FRSA). In this work, the physico-chemical characteristics of FA were modified by nanocrystallization and lyophilization to improve its therapeutic efficacy through the dermal route. FA-nanocrystals (NC) were prepared using a modified nanoprecipitation technique and the influence of several formulation/process variables on the prepared FA-NC characteristics were optimized using full factorial statistical design. The optimized FA-NC formulation was evaluated before and after lyophilization by several in-vitro, ex-vivo, and microbiological tests. Furthermore, the lyophilized FA-NC formulation was incorporated into a cream product and its topical antibacterial efficacy was assessed in vivo using a rat excision wound infection model. Surface morphology of optimized FA-NC showed spherical particles with a mean particle size of 115 nm, span value of 1.6 and zeta potential of −11.6 mV. Differential scanning calorimetry and powder X-ray diffractometry confirmed the crystallinity of FA following nanocrystallization and lyophilization. In-vitro results showed a 10-fold increase in the saturation solubility of FA-NC while ex-vivo skin permeation studies showed a 2-fold increase in FA dermal deposition from FA-NC compared to coarse FA. Microbiological studies revealed a 4-fofd decrease in the MIC against S. aureus and S. epidermidis from FA-NC cream compared to commercial Fucidin cream. In-vivo results showed that FA-NC cream improved FA distribution and enhanced bacterial exposure in the infected wound, resulting in increased therapeutic efficacy when compared to coarse FA marketed as Fucidin cream.

scholarly journals The F-Box Protein Fbp1 Regulates Sexual Reproduction and Virulence in Cryptococcus neoformans

2011 ◽  
Vol 10 (6) ◽  
pp. 791-802 ◽  
Author(s):  
Tong-Bao Liu ◽  
Yina Wang ◽  
Sabriya Stukes ◽  
Qing Chen ◽  
Arturo Casadevall ◽  
...  
Keyword(s):  
Sexual Reproduction ◽  
Cryptococcus Neoformans ◽  
Stress Responses ◽  
Membrane Integrity ◽  
Infection Model ◽  
Calcofluor White ◽  
Fungal Virulence ◽  
Content Type ◽  
F Box Protein

ABSTRACTCryptococcus neoformansis the leading cause of fungal meningitis in immunocomprised populations. Although extensive studies have been conducted on signal transduction pathways important for fungal sexual reproduction and virulence, how fungal virulence is regulated during infection is still not understood. In this study, we identified the F-box protein Fbp1, which contains a putative F-box domain and 12 leucine-rich repeats (LRR). Althoughfbp1mutants showed normal growth and produced normal major virulence factors, such as melanin and capsule, Fbp1 was found to be essential for fungal virulence, asfbp1mutants were avirulent in a murine systemic-infection model. Fbp1 is also important for fungal sexual reproduction. Basidiospore production was blocked in bilateral mating betweenfbp1mutants, even though normal dikaryotic hyphae were observed during mating.In vitroassays of stress responses revealed thatfbp1mutants are hypersensitive to SDS, but not calcofluor white (CFW) or Congo red, indicating that Fbp1 may regulate cell membrane integrity. Fbp1 physically interacts with Skp1 homologues in bothSaccharomyces cerevisiaeandC. neoformansvia its F-box domain, suggesting it may function as part of an SCF (Skp1, Cullins, F-box proteins) E3 ligase. Overall, our study revealed that the F-box protein Fbp1 is essential for fungal sporulation and virulence inC. neoformans, which likely represents a conserved novel virulence control mechanism that involves the SCF E3 ubiquitin ligase-mediated proteolysis pathway.

scholarly journals Targeting the Calcineurin Pathway Enhances Ergosterol Biosynthesis Inhibitors against Trichophyton mentagrophytes In Vitro and in a Human Skin Infection Model

2007 ◽  
Vol 51 (10) ◽  
pp. 3743-3746 ◽  
Author(s):  
Chiatogu Onyewu ◽  
Emily Eads ◽  
Wiley A. Schell ◽  
John R. Perfect ◽  
Yehuda Ullmann ◽  
...  
Keyword(s):  
Human Skin ◽  
Skin Infection ◽  
Ex Vivo ◽  
Day Treatment ◽  
Trichophyton Mentagrophytes ◽  
Ergosterol Biosynthesis ◽  
Infection Model ◽  
Drug Concentrations ◽  
Ergosterol Biosynthesis Inhibitors

ABSTRACT Fluconazole-FK506 or fluconazole-cyclosporine drug combinations were tested in an ex vivo Trichophyton mentagrophytes human skin infection model. Conidia colonization was monitored by scanning electron microscopy over a 7-day treatment period. The fluconazole-FK506 combination demonstrated the most obvious advantage over single-drug therapy by clearing conidia and protecting skin from damage at low drug concentrations.

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