Publications by Year: 2025

Fusidic acid is a translation inhibitor with activity against major Gram-positive bacterial pathogens such as S. aureus. However, its activity against Gram-negatives is poor based on an inability to access its cytoplasmic target in these organisms. Opportunities for functionalization of the fusidic acid scaffold to enhance activity against Gram-negative pathogens have not been explored. Using an activity-guided synthetic strategy, the tolerance of the tetracyclic natural product to derivatization at the A- and C-rings and its carboxylic acid side chain was explored with the goal of enhancing its activity spectrum and pharmacological properties. All side-chain carboxylic acid esters were inactive. Oxidation of the C-ring alcohol and oxime were not tolerated either. A number of esters of the A-ring alcohol retained modest activity against Gram-positive bacteria and were informative for future activity-guided studies. For the A-ring esters, differences in antibacterial activity relative to inhibitory activity in a ribosome in vitro translation assay suggested the possibility of a pro-druglike effect for the fusidic acid pyrazine-2-carboxylate. This study furthers the understanding of the activity of the fusidic acid scaffold against Gram-positive bacteria. These results suggest promise for future modification of the A-ring alcohol of fusidic acid in the advancement of its antibiotic properties.

Carbapenemase producing Enterobacterales (CPEs) represent a group of multidrug resistant pathogens for which few, if any, therapeutics options remain available. CPEs generally harbor plasmids that encode resistance to last resort carbapenems and many other antibiotics. We previously performed a high throughput screen to identify compounds that can disrupt the maintenance and replication of resistance conferring plasmids through use of a synthetic screening plasmid introduced into Escherichia coli K-12 tolC cells. Despite being identified as a potent and selective antiplasmid agent through this screening effort, CGS-15943 was inactive in wild-type E. coli, suggesting that it is susceptible to TolC-mediated efflux. Herein, a series of analogues were developed to confirm the activity of the triazoloquinazoline chemotype and overcome efflux observed in wild-type E. coli K-12. Two analogues demonstrated superior antiplasmid activity to CGS-15943 in E. coli tolC mutants, while one compound displayed moderate activity in wild-type E. coli at low concentrations.

BACKGROUND: Mycobacteroides abscessus are rapidly growing non-tuberculous mycobacteria that cause chronic lung and soft tissue infections. Treatment options are often severely limited due to intrinsic resistance to most antimicrobials. Amikacin has historically been a mainstay of combination treatment regimens. However, irreversible hearing loss and vestibular toxicity have led to a search for alternative agents. Apramycin is a novel aminoglycoside currently in phase I clinical trials that may offer lower potential for ototoxic and renal toxic side effects.

OBJECTIVES: The goal of this study was to compare apramycin's in vitro activity with amikacin and other aminoglycosides against a large collection of M. abscessus clinical isolates, both alone and in combination with clofazimine or linezolid. We also tested the activity of apramycin against a more limited collection of other species of rapidly growing mycobacteria.

METHODS: Analysis was performed using reference broth microdilution minimal inhibitory concentration testing, inkjet printer-assisted checkerboard assays, and time-kill assays.

RESULTS: Against M. abscessus, the MIC50/90 for apramycin (2 μg/mL) was 8-fold lower than for amikacin (16 μg/mL). Plazomicin was inactive, and organisms were rarely susceptible to tobramycin. Synergy was not detected by checkerboard assay. In time-kill studies, clofazimine modestly potentiated activity of apramycin and. to a lesser extent, amikacin. Apramycin and amikacin showed delayed bacterial killing that either achieved or approached a bactericidal threshold. Apramycin was similarly potent against other rapidly growing mycobacteria tested.

CONCLUSIONS: Apramycin exhibits more potent in vitro activity against a diverse set of M. abscessus and other rapidly growing mycobacteria than approved aminoglycosides.

Synergy of antimicrobial combinations was tested against contemporary Mycobacteroides abscessus isolates using 2D and 3D checkerboard assays. Triple combinations of omadacycline-azithromycin-clofazimine, tigecycline-azithromycin-clofazimine, omadacycline-azithromycin-linezolid, and omadacycline-azithromycin-contezolid demonstrated synergy (fractional inhibitory concentration index ≤ 0.5) against 33%, 31%, 62%, and 66% of isolates, respectively. Notably, in all triple combinations, macrolide-resistant M. abscessus subsp. abscessus and M. abscessus subsp. bolletii isolates were fully sensitized to azithromycin at the FIC index, as were isolates with elevated clofazimine MICs.

UNLABELLED: Pseudomonas aeruginosa, a Gram-negative pathogen, has emerged as one of the most highly antibiotic-resistant bacteria worldwide and subsequently has become a leading cause of healthcare-associated, life-threatening infections. P. aeruginosa multidrug efflux Y (MexY) is an efflux pump that belongs to the resistance-nodulation-cell division (RND) superfamily. It is a major determinant for resistance to aminoglycosides in this opportunistic pathogen. However, the detailed molecular mechanisms involved in aminoglycoside recognition and extrusion by MexY have not been elucidated. Here, we report the cryo-electron microscopy structure of MexY to a resolution of 3.63 Å. The structure directly indicates two plausible pathways for drug export. It also suggests that MexY is capable of picking up antibiotics via the ceiling of the central cavity formed by the MexY trimer. Molecular dynamics simulations depict that MexY is able to use a tunnel connecting the central cavity to the funnel of the trimer to export its substrates.

IMPORTANCE: Here, we report the cryo-electron microscopy structure of the MexY multidrug efflux pump, posing the possibility that this pump is capable of capturing antibiotics from both the central cavity and the periplasmic cleft of the pump. The results indicate that MexY may utilize charged residues to bind and export drugs, mediating resistance to these antibiotics.

Synergy of antimicrobial combinations was tested against contemporary Mycobacteroides abscessus isolates using 2-D and 3-D checkerboard assays. Triple combinations of omadacycline-azithromycin-clofazimine, tigecycline-azithromycin-clofazimine, omadacycline-azithromycin-linezolid and omadacycline-azithromycin-contezolid demonstrated synergy (FIC ≤ 0.5) against 33%, 31%, 62%, and 66% of isolates, respectively. Notably, in all triple combinations, macrolide resistant M. abscessus subsp. abscessus and M. abscessus subsp. bolletii isolates were fully sensitized to azithromycin at the FIC index, as were isolates with elevated clofazimine MICs.

Although artificial intelligence-particularly large-language models-receives daily attention, the application of AI to image-recognition challenges in clinical microbiology has been under development for several years. In the accompanying article, B. A. Mathison, K. Knight, J. Potts, B. Black, et al. (J Clin Microbiol 63:e01062-25, 2025, https://doi.org/10.1128/jcm.01062-25) (in collaboration with ARUP Laboratories and TechCyte) describe a trained convolutional neural network (CNN) that reviews wet-mount parasitology smears with accuracy and analytical sensitivity exceeding that of a cohort of highly trained medical technologists. The impressive results were enabled by an extensive, globally sourced training set. These findings constitute Part II of the authors' earlier Journal of Clinical Microbiology publication on CNN-based diagnosis of trichrome-stained smears and provide a robust proof-of-concept for integrating AI into clinical microbiology workflows. We comment on the translatability of this technology to routine clinical laboratories.

BACKGROUND: Mycobacteroides abscessus is a rapidly growing non-tuberculous mycobacterium that causes chronic lung and soft tissue infections. Treatment options are severely limited. Amikacin has historically been a mainstay of combination treatment regimens. However, irreversible hearing loss and vestibular toxicity have led to a search for alternative agents. Apramycin is a novel aminoglycoside currently in Phase I clinical trials that may offer lower potential for ototoxic and renal toxic side effects.

OBJECTIVES: The goal of this study was to compare apramycin's in vitro activity with amikacin and other aminoglycosides against a large collection of M. abscessus clinical isolates, alone, and for apramycin and amikacin, in combination with clofazimine or linezolid. We also examined activity against a more limited collection of other rapidly growing mycobacteria.

METHODS: Analysis was performed using reference broth microdilution MIC testing, inkjet printer-assisted checkerboard assays, and time-kill assays.

RESULTS: Against M. abscessus, the MIC50/90 for apramycin (2 mg/L) was one-eighth that of amikacin (16 mg/L). Plazomicin was inactive, and organisms were rarely susceptible to tobramycin. Synergy of either apramycin or amikacin with clofazimine or linezolid was not detected by checkerboard assay. In time-kill studies, clofazimine modestly increased activity of apramycin and, to a lesser extent, amikacin. Apramycin and amikacin showed delayed bacterial killing that either achieved or approached a bactericidal threshold. Apramycin was similarly potent against other rapidly growing mycobacteria tested.

CONCLUSIONS: Apramycin exhibits more potent in vitro activity against a diverse set of M. abscessus and other rapidly growing mycobacteria than currently recommended aminoglycosides.

RNA G-quadruplexes (rG4s) are unusual RNA secondary structures formed by stacking arrays of guanine tetrads. Although thousands of potential rG4-forming motifs occur throughout the mammalian transcriptome, many single-stranded RNA (ssRNA) viruses are thought to be depleted of rG4-forming sequences. Using in silico methods, we examine rG4-forming potential in single-stranded RNA (ssRNA) viruses and observe that, while canonical rG4 motifs are depleted, noncanonical rG4 motifs occur at comparable or higher frequencies relative to the mammalian transcriptome. We ask if the noncanonical rG4's can be leveraged to block viral replication and control infection using OC43, the coronavirus believed to be responsible for the 1889 "Russian flu" pandemic. Profiling with "d-rG4-seq" confirms a dearth of folded rG4 in the OC43 RNA genome during natural infection. Intriguingly, rG4 ligands induce synthetic rG4 structures of a noncanonical nature. Significantly, induced rG4 inhibits viral replication and reduces infectivity. We show that the rG4 ligands act by disrupting the unique pattern of OC43 "discontinuous transcription." Thus, rG4-targeting compounds present a potential therapeutic approach for targeting ssRNA viruses.

An in silico designed benzodioxin fused analogue of a des-(1-hydroxyethyl)-lincomycin analogue was synthesized in a de novo asymmetric fashion from an achiral acylfuran, a 4-(n-Pr)-N-methyl-proline, and catechol. The synthesis of the 6-amino-galactose portion of the lincomycin analogue necessitated the development of a novel stereospecific tandem Pd-glycosylation/1,4-addition reaction between catechol and an N-Cbz-protected 6-amino-pyranone with a Pd-π-allyl leaving group at the C-1 position. The desired galacto-stereochemistry was installed by a subsequent stereoselective ketone reduction, alcohol elimination, and diastereoselective dihydroxylation of the C-3/4 alkene.