Now a large international team spanning Spain and Slovenia has delivered what the field has lacked for decades: a single, rigorously controlled comparison of 26 aminoglycosides and their molecular components, tested side by side under identical conditions. The study, led by Nadia García-Mateo, Hrvoje Petković, and M. Beatriz Durán-Alonso, appears in Frontiers in Pharmacology. The researchers evaluated antibacterial potency against 61 well-characterized clinical isolates from the ESKAPE panel, a collection that includes carbapenemase-producing enterobacteria, multidrug-resistant Pseudomonas aeruginosa, and multidrug-resistant Acinetobacter baumannii, the pathogens that pose the gravest threat to hospitalized patients. In parallel, they measured toxicity using two otic cell lines derived from the organ of Corti, the OC-k3 and HEI-OC1 lines, plus the porcine kidney cell line LLC-PK1, and they extended key findings to organotypic cochlear explants from newborn mice.

The antibacterial screen produced clear winners. Eight commercial aminoglycosides demonstrated strong activity against the multidrug-resistant isolates: amikacin, apramycin, gentamicin, isepamicin, neomycin, paromomycin, sisomicin, and tobramycin. Apramycin and amikacin emerged as the most broadly active agents, each inhibiting 90 percent of the isolates at 32 milligrams per liter, while several competitors failed to reach meaningful inhibition even at 64 milligrams per liter. The lowest MIC50 value, just 2 milligrams per liter, belonged to the neomycin preparations, and the neomycin B congener showed the strongest individual performance. Four isolated congeners, gentamicin C1a, C2, C2a, and neomycin B, also proved potent, alongside the intriguing observation that kanamycin B slightly outperformed both its parental mixture and the kanamycin A congener.

Toxicity testing revealed a far more complicated picture. Hygromycin B was the most toxic compound on both otic cell lines even at low concentrations and short exposures, while kasugamycin and streptomycin also damaged both lines significantly. Kanamycin, ribostamycin, and tobramycin showed no significant loss of cell viability in the MTT assays, and apramycin, isepamicin, paromamine, and paromomycin were toxic only at the higher 5 millimolar concentration. Notably, the two otic cell lines sometimes disagreed: sisomicin selectively damaged OC-k3 cells while sparing HEI-OC1 cultures, and amikacin’s apparent toxicity to HEI-OC1 cells in the MTT assay vanished when the researchers used an ATP-based viability test instead. The cochlear explant data suggested the ATP assay and the OC-k3 line together offer the more reliable predictions.

The most striking results came from dissecting gentamicin itself. Commercial gentamicin is not a single molecule but a fermentation-derived mixture of structurally related congeners, dominated by the C-subtypes C1, C1a, C2, C2a, and C2b, with minor quantities of A, B, B1, and X2 variants. For the first time, the team directly compared seven of the most common gentamicin congeners for both antibacterial activity and toxicity. At 2 millimolar concentrations, gentamicin B1 and gentamicin X2 devastated the otic cell lines, dropping viability to single digits or low double digits on the OC-k3 line, an outcome dramatically worse than the gentamicin mixture itself, which left more than 80 percent of cells viable. These congeners also showed very weak antibacterial activity, with gentamicin X2 among the poorest inhibitors of the bacterial ribosome, and gentamicin X2 proved the most nephrotoxic of all the gentamicin congeners on the LLC-PK1 kidney cell line.

The implications for clinical practice are sobering. Because the congener composition of commercial gentamicin varies across manufacturers and even between fermentation batches, even small amounts of B1 or X2 contamination could meaningfully alter the overall toxicity of a given formulation. European regulators already require gentamicin B1 to be identified as a specified impurity, but gentamicin X2 has not received the same scrutiny. The researchers point to structural clues: gentamicin B1 and X2 differ from the C-group at specific positions on the first sugar ring, echoing the arrangement found in sisomicin, which also showed high ototoxicity. They suggest that the exact geometry of this ring likely modulates how these compounds interact with cellular targets, though the precise mechanisms behind the extreme toxicity of B1 and X2 remain unresolved and are probably multifactorial.

The ex vivo work on murine cochlear explants sharpened the picture further. Amikacin and isepamicin left both outer and inner hair cells essentially unscathed, with survival rates slightly above untreated controls, while tobramycin and sisomicin destroyed most hair cells in the cultured cochlear epithelium. Interestingly, applying plain neomycin caused a significant loss of outer hair cells that was not observed with the neomycin B congener, even though the two performed identically in cell-line assays. The explants also reproduced a well-known vulnerability gradient, with hair cells at the basal end of the cochlea, which encode high frequencies, dying first. The concentration used, 0.1 millimolar, sits within the range of peak plasma levels measured in patients treated with gentamicin, isepamicin, or amikacin, lending physiological relevance to the model.

On the kidney side, amikacin earned a singular distinction: it was the only strongly antibacterial aminoglycoside that produced no significant nephrotoxicity in the LLC-PK1 proximal tubule cell line. This aligns with clinical experience, in which amikacin is generally regarded as less nephrotoxic than gentamicin. Surprisingly, every isolated gentamicin congener, including the least toxic C1a and C2a variants, proved more nephrotoxic than the commercial mixture, and neomycin B was significantly more toxic to kidney cells than its parental mixture. Aminoglycosides accumulate in kidney lysosomes and bind negatively charged phospholipids, but the researchers could not find a simple correlation between charge or structure and the observed nephrotoxicity ranking, underscoring how much remains unknown about these mechanisms.

The authors are careful about the limits of their work. All toxicity measurements were performed on cell lines and explants at concentrations well above those typically found in patients’ plasma, which vary with dosing regimens, tissue distribution, and the infectious agent, so confirmation in living animal models, such as guinea pig cochlear studies, will be essential before the findings reshape clinical practice. Still, the methodological validation is strong: this same in vitro pipeline previously flagged gentamicin C1a as less ototoxic than the mixture, a result later confirmed in vivo, and the consistent toxicity signals from gentamicin B1 and X2 across every assay lend credibility to the alarm. As biosynthetic engineering and scalable purification now make it economically feasible to isolate individual congeners at industrial scale, the study suggests a dual path forward: refining existing formulations to exclude the most dangerous minor components, and mining natural aminoglycoside diversity, exemplified by semisynthetic successes like plazomicin and etimicin, for new drug leads that separate bacterial killing from cochlear and renal destruction.

Subject of Research: Comparative antibacterial activity, ototoxicity and nephrotoxicity of aminoglycoside antibiotics and their congeners

Article Title: Comparative evaluation of the antibacterial activity and toxicity of a large panel of aminoglycoside antibiotics

Article References: García-Mateo, N., López-Hernández, I., Čusak, A., Fernández-Martínez, M., Šala, M., Schimmang, T., Kosec, G., Fujs, Š., Martínez-Martínez, L., Petković, H., & Durán-Alonso, M. B. (2026). Comparative evaluation of the antibacterial activity and toxicity of a large panel of aminoglycoside antibiotics. Frontiers in Pharmacology, 17, Article 1930759. https://doi.org/10.3389/fphar.2026.1930759

Image Credits: AI Generated

DOI: 10.3389/fphar.2026.1930759

Keywords: aminoglycosides, gentamicin, antibiotic resistance, ototoxicity, nephrotoxicity, ESKAPE pathogens, congeners, cochlear explants, kidney cell line, MIC90, multidrug resistance, antimicrobial susceptibility