Apramycin

Antimicrobial resistance is a major global health concern that requires innovative therapeutic strategies. This study aimed to address this challenge by designing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein (CRISPR-Cas)-based plasmid systems for potential genome editing applications in multidrug-resistant (MDR) Klebsiella pneumoniae clinical isolates. Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem were determined according to European Committee on Antimicrobial Susceptibility Testing guidelines. All isolates (n = 5) were resistant, with MIC ranges of 4–128 μg/ml for imipenem, 8–64 μg/ml for meropenem, and 8–256 μg/ml for ertapenem. Resistance gene analysis revealed blaOXA-48-like and blaCTX-M-15 in all isolates, while blaNDM-1 was detected in one isolate. Two CRISPR-based plasmid systems, CRISPR-Cas9 and CRISPR-assisted cytidine deaminase, were designed. Target genes were amplified by polymerase chain reaction, and guide RNA (gRNA) sequences were designed from selected regions. Apramycin (50 μg/ml) was identified as a suitable selection marker. The pSGKP–AmpR(Pro)–ApmR plasmid was successfully constructed, whereas Cas9 and APOBEC constructs could not be cloned. Overall, this study highlights technical challenges in developing CRISPR-based tools for MDR K. pneumoniae and emphasizes the need for isolate-specific plasmid design and gRNA optimization.

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.

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.

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

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.

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