Lower ototoxicity and absence of hidden hearing loss point to gentamicin C1a and apramycin as promising antibiotics for clinical use

Abstract

Spread of antimicrobial resistance and shortage of novel antibiotics have led to an urgent need for new antibacterials. Although aminoglycoside antibiotics (AGs) are very potent anti-infectives, their use is largely restricted due to serious side-effects, mainly nephrotoxicity and ototoxicity. We evaluated the ototoxicity of various AGs selected from a larger set of AGs on the basis of their strong antibacterial activities against multidrug-resistant clinical isolates of the ESKAPE panel: gentamicin, gentamicin C1a, apramycin, paromomycin and neomycin. Following local round window application, dose-dependent effects of AGs on outer hair cell survival and compound action potentials showed gentamicin C1a and apramycin as the least toxic. Strikingly, although no changes were observed in compound action potential thresholds and outer hair cell survival following treatment with low concentrations of neomycin, gentamicin and paromomycin, the number of inner hair cell synaptic ribbons and the compound action potential amplitudes were reduced. This indication of hidden hearing loss was not observed with gentamicin C1a or apramycin at such concentrations. These findings identify the inner hair cells as the most vulnerable element to AG treatment, indicating that gentamicin C1a and apramycin are promising bases for the development of clinically useful antibiotics.

Figure 1

Viability of OC-k3 and HEI-OC1 cell cultures following AG treatments. Selected AGs were applied to the OC-k3 and HEI-OC1 otic cell lines at 2 mM (a) and 5 mM (b). After 48-h, the viability of the cells were determined using the MTT test, comparing the treated cells to the control cells (100%). Data are presented as means ± SD. *p < 0.05; **p < 0.01 (two-tailed unpaired Student’s t-tests used to compare each treatment to controls). (a) 2 mM AGs, OC-k3 cells: GM C1a, p = 0.3; Apra, p = 0.87; GM, p = 0.00000; Paro, p = 0.8; Neo, p = 0.016; (a) 2 mM AGs, HEI-OC1 cells: GM C1a, p = 0.01; Apra, p = 0.18; GM, p = 0.016; Paro, p = 0.051; Neo, p = 1.99E-5; (b) 5 mM AGs, OC-k3 cells: GM C1a, p = 0.003; Apra, p = 0.009; GM, p = 0.01; Paro, p = 0.043; Neo, p = 0.0005; (b) 5 mM AGs, HEI-OC1 cells: GM C1a, p = 0.006; Apra, p = 3.3E-6; GM, p = 0.001; Paro, p = 0.0001; Neo, p = 5.5E-5). Abbreviations: AG, aminoglycoside; Apra, apramycin; GM, gentamicin; GM C1a, gentamicin C1a; MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; Neo, neomycin; Paro, paromomycin.

Figure 2

Evaluation of AG-induced ototoxicity on organotypic cultures of cochlear epithelia. (a) Explant cultures were treated with 0.1 mM AG for 23 h, and surviving HCs were detected following immunostaining of their stereocillia with phalloidin (a; from left to right: apical, middle, basal zones). Scale bar, 50 µm. (b) Proportions of surviving OHCs (full bars) and IHCs (striped bars) in AG-treated explants, relative to controls (100%). Data are presented as means ± SD. *p < 0.05; **p < 0.01 (two-tailed unpaired Student’s t-tests used to compare numbers of surviving HCs in each treatment group, to those in untreated controls). OHC survival: GM C1a, p = 0.01; Apra, p = 0.52; GM, p = 3.7E-8; Paro, p = 0.06; Neo, p = 0.044; IHC survival: GM C1a, p = 1.44E-8; Apra, p = 0.92; GM, p = 5.49E-7; Paro, p = 0.98; Neo, p = 0.36). A clear reduction in the numbers of surviving IHCs was observed in explants treated with GM and GM C1a. Here, IHC loss was similar for both AGs (26.5% ± 7.7%; 16.5% ± 6.3%; of surviving IHCs following GM and GM C1a treatments, respectively (p > 0.05)). Abbreviations: AG, aminoglycoside; Apra, apramycin; GM, gentamicin; GM C1a, gentamicin C1a; HC, hair cell; IHC, inner hair cell; Neo, neomycin; OHC, outer hair cell; Paro, paromomycin.

Figure 3

AG-induced cochleotoxicity on CAP threshold and OHC survival. (a) Comparison of CAP threshold shifts for the ‘basal region’ on day 21, in 60, 210 and 420 mg/mL groups. (b) Representative immunohistochemistry images of the basal cochlear turn showing HC loss following AG application, on day 21. Scale bar, 40 μm. (c) Comparison of surviving OHCs among six groups at three AG concentrations, on day 21. Surviving OHCs were counted in whole mounts of the basal cochlear turn along the basilar membrane. Data are shown as mean number of surviving OHCs per 100 µm distance of basilar membrane. (d) Analysis of the correlation between CAP threshold and OHC survival. X-axis, number of surviving OHCs per 10 µm distance of basilar membrane; Y-axis, CAP threshold shift (dB). The data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey’s multiple comparison tests). (a) 60 mg/mL p = 0.2082; 210 mg/mL p = 0.0044, 420 mg/mL p = 0.0024; (c) 60 mg/mL p = 0.2842, 210 mg/mL p = 0.1123, 420 mg/mL p = 0.0086. Abbreviations: AG, aminoglycoside; Apra, apramycin; CAP, compound action potential; GM, gentamicin; GM C1a, gentamicin C1a; IHC, inner hair cell; Neo, neomycin; O or OHC, outer hair cell; Paro, paromomycin.

Figure 4

AG-induced cochleotoxicity on CAP amplitude and survival of IHC elements. (a) Comparison of ratios of CAP amplitudes (day 21/ before application) for the ‘basal region’ (60, 420 mg/mL). (b) Quantitative analysis of surviving IHCs, on day 21. (c) Survival of synaptic ribbons, on day 21, following administration of 60 or 420 mg/mL AGs. Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey’s multiple comparison tests). (a) 60 mg/mL p < 0.0001, 420 mg/mL p = 0.0009; (b) 60 mg/mL p = 0.0764, 420 mg/mL p = 0.1194; (c) 60 mg/mL p = 0.0051, 420 mg/mL p = 0.0011). Correlations between the ratio of CAP amplitude (day 21/before application)/CAP threshold shifts and synaptic ribbon number in animals receiving 60 mg/mL AGs (n = 34) (d). Representative immunohistochemistry images showing synaptic ribbon loss in the IHCs after applying either control solution or AGs. (e) Scale bar, 20 µm. Abbreviations: AG, aminoglycoside; Apra, apramycin; CAP, compound action potential; CtBP2, C-terminal-binding protein 2; GM, gentamicin; GM C1a, gentamicin C1a; Neo, neomycin; Paro, paromomycin.

Figure 5

AG-induced effects on SP-, CAP- and SP/CAP amplitudes. (a) SP and CAP measurements in the control group (left) and the AG-treated group, before and 21 days after the application of 60 mg/mL GM (right), recorded at 80 dB sound pressure level of 22.6 kHz. (b) SP amplitude ratio among six groups on day 21. (c) SP/CAP amplitude ratio among seven groups (before application in all groups (n = 36), control and those receiving AGs). (d) Correlation between SP/ CAP amplitude ratio and synaptic ribbon number in the groups of animals treated with 60 mg/mL AGs (n = 32). Data are means ± SD. *p < 0.05; **p < 0.01; ***p < 0.001 (one-way ANOVA with Tukey’s multiple comparison tests). (b) p = 0.0001. (c) p = 0.846). Abbreviations: CAP, compound action potential; Apra, apramycin; GM, gentamicin; GM C1a, gentamicin C1a; Neo, neomycin; SP, Summating potential; Paro, paromomycin.