Exploitation of the bilosome platform technology to formulate antibiotics and enhance efficacy of melioidosis treatments

Burkholderia pseudomallei is a Gram-negative intracellular bacterium which is recalcitrant to antibiotic therapy. There also is currently no licensed vaccine for this potentially fatal pathogen, further highlighting the requirement for better therapeutics to treat the disease melioidosis. Here we use an oral delivery platform, the bilosome to entrap already- licensed antibiotics. Bilosome-entrapped antibiotics were used to treat mice infected via the aerosol route with B. pseudomallei. When treatment was started by the oral route at 6 h post-infection and continued for 7 days, bilosome levofloxacin and bilosome doxycycline formulations were significantly more efficacious than free antibiotics in terms of survival rates. Additionally, bilosome formulated levofloxacin protected mice from antibiotic and infection induced weight loss following B. pseudomallei infection. The microbiomes of mice treated with levofloxacin were depleted of all phyla with the exception of Firmicutes, but doxycycline treatment had minimal effect on the microbiome. Encapsulation of either drug in bilosomes had no deleterious or clear advantageous effect on microbiome. This indicates that the ability of bilosomes to ameliorate antibiotic induced weight loss is not due to microbiome effects. The bilosome platform not only has potential to reduce adverse effects of orally delivered antimicrobials, but has potential for other therapeutics which may cause detrimental side-effects or require enhanced delivery

The effect of environmental stress on antibiotic production by streptomyces

Antibiotic Resistance is a world-wide issue that increases with every year. The World Health Organisation (WHO) has identified priority pathogenic microorganisms with dangerous resistance to 'last-resort' antibiotics, these are the 'ESKAPE' pathogens.To face this problem, we investigated novel antibiotic-producing bacteria. The majority of currently available originate from organisms belonging to the Actinobacteria Phylum. Unfortunately, no significant compounds have been discovered in decades and so this has lead us to search in sparsely researched locations such as the hyper-arid Atacama Desert in Chile and compare them to more commonly researched North European soil samples which are naturally more hydrated. In this project we studied bacteria isolated from soil samples from Chiles Atacama Desert and the west of Scotland. Relevant strains were selected by unique morphology and by antimicrobial-activity. Bioactivity tests were performed on all isolates grown on 4 different media against 'ESKAPE' pathogens, Bacillus subtilis and Saccharomyces cerevisiae across which all were inhibited in the streak assays and all but Klebsiella pneumoniae and Pseudomonas aeruginosa in plug assays. Those presenting relevant bioactive profiles were taxonomically identified using 16S rRNAgene sequencing as members of the genera; Streptomyces, Micromonospora and Methylobacterium. Amongst those, isolate identified as Streptomyces narbones is presented activity against a clinical isolate of carbapenem-resistant Acinetobacterbaumannii. In Addition, Streptomyces puniceus presented a unique morphology that was not seen before in Streptomyces. Moreover, elicitation of bio-active compounds from antimicrobial-producing isolates was achieved by increasing osmotic stress or supplementing with Nitrogen or Humic acid. Further studies to compliment this project would include whole-genome sequencing of the most interesting strains in tandem with mass spectrometry to identify the active compounds and activation of crypticbiosynthetic gene clusters through elicitation.Antibiotic Resistance is a world-wide issue that increases with every year. The World Health Organisation (WHO) has identified priority pathogenic microorganisms with dangerous resistance to 'last-resort' antibiotics, these are the 'ESKAPE' pathogens.To face this problem, we investigated novel antibiotic-producing bacteria. The majority of currently available originate from organisms belonging to the Actinobacteria Phylum. Unfortunately, no significant compounds have been discovered in decades and so this has lead us to search in sparsely researched locations such as the hyper-arid Atacama Desert in Chile and compare them to more commonly researched North European soil samples which are naturally more hydrated. In this project we studied bacteria isolated from soil samples from Chiles Atacama Desert and the west of Scotland. Relevant strains were selected by unique morphology and by antimicrobial-activity. Bioactivity tests were performed on all isolates grown on 4 different media against 'ESKAPE' pathogens, Bacillus subtilis and Saccharomyces cerevisiae across which all were inhibited in the streak assays and all but Klebsiella pneumoniae and Pseudomonas aeruginosa in plug assays. Those presenting relevant bioactive profiles were taxonomically identified using 16S rRNAgene sequencing as members of the genera; Streptomyces, Micromonospora and Methylobacterium. Amongst those, isolate identified as Streptomyces narbones is presented activity against a clinical isolate of carbapenem-resistant Acinetobacterbaumannii. In Addition, Streptomyces puniceus presented a unique morphology that was not seen before in Streptomyces. Moreover, elicitation of bio-active compounds from antimicrobial-producing isolates was achieved by increasing osmotic stress or supplementing with Nitrogen or Humic acid. Further studies to compliment this project would include whole-genome sequencing of the most interesting strains in tandem with mass spectrometry to identify the active compounds and activation of crypticbiosynthetic gene clusters through elicitation

Investigating the anti-inflammatory effects of non-ionic surfactant vesicles

Error on title page – year of award is 2022.Inflammation can be an unwanted consequence or cause of debilitating diseases of infectious and non-infectious aetiologies. Sepsis can be a result of an uncontrolled immune response to bacterial infections (including MRSA, E. coli and P. Aeruginosa) or viral infections (including SARS-CoV-2 and Influenza). Immune dysfunction can also cause autoimmune diseases including rheumatoid arthritis, ulcerative colitis and Crohn’s disease. Current anti-inflammatory medications have a number of deficiencies including lack of specificity and undesirable side effects. Herein the potential of Non-ionic surfactant vesicles (NISV) as an anti-inflammatory drug and their mode of action is investigated. NISV were found to have a wide array of anti-inflammatory effects on macrophages in vitro including down-regulation of IL-6, IL-12 and multiple chemokines regardless of whether cells were stimulated with LPS, Poly(I:C) or Pam3csk4. The individual components of NISV, monopalmityol glycerol (MPG), dicetyl phosphate (DCP) and cholesterol) did not replicate the immunomodulatory effects found in macrophages, proving the formulation of NISV is essential for the anti-inflammatory effects. Liposomes were shown to augment LPS and Poly(I:C) stimulation of macrophages, inducing upregulation of pro-inflammatory cytokines including IL-6 and TNF-α, demonstrating that the anti-inflammatory effects of NISV are not a common feature of all vesicular formulations. Transcriptomic analyses showed consistent anti-inflammatory effects, and indicated down-regulation of NF-κB as an important aspect of the anti-inflammatory effects mediated by NISV. Metabolomic analysis show NISV disrupt the Warburg effect by reducing production of itaconate and succinate, indicating anti-inflammatory downstream effects. The mechanism through which NISV down-regulate NF-κB is unknown. However, the NISV’s primary component, MPG, demonstrates structural similarity to the sphingolipid, sphingosine-1-phosphate (S1P). As S1P is an immune mediator that acts through NF-κB, future work should explore the hypothesis that MPG disrupts S1P signalling.Inflammation can be an unwanted consequence or cause of debilitating diseases of infectious and non-infectious aetiologies. Sepsis can be a result of an uncontrolled immune response to bacterial infections (including MRSA, E. coli and P. Aeruginosa) or viral infections (including SARS-CoV-2 and Influenza). Immune dysfunction can also cause autoimmune diseases including rheumatoid arthritis, ulcerative colitis and Crohn’s disease. Current anti-inflammatory medications have a number of deficiencies including lack of specificity and undesirable side effects. Herein the potential of Non-ionic surfactant vesicles (NISV) as an anti-inflammatory drug and their mode of action is investigated. NISV were found to have a wide array of anti-inflammatory effects on macrophages in vitro including down-regulation of IL-6, IL-12 and multiple chemokines regardless of whether cells were stimulated with LPS, Poly(I:C) or Pam3csk4. The individual components of NISV, monopalmityol glycerol (MPG), dicetyl phosphate (DCP) and cholesterol) did not replicate the immunomodulatory effects found in macrophages, proving the formulation of NISV is essential for the anti-inflammatory effects. Liposomes were shown to augment LPS and Poly(I:C) stimulation of macrophages, inducing upregulation of pro-inflammatory cytokines including IL-6 and TNF-α, demonstrating that the anti-inflammatory effects of NISV are not a common feature of all vesicular formulations. Transcriptomic analyses showed consistent anti-inflammatory effects, and indicated down-regulation of NF-κB as an important aspect of the anti-inflammatory effects mediated by NISV. Metabolomic analysis show NISV disrupt the Warburg effect by reducing production of itaconate and succinate, indicating anti-inflammatory downstream effects. The mechanism through which NISV down-regulate NF-κB is unknown. However, the NISV’s primary component, MPG, demonstrates structural similarity to the sphingolipid, sphingosine-1-phosphate (S1P). As S1P is an immune mediator that acts through NF-κB, future work should explore the hypothesis that MPG disrupts S1P signalling

Dataset supporting the University of Southampton Doctoral Thesis "Label Free Imaging for Drug Delivery Across Biophysical Barriers".

Data underpinning thesis chapters 5 and 6 Chapter 5 data contains MATLAB Raman spectroscopic data of NISV samples Chapter 6 contains CARS cellular images of NISVs</span