ABSTRACT
BACKGROUND: The aminoglycoside apramycin has been proposed as a drug candidate for the treatment of critical Gram-negative systemic infections. However, the potential of apramycin in the treatment of drug-resistant bloodstream infections (BSIs) has not yet been assessed. METHODS: The resistance gene annotations of 40â888 blood-culture isolates were analysed. In vitro profiling of apramycin comprised cell-free translation assays, broth microdilution, and frequency of resistance determination. The efficacy of apramycin was studied in a mouse peritonitis model for a total of nine Escherichia coli and Klebsiella pneumoniae isolates. RESULTS: Genotypic aminoglycoside resistance was identified in 87.8% of all 6973 carbapenem-resistant Enterobacterales blood-culture isolates, colistin resistance was shown in 46.4% and apramycin in 2.1%. Apramycin activity against methylated ribosomes was > 100-fold higher than that for other aminoglycosides. Frequencies of resistance were < 10-9 at 8 × minimum inhibitory concentration (MIC). Tentative epidemiological cut-offs (TECOFFs) were determined as 8 µg/mL for E. coli and 4 µg/mL for K. pneumoniae. A single dose of 5 to 13 mg/kg resulted in a 1-log colony-forming unit (CFU) reduction in the blood and peritoneum. Two doses of 80 mg/kg resulted in an exposure that resembles the AUC observed for a single 30 mg/kg dose in humans and led to complete eradication of carbapenem- and aminoglycoside-resistant bacteraemia. CONCLUSION: Encouraging coverage and potent in vivo efficacy against a selection of highly drug-resistant Enterobacterales isolates in the mouse peritonitis model warrants the conduct of clinical studies to validate apramycin as a drug candidate for the prophylaxis and treatment of BSI.
Subject(s)
Aminoglycosides , Anti-Bacterial Agents , Carbapenems , Disease Models, Animal , Escherichia coli , Klebsiella Infections , Klebsiella pneumoniae , Microbial Sensitivity Tests , Nebramycin , Animals , Klebsiella pneumoniae/drug effects , Klebsiella pneumoniae/genetics , Nebramycin/analogs & derivatives , Nebramycin/pharmacology , Nebramycin/therapeutic use , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Escherichia coli/drug effects , Escherichia coli/genetics , Mice , Carbapenems/pharmacology , Carbapenems/therapeutic use , Aminoglycosides/pharmacology , Aminoglycosides/therapeutic use , Klebsiella Infections/drug therapy , Klebsiella Infections/microbiology , Peritonitis/drug therapy , Peritonitis/microbiology , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Bacteremia/drug therapy , Bacteremia/microbiology , Humans , Female , Carbapenem-Resistant Enterobacteriaceae/drug effects , Drug Resistance, BacterialABSTRACT
Aminoglycoside antibiotics are powerful bactericidal therapeutics that are often used in the treatment of critical Gram-negative systemic infections. The emergence and global spread of antibiotic resistance, however, has compromised the clinical utility of aminoglycosides to an extent similar to that found for all other antibiotic-drug classes. Apramycin, a drug candidate currently in clinical development, was suggested as a next-generation aminoglycoside antibiotic with minimal cross-resistance to all other standard-of-care aminoglycosides. Here, we analyzed 591,140 pathogen genomes deposited in the NCBI National Database of Antibiotic Resistant Organisms (NDARO) for annotations of apramycin-resistance genes, and compared them to the genotypic prevalence of carbapenem resistance and 16S-rRNA methyltransferase (RMTase) genes. The 3-N-acetyltransferase gene aac(3)-IV was found to be the only apramycin-resistance gene of clinical relevance, at an average prevalence of 0.7%, which was four-fold lower than that of RMTase genes. In the important subpopulation of carbapenemase-positive isolates, aac(3)-IV was nine-fold less prevalent than RMTase genes. The phenotypic profiling of selected clinical isolates and recombinant strains expressing the aac(3)-IV gene confirmed resistance to not only apramycin, but also gentamicin, tobramycin, and paromomycin. Probing the structure-activity relationship of such substrate promiscuity by site-directed mutagenesis of the aminoglycoside-binding pocket in the acetyltransferase AAC(3)-IV revealed the molecular contacts to His124, Glu185, and Asp187 to be equally critical in binding to apramycin and gentamicin, whereas Asp67 was found to be a discriminating contact. Our findings suggest that aminoglycoside cross-resistance to apramycin in clinical isolates is limited to the substrate promiscuity of a single gene, rendering apramycin best-in-class for the coverage of carbapenem- and aminoglycoside-resistant bacterial infections.
Subject(s)
Acetyltransferases/genetics , Aminoglycosides/pharmacology , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Gram-Negative Bacteria/genetics , Gram-Negative Bacterial Infections/microbiology , Acetyltransferases/chemistry , Acetyltransferases/metabolism , Aminoglycosides/chemistry , Anti-Bacterial Agents/chemistry , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Carbapenems/pharmacology , Catalytic Domain , Databases, Genetic , Genome, Bacterial/drug effects , Gram-Negative Bacteria/drug effects , Gram-Negative Bacterial Infections/drug therapy , Humans , Methyltransferases/chemistry , Methyltransferases/genetics , Methyltransferases/metabolism , Molecular Epidemiology , Mutagenesis, Site-Directed , Nebramycin/analogs & derivatives , Nebramycin/pharmacology , Standard of Care , Structure-Activity RelationshipABSTRACT
R-type bacteriocins are minimal contractile nanomachines that hold promise as precision antibiotics1-4. Each bactericidal complex uses a collar to bridge a hollow tube with a contractile sheath loaded in a metastable state by a baseplate scaffold1,2. Fine-tuning of such nucleic acid-free protein machines for precision medicine calls for an atomic description of the entire complex and contraction mechanism, which is not available from baseplate structures of the (DNA-containing) T4 bacteriophage5. Here we report the atomic model of the complete R2 pyocin in its pre-contraction and post-contraction states, each containing 384 subunits of 11 unique atomic models of 10 gene products. Comparison of these structures suggests the following sequence of events during pyocin contraction: tail fibres trigger lateral dissociation of baseplate triplexes; the dissociation then initiates a cascade of events leading to sheath contraction; and this contraction converts chemical energy into mechanical force to drive the iron-tipped tube across the bacterial cell surface, killing the bacterium.
Subject(s)
Pseudomonas aeruginosa , Pyocins/chemistry , Pyocins/metabolism , Bacteriophage T4/chemistry , Bacteriophage T4/metabolism , Cryoelectron Microscopy , Crystallography, X-Ray , Genes, Bacterial/genetics , Models, Molecular , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , Pseudomonas aeruginosa/chemistry , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/metabolism , Substrate Specificity , Type VI Secretion Systems/chemistry , Type VI Secretion Systems/metabolismABSTRACT
Bacteriophages recognize their host cells with the help of tail fiber and tailspike proteins that bind, cleave, or modify certain structures on the cell surface. The spectrum of ligands to which the tail fibers and tailspikes can bind is the primary determinant of the host range. Bacteriophages with multiple tailspike/tail fibers are thought to have a wider host range than their less endowed relatives but the function of these proteins remains poorly understood. Here, we describe the structure, function, and substrate specificity of three tailspike proteins of bacteriophage CBA120-TSP2, TSP3 and TSP4 (orf211 through orf213, respectively). We show that tailspikes TSP2, TSP3 and TSP4 are hydrolases that digest the O157, O77, and O78 Escherichia coli O-antigens, respectively. We demonstrate that recognition of the E. coli O157:H7 host by CBA120 involves binding to and digesting the O157 O-antigen by TSP2. We report the crystal structure of TSP2 in complex with a repeating unit of the O157 O-antigen. We demonstrate that according to the specificity of its tailspikes TSP2, TSP3, and TSP4, CBA120 can infect E. coli O157, O77, and O78, respectively. We also show that CBA120 infects Salmonella enterica serovar Minnesota, and this host range expansion is likely due to the function of TSP1. Finally, we describe the assembly pathway and the architecture of the TSP1-TSP2-TSP3-TSP4 branched complex in CBA120 and its related ViI-like phages.
Subject(s)
Bacteriophages/metabolism , Viral Proteins/chemistry , Viral Proteins/metabolism , Crystallography, X-Ray , Escherichia coli O157/metabolism , Escherichia coli Proteins/metabolism , Host Specificity , Models, Molecular , Peptides/chemistry , Peptides/metabolism , Protein Binding , Protein Domains , Proteolysis , Salmonella enterica/virology , Static Electricity , Structure-Activity Relationship , Substrate SpecificityABSTRACT
The feasibility of (1)H-High Resolution-Magic Angle Spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy for the direct analysis of viscous cosmetic and pharmaceutical formulations such as creams, gels, and pastes is presented. Three examples are described: (i) the detection of chitosan in toothpaste, (ii) the analysis of dexamethasone acetate (DMA) in a cream, and (iii) the analysis of the local anesthetics, lidocaine and prilocaine, in a gel and a cream. All active components could be directly detected in their original commercial formulations without the need for laborious sample preparation steps. In addition, the possibility for HR-MAS-based quantifications and the analysis of dynamic properties of active components in different formulations applying HR-MAS diffusion-ordered NMR spectroscopy are shown.