rat-burn-model-to-study-full-thickness-cutaneous-thermal-burn

Rat Burn Model to Study Full-Thickness Cutaneous Thermal Burn and Infection

Burn induction methodologies are inconsistently described in rat models. A uniform burn wound model, which represents the clinical scenario, is necessary ...

intraventricular-drug-delivery-sampling-for-pharmacokinetics

Intraventricular Drug Delivery and Sampling for Pharmacokinetics and Pharmacodynamics Study

Although the blood-brain barrier (BBB) protects the brain from foreign entities, it also prevents some therapeutics from crossing into the central nervous ...

The University of Melbourne

Gram-negative bacteria produce outer membrane vesicles (OMVs) as delivery vehicles for nefarious bacterial cargo such as virulence factors, which are antibiotic resistance determinants. This study aimed to investigate the impact of polymyxin B treatment on the OMV lipidome from paired polymyxin-susceptible and -resistant  isolates.  ATCC 700721 was employed as a reference strain in addition to two clinical strains,  FADDI-KP069 and  BM3. Polymyxin B treatment of the polymyxin-susceptible strains resulted in a marked reduction in the glycerophospholipid, fatty acid, lysoglycerophosphate and sphingolipid content of their OMVs. Conversely, the polymyxin-resistant strains expressed OMVs richer in all of these lipid species, both intrinsically and increasingly under polymyxin treatment. The average diameter of the OMVs derived from the  ATCC 700721 polymyxin-susceptible isolate, measured by dynamic light scattering measurements, was ~90.6 nm, whereas the average diameter of the OMVs isolated from the paired polymyxin-resistant isolate was ~141 nm. Polymyxin B treatment (2 mg/L) of the  ATCC 700721 cells resulted in the production of OMVs with a larger average particle size in both the susceptible (average diameter ~124 nm) and resistant (average diameter ~154 nm) strains. In light of the above, we hypothesize that outer membrane remodelling associated with polymyxin resistance in  may involve fortifying the membrane structure with increased glycerophospholipids, fatty acids, lysoglycerophosphates and sphingolipids. Putatively, these changes serve to make the outer membrane and OMVs more impervious to polymyxin attack.

Lipidomic Analysis of the Outer Membrane Vesicles from Paired Polymyxin-Susceptible and -Resistant  Clinical Isolates.

In the present report we characterized the outer membrane proteome, genomic, and lipid A remodelling changes following the evolution of a colistin-susceptible K. pneumoniae ATCC 13883 strain into an extremely colistin-resistant strain. Lipid A profiling revealed the outer membrane of the colistin-susceptible strain is decorated primarily by hexa- and hepta-acylated lipid A species and a minor tetra-acylated species. In the lipid A profile of the extremely colistin-resistant strain, in addition to the aforementioned lipid A species, the obligatory 4-amino-4-deoxy-l-arabinose modification of the hexa-acylated lipid A was detected. Comparative genomic analysis revealed that the mgrB gene of the colistin-resistant strain is inactivated by a single nucleotide insertion which produces a frame-shift, resulting in premature termination. We also detected two synonymous mutations in the two-component system genes phoP and phoQ. Comparative profiling of the outer membrane proteome of each strain revealed that outer membrane proteins from bacterial stress response, glutamine degradation, pyruvate, aspartate, and asparagine metabolic pathways were over-represented in the extremely colistin-resistant K. pneumoniae ATCC 13883 strain. In comparison, in the sensitive strain, outer membrane proteins from carbohydrate metabolism, H-ATPase, cell division, and peptidoglycan biosynthesis were over-represented. Notably, there were no discernible differences between the OmpK35 and OmpK36 major outer membrane porins between the polymyxin-susceptible and -resistant strains suggesting porin deficiency is not involved in the colistin resistance in the ATCC 13883 strain. These findings shed new light on the outer membrane remodelling events accompanying the development of extremely high levels of colistin resistance in K. pneumoniae.

A Comparative Study of Outer Membrane Proteome between Paired Colistin-Susceptible and Extremely Colistin-Resistant Klebsiella pneumoniae Strains.

Polymyxins are amongst the most important antibiotics in modern medicine, in recent times their clinical utility has been overshadowed by nosocomial outbreaks of polymyxin resistant MDR Gram-negative 'superbugs'. An effective strategy to surmount polymyxin resistance is combination therapy with FDA-approved non-antibiotic drugs. Herein we used untargeted metabolomics to investigate the mechanism(s) of synergy between polymyxin B and the selective estrogen receptor modulator (SERM) tamoxifen against a polymyxin-resistant MDR cystic fibrosis (CF)  FADDI-PA006 isolate (polymyxin B MIC=8 mg/L , it is an MDR polymyxin resistant  isolated from the lungs of a CF patient). The metabolome of FADDI-PA006 was profiled at 15 min, 1 and 4 h following treatment with polymyxin B (2 mg/L), tamoxifen (8 mg/L) either as monotherapy or in combination. At 15 min, the combination treatment induced a marked decrease in lipids, primarily fatty acid and glycerophospholipid metabolites that are involved in the biosynthesis of bacterial membranes. In line with the polymyxin-resistant status of this strain, at 1 h, both polymyxin B and tamoxifen monotherapies produced little effect on bacterial metabolism. In contrast to the combination which induced extensive reduction (≥ 1.0-log2-fold, p ≤ 0.05; FDR ≤ 0.05) in the levels of essential intermediates involved in cell envelope biosynthesis. Overall, these novel findings demonstrate that the primary mechanisms underlying the synergistic bactericidal effect of the combination against the polymyxin-resistant  CF isolate FADDI-PA006 involves a disruption of the cell envelope biogenesis and an inhibition of aminoarabinose LPS modifications that confer polymyxin resistance.

Mechanistic Insights From Global Metabolomics Studies into Synergistic Bactericidal Effect of a Polymyxin B Combination With Tamoxifen Against Cystic Fibrosis MDR .

Antimicrobial resistance is a serious threat to global human health; therefore, new anti-infective therapeutics are required. The cyclic depsi-peptide teixobactin exhibits potent antimicrobial activity against several Gram-positive pathogens. To study the natural product's mechanism of action and improve its pharmacological properties, efficient chemical methods for preparing teixobactin analogues are required to expedite structure-activity relationship studies. Described herein is a synthetic route that enables rapid access to analogues. Furthermore, our new N-methylated analogues highlight that hydrogen bonding along the N-terminal tail is likely to be important for antimicrobial activity.

The impact of backbone N-methylation on the structure-activity relationship of Leu -teixobactin.

In the present study, we employed untargeted metabolomics to investigate the synergistic killing mechanism of polymyxin B in combination with an aminoglycoside, amikacin, against a polymyxin-susceptible isolate of , FADDI-PA111 (MIC = 2 mg/liter for both polymyxin B and amikacin), and a polymyxin-resistant Liverpool epidemic strain (LES), LESB58 (the corresponding MIC for both polymyxin B and amikacin is 16 mg/liter). The metabolites were extracted 15 min, 1 h, and 4 h following treatment with polymyxin B alone (2 mg/liter for FADDI-PA111; 4 mg/liter for LESB58), amikacin alone (2 mg/liter), and both in combination and analyzed using liquid chromatography-mass spectrometry (LC-MS). At 15 min and 1 h, polymyxin B alone induced significant perturbations in glycerophospholipid and fatty acid metabolism pathways in FADDI-PA111 and, to a lesser extent, in LESB58. Amikacin alone at 1 and 4 h induced significant perturbations in peptide and amino acid metabolism, which is in line with the mode of action of aminoglycosides. Pathway analysis of FADDI-PA111 revealed that the synergistic effect of the combination was largely due to the inhibition of cell envelope biogenesis, which was driven initially by polymyxin B via suppression of key metabolites involved in lipopolysaccharide, peptidoglycan, and membrane lipids (15 min and 1 h) and later by amikacin (4 h). Overall, these novel findings demonstrate that the disruption of cell envelope biogenesis and central carbohydrate metabolism, decreased levels of amino sugars, and a downregulated nucleotide pool are the metabolic pathways associated with the synergistic killing of the polymyxin-amikacin combination against  This mechanistic study might help optimize synergistic polymyxin B combinations in the clinical setting.

Metabolomics Study of the Synergistic Killing of Polymyxin B in Combination with Amikacin against Polymyxin-Susceptible and -Resistant Pseudomonas aeruginosa.

The discovery of antibiotics has led to the effective treatment of bacterial infections that were otherwise fatal and has had a transformative effect on modern medicine. Teixobactin is an unusual depsipeptide natural product that was recently discovered from a previously unculturable soil bacterium and found to possess potent antibacterial activity against several Gram positive pathogens, including methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci. One of the key features of teixobactin as an antibiotic lead is that resistance could not be generated in a laboratory setting. This is proposed to be a result of a mechanism of action that involves binding to essential cell wall synthesis building blocks, lipid II and lipid III. Since the initial isolation report in 2015, significant efforts have been made to understand its unique mechanism of action, develop efficient synthetic routes for its production, and thus enable the generation of analogues for structure-activity relationship studies and optimization of its pharmacological properties. Our review provides a comprehensive treatise on the progress in understanding teixobactin chemistry, structure-activity relationships, and mechanisms of antibacterial activity. Teixobactin represents an exciting starting point for the development of new antibiotics that can be used to combat multidrug-resistant bacterial ("superbug") infections.

Synthesis and structure-activity relationships of teixobactin.

This study aimed to investigate synergistic antibacterial activity of polymyxin B in combination with the selective serotonin reuptake inhibitor, sertraline, against the Gram-negative pathogens , , and . The combination of polymyxin B and sertraline showed synergistic antibacterial activity in checkerboard and static time-kill assays at clinically relevant concentrations against both polymyxin-susceptible and polymyxin-resistant isolates. The potential antimicrobial mode of action of the combination was investigated against  FADDI-PA024 using untargeted metabolomics alongside scanning and transmission electron microscopy (EM). Scanning and transmission EM revealed that the polymyxin B and sertraline combination resulted in greater damage to the bacterial cell compared to each drug alone. Metabolomics results showed that the combination significantly affected the bacterial ability to remodel its outer membrane. This was reflected by the major perturbation of glycerophospholipids and fatty acids and the pantothenate and coenzyme A (CoA) pathways, which feed fatty acid elongation (e.g., -hexadec-2-enoyl-CoA) as well as inhibit the biosynthesis of lipopolysaccharide and peptidoglycan. The combination also inhibited the polymyxin resistance phosphoethanolamine (pEtN) lipid A modification pathway, indicated by the declined levels of phosphoethanolamine. In summary, the present study highlights the potential possibilities of a polymyxin-sertraline combination for the treatment of infections caused by multidrug resistant Gram-negative bacteria such as central nervous system (CNS) infections via direct intraventricular/intrathecal delivery.

Effective Strategy Targeting Polymyxin-Resistant Gram-Negative Pathogens: Polymyxin B in Combination with the Selective Serotonin Reuptake Inhibitor Sertraline.

Antibiotics have served humankind through their use in modern medicine as effective treatments for otherwise fatal bacterial infections. Teixobactin is a first member of newly discovered natural antibiotics that was recently identified from a hitherto-unculturable soil bacterium, , and recognized as a potent antibacterial agent against various Gram-positive bacteria, including methicillin-resistant  (MRSA) and vancomycin-resistant enterococci. The most distinctive characteristic of teixobactin as an effective antibiotic is that teixobactin resistance could not be evolved in a laboratory setting. It is purported that teixobactin's "resistance-resistant" mechanism of action includes binding to the essential bacterial cell wall synthesis building blocks lipid II and lipid III. In the present study, metabolomics was used to investigate the potential metabolic pathways involved in the mechanisms of antibacterial activity of the synthetic teixobactin analogue Leu-teixobactin against a MRSA strain,  ATCC 700699. The metabolomes of  ATCC 700699 cells 1, 3, and 6 h following treatment with Leu-teixobactin (0.5 μg/ml, i.e., 0.5× MIC) were compared to those of the untreated controls. Leu-teixobactin significantly perturbed bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan (lipid I and II) metabolism, and cell wall teichoic acid (lipid III) biosynthesis as early as after 1 h of treatment, reflecting an initial activity on the cell envelope. Concordant with its time-dependent antibacterial killing action, Leu-teixobactin caused more perturbations in the levels of key intermediates in pathways of amino-sugar and nucleotide-sugar metabolism and their downstream peptidoglycan and teichoic acid biosynthesis at 3 and 6 h. Significant perturbations in arginine metabolism and the interrelated tricarboxylic acid cycle, histidine metabolism, pantothenate, and coenzyme A biosynthesis were also observed at 3 and 6 h. To conclude, this is the first study to provide novel metabolomics mechanistic information, which lends support to the development of teixobactin as an antibacterial drug for the treatment of multidrug-resistant Gram-positive infections. Antimicrobial resistance is one of the greatest threats to the global health system. It is imperative that new anti-infective therapeutics be developed against problematic "superbugs." The cyclic depsipeptide teixobactin holds much promise as a new class of antibiotics for highly resistant Gram-positive pathogens (e.g., methicillin-resistant  [MRSA]). Understanding its molecular mechanism(s) of action could lead to the design of new compounds with a broader activity spectrum. Here, we describe the first metabolomics study to investigate the killing mechanism(s) of teixobactin against MRSA. Our findings revealed that teixobactin significantly disorganized the bacterial cell envelope, as reflected by a profound perturbation in the bacterial membrane lipids and cell wall biosynthesis (peptidoglycan and teichoic acid). Importantly, teixobactin significantly suppressed the main intermediate d-alanyl-d-lactate involved in the mechanism of vancomycin resistance in  These novel results help explain the unique mechanism of action of teixobactin and its lack of cross-resistance with vancomycin.

The Killing Mechanism of Teixobactin against Methicillin-Resistant Staphylococcus aureus: an Untargeted Metabolomics Study.

A post-antibiotic world is fast becoming a reality, given the rapid emergence of pathogens that are resistant to current drugs. Therefore, there is an urgent need to discover new classes of potent antimicrobial agents with novel modes of action.  is an herbaceous plant that has been used for millennia for medicinal and recreational purposes. Its bioactivity is largely due to a class of compounds known as cannabinoids. Recently, these natural products and their analogs have been screened for their antimicrobial properties, in the quest to discover new anti-infective agents. This paper seeks to review the research to date on cannabinoids in this context, including an analysis of structure-activity relationships. It is hoped that it will stimulate further interest in this important issue.

The Antimicrobial Activity of Cannabinoids.

Based on its clinical benefits, Trikafta - the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) - was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661 plus VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to approximately 62% of WT CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K, and N1303K), confined to MSD1, MSD2, NBD1, and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.

Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination.

The status quo for combating uprising antibacterial resistance is to employ synergistic combinations of antibiotics. Nevertheless, the currently available combination therapies are fast becoming untenable. Combining antibiotics with various FDA-approved non-antibiotic drugs has emerged as a novel strategy against otherwise untreatable multi-drug resistant (MDR) pathogens. The apex of this study was to investigate the mechanisms of antibacterial synergy of the combination of polymyxin B with the phenothiazines against the MDR Gram-negative pathogens Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa. The synergistic antibacterial effects were tested using checkerboard and static time-kill assays. Electron microscopy (EM) and untargeted metabolomics were used to ascertain the mechanism(s) of the antibacterial synergy. The combination of polymyxin B and the phenothiazines showed synergistic antibacterial activity in checkerboard and static time-kill assays at clinically relevant concentrations against both polymyxin-susceptible and polymyxin-resistant isolates. EM revealed that the polymyxin B-prochlorperazine combination resulted in greater damage to the bacterial cell compared to each drug monotherapy. In metabolomics, at 0.5 h, polymyxin B monotherapy and the combination (to a greatest extent) disorganised the bacterial cell envelope as manifested by a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids), peptidoglycan and lipopolysaccharide (LPS) biosynthesis. At the late time exposure (4 h), the aforementioned effects (except LPS biosynthesis) perpetuated mainly with the combination therapy, indicating the disorganising bacterial membrane biogenesis is potentially behind the mechanisms of antibacterial synergy. In conclusion, the study highlights the potential usefulness of the combination of polymyxin B with phenothiazines for the treatment of polymyxin-resistant Gram-negative infections (e.g. CNS infections).

Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens.

 is an opportunistic Gram-negative pathogen causing nosocomial infections.  rapidly acquires antibiotic resistance and is known as a reservoir for resistance genes. Polymyxins remain effective as a last-line therapy against infections caused by multidrug-resistant (MDR) ; however, resistance to polymyxins emerges rapidly with monotherapy. Synergistic combinations of polymyxins with FDA-approved non-antibiotics are a novel approach to preserve its efficacy whilst minimising the emergence of polymyxin resistance in . This study aimed to investigate the synergistic antibacterial activity of polymyxin B in combination with the anti-fungal caspofungin against . The combination of polymyxin B and caspofungin showed marked synergistic antibacterial killing activity in checkerboard broth microdilution and static time-kill assays at clinically relevant concentrations at early (0.5 and 1 h) and later (4 h) time points. The potential bacterial killing mechanism of the combination was studied against  FADDI-KP001 using metabolomics and transcriptomics studies at 0.5, 1 and 4 h. The key pathways involved in the synergistic killing action of the combination were cell wall assembly (peptidoglycan and lipopolysaccharide biosynthesis), central carbon metabolism (glycolysis, pentose phosphate pathway and tricarboxylic acid cycle) and fatty acid biosynthesis. Moreover, the combination inhibited the most common bacterial virulence pathway (phosphotransferase system) as well as the multi-resistant efflux mechanisms, including ATP-binding cassette (ABC) transporter pathway. Overall, this study sheds light on the possibility of a polymyxin-caspofungin combination for the treatment of infections caused by  and may help repurpose FDA-approved caspofungin against MDR  infections.

Unique mechanistic insights into pathways associated with the synergistic activity of polymyxin B and caspofungin against multidrug-resistant .

Polymyxins have resurged as the last-resort antibiotics against multidrug-resistant . As reports of polymyxin resistance in  with monotherapy have become increasingly common, combination therapy is usually the only remaining treatment option. A novel and effective strategy is to combine polymyxins with non-antibiotic drugs. This study aimed to investigate, using untargeted metabolomics, the mechanisms of antibacterial killing synergy of the combination of polymyxin B with a synthetic cannabidiol against  ATCC 19606. The antibacterial synergy of the combination against a panel of Gram-negative pathogens (,  and ) was also explored using checkerboard and static time-kill assays. The polymyxin B-cannabidiol combination showed synergistic antibacterial activity in checkerboard and static time-kill assays against both polymyxin-susceptible and polymyxin-resistant isolates. The metabolomics study at 1 h demonstrated that polymyxin B monotherapy and the combination (to the greatest extent) significantly perturbed the complex interrelated metabolic pathways involved in the bacterial cell envelope biogenesis (amino sugar and nucleotide sugar metabolism, peptidoglycan, and lipopolysaccharide (LPS) biosynthesis), nucleotides (purine and pyrimidine metabolism) and peptide metabolism; notably, these pathways are key regulators of bacterial DNA and RNA biosynthesis. Intriguingly, the combination caused a major perturbation in bacterial membrane lipids (glycerophospholipids and fatty acids) compared to very minimal changes induced by monotherapies. At 4 h, polymyxin B-cannabidiol induced more pronounced effects on the abovementioned pathways compared to the minimal impact of monotherapies. This metabolomics study for the first time showed that in disorganization of the bacterial envelope formation, the DNA and RNA biosynthetic pathways were the most likely molecular mechanisms for the synergy of the combination. The study suggests the possibility of cannabidiol repositioning, in combination with polymyxins, for treatment of MDR polymyxin-resistant Gram-negative infections.

ArticleTotal : 2	Year
Publication title Cited by 3	2022
Publication title Cited by 5	2022

Article	Year
Lipidomic Analysis of the Outer Membrane Vesicles from Paired Polymyxin-Susceptible and -Resistant Clinical Isolates. International journal of molecular sciences\| PubMed ID: 30103446	2018
A Comparative Study of Outer Membrane Proteome between Paired Colistin-Susceptible and Extremely Colistin-Resistant Klebsiella pneumoniae Strains. ACS infectious diseases\| PubMed ID: 30232886	2018
Mechanistic Insights From Global Metabolomics Studies into Synergistic Bactericidal Effect of a Polymyxin B Combination With Tamoxifen Against Cystic Fibrosis MDR . Computational and structural biotechnology journal\| PubMed ID: 30546859	2018
The impact of backbone N-methylation on the structure-activity relationship of Leu -teixobactin. Journal of peptide science : an official publication of the European Peptide Society\| PubMed ID: 31389086	2019
Metabolomics Study of the Synergistic Killing of Polymyxin B in Combination with Amikacin against Polymyxin-Susceptible and -Resistant Pseudomonas aeruginosa. Antimicrobial agents and chemotherapy\| PubMed ID: 31611351	2019
Synthesis and structure-activity relationships of teixobactin. Annals of the New York Academy of Sciences\| PubMed ID: 31792983	2020
Effective Strategy Targeting Polymyxin-Resistant Gram-Negative Pathogens: Polymyxin B in Combination with the Selective Serotonin Reuptake Inhibitor Sertraline. ACS infectious diseases\| PubMed ID: 32427476	2020
The Killing Mechanism of Teixobactin against Methicillin-Resistant Staphylococcus aureus: an Untargeted Metabolomics Study. mSystems\| PubMed ID: 32457238	2020
The Antimicrobial Activity of Cannabinoids. Antibiotics (Basel, Switzerland)\| PubMed ID: 32668669	2020
Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination. JCI insight\| PubMed ID: 32853178	2020
Polymyxin B combinations with FDA-approved non-antibiotic phenothiazine drugs targeting multi-drug resistance of Gram-negative pathogens. Computational and structural biotechnology journal\| PubMed ID: 32952938	2020
Unique mechanistic insights into pathways associated with the synergistic activity of polymyxin B and caspofungin against multidrug-resistant . Computational and structural biotechnology journal\| PubMed ID: 35284046	2022
Mechanisms Underlying Synergistic Killing of Polymyxin B in Combination with Cannabidiol against : A Metabolomic Study. Pharmaceutics\| PubMed ID: 35456620	2022

Maytham Hussein

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