JMM profile: rifampicin: a broad-spectrum antibiotic

Rifampicin (also known as rifampin) inhibits RNA synthesis, and is used to treat tuberculosis, leprosy, staphylococcal infections and legionnaires' disease. It can also protect at-risk populations from Haemophilus influenzae type b and Neisseria meningitidis. It is a polyketide antibiotic and is on the World Health Organization (WHO) list of essential medicines due to its critical importance to human medicine. The adverse effect of liver toxicity is controlled by testing during prolonged treatment regimes. Rifampicin's red-orange colour can result in the colouration of sweat, tears and urine. Resistance to rifampicin arises from mutation of the target RNA polymerase or ADP ribosylation of the antibiotic or efflux. Mycobacteria may become singularly resistant to rifampicin or as part of multidrug or extensive drug resistance

JMM Profile: Carbapenems: a broad-spectrum antibiotic

Carbapenems are potent members of the β-lactam family that inhibit bacterial cell-wall biosynthesis inhibitors . They are highly effective against Gram-negative and Gram-positive drug-resistant infections . As such, carbapenems are typically reserved as an antibiotic of last resort. The WHO lists meropenem as an essential medicine. Nausea and vomiting are reported in ≤20% of carbapenem recipients, with 1.5% suffering seizures. Enzymatic hydrolysis of the β-lactam ring is the main driver of clinical resistance. These enzymes can be classified as Class A, B and D. Classes A and D are serine β-lactamases, whereas Class B rely on metal-mediated hydrolysis, typically through zinc

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

NirA is an alternative nitrite reductase from Pseudomonas aeruginosa with potential as an anti-virulence target

Pseudomonas aeruginosa is capable of causing a wide-range of diseases due to production of an extensive arsenal of virulence factors. This opportunistic pathogen is capable of causing both acute and chronic infection, with treatment complicated due to its intrinsic resistance and tolerance of antibiotics. With the antibacterial pipeline drying up, anti-virulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. The work presented in this thesis builds upon the successful ‘integrated whole-genome screening for Pseudomonas aeruginosa virulence genes using multiple disease models’, performed in our lab as part of the NABATIVI project to combat anti-virulence target identification. In this previous study, Tn5 mutant was identified to be inserted into the hypothetical protein PA4130, causing attenuation in pyocyanin production; swarming motility; Drosophila melanogaster; Caenorhabditis elegans; and A549 human epithelial cell culture. The work from this screen was validated through generation of in-frame PA4130 deletion mutants in multiple phylogenetically distinct clinical strains of P. aeruginosa, demonstrating that the observed reductions in pyocyanin and swarming motility were conserved. Subsequent re-screening of PAO1-L ΔPA4130 through invertebrate infection models and tissue culture confirmed the phenotypes observed in a PA4130::Tn5 mutant, and further screening in acute murine lung models revealing an 80% increase in survival as compared to the isogenic wild-type strain. Sequence analysis of PA4130 revealed resemblance to nitrite or sulphite reductase hemoprotein sub-units with successful over-expression and purification reliant on siroheme synthase co-overexpression. Structural characterisation of PA4130 by crystallography failed, however, methyl viologen oxidation assays with purified PA4130 demonstrated that this enzyme is an ammonium-forming nitrite reductase operating in a ferredoxin-dependent manner. PA4130 was subsequently renamed NirA to fit with current nomenclature. With P. aeruginosa encoding a second siroheme-dependent assimilatory nitrite reductase in NirB, functional redundancy and overlap was explored between NirA and NirB. When grown with nitrate or nitrite as a single nitrogen source under aerobic conditions, nirB mutants were unable to grow, whilst deletion in nirA had no effect, suggesting NirA is not an assimilatory nitrite reductase. However, under micro-aerobic conditions, deletion of nirA resulted in attenuated growth. Previous work demonstrated that nirA is upregulated by cyanogenesis and functions alongside CioAB and the PA4129-34 gene cluster in protection from cyanide intoxication. Cyanide is normally a potent inhibitor of heme-containing enzymes; therefore it was hypothesized that NirA encodes a cyanide resistant nitrite reductase that supports the function of NirB under increasing cyanide concentrations. Functional assays revealed NirA demonstrates significant nitrite reductase activity at 600μm cyanide, with the E. coli sulphite-reductase homologue CysI completely inhibited by 50μm. With high levels of bacterial or host-derived cyanide and nitrite/nitrate found during infection, we propose a model by which NirA is essential for detoxification of nitrite due to NirB/NirS inactivation by cyanide. Whilst further work is required to confirm this assessment in conjunction with NirS, potential inhibition of NirA could sensitise P. aeruginosa to cyanide or nitrite self-intoxication. This work supports the increasingly recognised role of reduced oxygenation and nitrate metabolism during P. aeruginosa infection

NirA is an alternative nitrite reductase from Pseudomonas aeruginosa with potential as an anti-virulence target

Pseudomonas aeruginosa is capable of causing a wide-range of diseases due to production of an extensive arsenal of virulence factors. This opportunistic pathogen is capable of causing both acute and chronic infection, with treatment complicated due to its intrinsic resistance and tolerance of antibiotics. With the antibacterial pipeline drying up, anti-virulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. The work presented in this thesis builds upon the successful ‘integrated whole-genome screening for Pseudomonas aeruginosa virulence genes using multiple disease models’, performed in our lab as part of the NABATIVI project to combat anti-virulence target identification. In this previous study, Tn5 mutant was identified to be inserted into the hypothetical protein PA4130, causing attenuation in pyocyanin production; swarming motility; Drosophila melanogaster; Caenorhabditis elegans; and A549 human epithelial cell culture. The work from this screen was validated through generation of in-frame PA4130 deletion mutants in multiple phylogenetically distinct clinical strains of P. aeruginosa, demonstrating that the observed reductions in pyocyanin and swarming motility were conserved. Subsequent re-screening of PAO1-L ΔPA4130 through invertebrate infection models and tissue culture confirmed the phenotypes observed in a PA4130::Tn5 mutant, and further screening in acute murine lung models revealing an 80% increase in survival as compared to the isogenic wild-type strain. Sequence analysis of PA4130 revealed resemblance to nitrite or sulphite reductase hemoprotein sub-units with successful over-expression and purification reliant on siroheme synthase co-overexpression. Structural characterisation of PA4130 by crystallography failed, however, methyl viologen oxidation assays with purified PA4130 demonstrated that this enzyme is an ammonium-forming nitrite reductase operating in a ferredoxin-dependent manner. PA4130 was subsequently renamed NirA to fit with current nomenclature. With P. aeruginosa encoding a second siroheme-dependent assimilatory nitrite reductase in NirB, functional redundancy and overlap was explored between NirA and NirB. When grown with nitrate or nitrite as a single nitrogen source under aerobic conditions, nirB mutants were unable to grow, whilst deletion in nirA had no effect, suggesting NirA is not an assimilatory nitrite reductase. However, under micro-aerobic conditions, deletion of nirA resulted in attenuated growth. Previous work demonstrated that nirA is upregulated by cyanogenesis and functions alongside CioAB and the PA4129-34 gene cluster in protection from cyanide intoxication. Cyanide is normally a potent inhibitor of heme-containing enzymes; therefore it was hypothesized that NirA encodes a cyanide resistant nitrite reductase that supports the function of NirB under increasing cyanide concentrations. Functional assays revealed NirA demonstrates significant nitrite reductase activity at 600μm cyanide, with the E. coli sulphite-reductase homologue CysI completely inhibited by 50μm. With high levels of bacterial or host-derived cyanide and nitrite/nitrate found during infection, we propose a model by which NirA is essential for detoxification of nitrite due to NirB/NirS inactivation by cyanide. Whilst further work is required to confirm this assessment in conjunction with NirS, potential inhibition of NirA could sensitise P. aeruginosa to cyanide or nitrite self-intoxication. This work supports the increasingly recognised role of reduced oxygenation and nitrate metabolism during P. aeruginosa infection