Antibiotic Efficacy Testing in an Ex vivo Model of <em>Pseudomonas aeruginosa</em> and <em>Staphylococcus aureus</em> Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

University of Warwick

A range of new water-compatible optically pure metallohelices - made by self-assembly of simple non-peptidic organic components around Fe ions - exhibit similar architecture to some natural cationic antimicrobial peptides (CAMPs) and are found to have high, structure-dependent activity against bacteria, including clinically problematic Gram-negative pathogens. A key compound is shown to freely enter rapidly dividing  cells without significant membrane disruption, and localise in distinct foci near the poles. Several related observations of CAMP-like mechanisms are made  biophysical measurements, whole genome sequencing of tolerance mutants and transcriptomic analysis. These include: high selectivity for binding of G-quadruplex DNA over double stranded DNA; inhibition of both DNA gyrase and topoisomerase I ; curing of a plasmid that contributes to the very high virulence of the  strain used; activation of various two-component sensor/regulator and acid response pathways; and subsequent attempts by the cell to lower the net negative charge of the surface. This impact of the compound on multiple structures and pathways corresponds with our inability to isolate fully resistant mutant strains, and supports the idea that CAMP-inspired chemical scaffolds are a realistic approach for antimicrobial drug discovery, without the practical barriers to development that are associated with natural CAMPS.

Metallohelices that kill Gram-negative pathogens using intracellular antimicrobial peptide pathways.

There is a dire need for new antimicrobial compounds to combat the growing threat of widespread antibiotic resistance. With a currently very scarce drug pipeline, consisting mostly of derivatives of known antibiotics, new classes of antibiotics are urgently required. Metal complexes are currently in clinical development for the treatment of cancer, malaria and neurodegenerative diseases. However, only little attention has been paid to their application as potential antimicrobial compounds. We report the evaluation of 906 metal-containing compounds that have been screened by the Community for Open Antimicrobial Drug Discovery (CO-ADD) for antimicrobial activity. Metal-bearing compounds display a significantly higher hit-rate (9.9%) when compared to the purely organic molecules (0.87%) in the CO-ADD database. Out of 906 compounds, 88 show activity against at least one of the tested strains, including fungi, while not displaying any cytotoxicity against mammalian cell lines or haemolytic properties. Herein, we highlight the structures of the 30 compounds with activity against Gram-positive and/or Gram-negative bacteria containing Mn, Co, Zn, Ru, Ag, Eu, Ir and Pt, with activities down to the nanomolar range against methicillin resistant  (MRSA). 23 of these complexes have not been reported for their antimicrobial properties before. This work reveals the vast diversity that metal-containing compounds can bring to antimicrobial research. It is important to raise awareness of these types of compounds for the design of truly novel antibiotics with potential for combatting antimicrobial resistance.

Metal complexes as a promising source for new antibiotics.

The multi-drug resistance of the opportunistic pathogen  is of growing concern, with many clinical isolates proving to be resistant to last resort as well as front line antibiotic treatments. The use of bacteriophages is an attractive alternative to controlling and treating this emerging nosocomial pathogen. In this study, we have investigated bacteriophages collected from hospital wastewater in Thailand and we have explored their activity against clinical isolates of . Bacteriophage vB_AbaM_PhT2 showed 28% host range against 150 multidrug resistant (MDR) isolates and whole genome sequencing did not detect any known virulence factors or antibiotic resistance genes. Purified vB_AbaM_PhT2 samples had endotoxin levels below those recommended for preclinical trials and were not shown to be directly cytotoxic to human cell lines in vitro. The treatment of human brain and bladder cell lines grown in the presence of  with this bacteriophage released significantly less lactate dehydrogenase compared to samples with no bacteriophage treatment, indicating that vB_AbaM_PhT2 can protect from  induced cellular damage. Our results have also indicated that there is synergy between this bacteriophage and the end line antibiotic colistin. We therefore propose bacteriophage vB_AbaM_PhT2 as a good candidate for future research and for its potential development into a surface antimicrobial for use in hospitals.

Investigating Bacteriophages Targeting the Opportunistic Pathogen .

Novel antimicrobials are urgently needed to combat drug-resistant bacteria and to overcome the inherent difficulties in treating biofilm-associated infections. Studying plants and other natural materials used in historical infection remedies may enable further discoveries to help fill the antibiotic discovery gap. We previously reconstructed a 1,000-year-old remedy containing onion, garlic, wine, and bile salts, known as 'Bald's eyesalve', and showed it had promising antibacterial activity. In this current paper, we have found this bactericidal activity extends to a range of Gram-negative and Gram-positive wound pathogens in planktonic culture and, crucially, that this activity is maintained against Acinetobacter baumannii, Stenotrophomonas maltophilia, Staphylococcus aureus, Staphylococcus epidermidis and Streptococcus pyogenes in a soft-tissue wound biofilm model. While the presence of garlic in the mixture can explain the activity against planktonic cultures, garlic has no activity against biofilms. We have found the potent anti-biofilm activity of Bald's eyesalve cannot be attributed to a single ingredient and requires the combination of all ingredients to achieve full activity. Our work highlights the need to explore not only single compounds but also mixtures of natural products for treating biofilm infections and underlines the importance of working with biofilm models when exploring natural products for the anti-biofilm pipeline.

Anti-biofilm efficacy of a medieval treatment for bacterial infection requires the combination of multiple ingredients.

Delafloxacin, a fourth-generation anionic fluoroquinolone (FQ) was approved in 2019 for community acquired bacterial pneumonia (CARP). It has broad spectrum activity and an improved class-related toxicity profile. However, it has recently failed a Phase 3 clinical trial for treatment of N. gonorrhoeae infections due to the lack of sufficient efficacy at the dose administered. Inspired by the microbiological and safety profile of delafloxacin, we have developed and profiled the first reported delafloxacin carbon analogue whereby a Nitrogen-for-Carbon swap has been successfully carried out at the C7 position. Not only have we shown that compounds with this modification maintain activity against N. gonorrhoeae (plus other gram-positive and gram-negative bacteria) but they also demonstrate a differentiated physicochemical profile. A zwitterionic derivative of delafloxacin was also profiled and demonstrated a superior microbiological profile against gram-negative strains, whilst maintaining favorable selected ADMET properties.

Novel C-7 carbon substituted fourth generation fluoroquinolones targeting N. Gonorrhoeae infections.

 infections, including catheter associated urinary tract infections, are a considerable burden on health care systems. This is due to their difficulty to treat, caused by antimicrobial resistance and their ability to form biofilms. In this study, we investigated the use of a  phage cocktail to reduce biofilm viability. We used two methodologies to investigate this, a standard 96-well plate assay and a more complicated Foley catheter-based model. The phage cocktail was used alone and in combination with clinically relevant antibiotic treatments. Viability was measured by both a resazurin based stain and colony forming unit counts, of cells sloughed off from the biofilm. We showed that phage infection dynamics and host survival vary significantly in different standard laboratory media, presumably due to the expression of different surface receptors and capsule composition by the bacteria effecting phage binding. This underscores the importance of a realistic model for developing phage therapy. We demonstrate that bacteriophage-based treatments are a viable option for preventing  colonisation and biofilm formation on urinary catheters. Phage cocktails were able to significantly reduce the amount of biofilm that formed when they were present during early biofilm formation. The phages used in this study were unable to significantly reduce a pre-formed mature biofilm, despite encoding depolymerases. Phages applied together with antimicrobial treatments, showed synergistic interactions, in some cases the combined treatment was much more effective than antimicrobial treatments alone. We show that phage cocktails have the potential to prevent  biofilms in catheters, if used early or as a preventative treatment and will work well alongside standard antibiotics in the treatment of catheter-associated urinary tract infections (CAUTI).

John Moat

School of Life Science,
Warwick Antimicrobial Screening Facility,
Warwick Antimicrobial Screening Facility, School of Life Science

University of Warwick

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

John Moat

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University of Warwick

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

School of Life Science,
Warwick Antimicrobial Screening Facility,
Warwick Antimicrobial Screening Facility, School of Life Science