Antibiotic resistance is rapidly becoming a clinically significant problem to the healthcare system. Treatment of chronic otitis media caused by bacteria, resistant to multiple antimicrobial agents, has emerged as one of the greatest challenges faced by clinicians. Consequently, Methicillin-Resistant Staphylococcus Aureus (MRSA) infections cannot be treated with commercially available ciprofloxacin ototopical drops.1,2 The prevalence of MRSA in chronic suppurative otitis media (CSOM) is known to be more than ∼60–70% among S. Aureus isolates from hospitals in Korea.3 Besides CSOM, MRSA is an emerging problem related to chronic pediatric tympanostomy tube otorrhea. To date, standard treatment protocols have not been developed, and there is a diverse spectrum of reported medical treatments. The additional use of oral antibiotics such as linezolid,4 trimethoprim-sulfamethoxazole,5 fusidic acid,6 or rifampin to treat MRSA otorrhea may be helpful, but after systemic administration, the effective bioavailable concentration in the middle ear to eradicate infection cannot be accurately measured or described. Ototopical drug delivery presents a highly promising alternative to oral antibiotics for the administration of therapeutics to the middle ear.

The use of vancomycin for the treatment of MRSA infections has increased.7 One of the authors of this paper used topical vancomycin to treat patients with MRSA CSOM and obtained good results.8 However, the concern regarding the use of vancomycin is the possible emergence of vancomycin-resistant strains such as Vancomycin-Intermediate S. aureus (VISA) and Vancomycin-Resistant Enterococcus (VRE). In clinical settings, its less frequent use may facilitate decreases in the appearance of vancomycin-resistant strains. Therefore, it is necessary to develop new antimicrobial compounds against the MRSA. Recently, Antimicrobial Peptides (AMPs) have been considered as novel antibiotics against drug-resistant microorganisms, such as MRSA, and some of them are being evaluated in clinical trials.9 AMPs are fundamental components of the mammalian innate immunity that control microbial infections and represent the first line of defense against different pathogens on epidermal and mucosal epithelial surfaces. AMPs play a significant role in host innate immunity defense to prevent first infections10 and are produced by macrophages, blood cells, and epithelial cells in mammalians. The AMPs identified in humans include members of the cathelicidin, defensin, thrombocidin, and histatin families.11

Expression of an AMP in the skin of the external auditory canal skin and in the middle ear has been reported.12–14 Human cathelicidin LL-37 is a 37 residue-long cationic, amphipathic α-helical antimicrobial peptide.15 Besides its direct bactericidal action, LL-37 is known to have a potent Lipopolysaccharide (LPS) neutralizing activity in various cell types.16–18 KR-12 (residues 18–29 of LL-37) is the smallest peptide of human cathelicidin LL-37 possessing antimicrobial activity.19 Recently, we developed novel KR-12 analogs, designed based on the structure–activity analysis of KR-12 (KRIVQRIKDFLR-NH2),20 which showed potent antimicrobial and anti-endotoxic activities.

Purulent otorrhea is a common disease in patients suffering from CSOM. Patients with CSOM and MRSA infection generally have a chronic inflammation of the middle ear and mastoid, with a persistent perforation of the tympanic membrane, associated with recurrent otorrhea. Ototopical medication leads more likely to the use of lower doses compared to systemic antibiotics. Moreover, the ability of systemic antibiotics for the treatment of infections associated with infected implants in the middle ear is not well elucidated. However, ototopical drops against MRSA are not commercially yet available. In this study, we evaluated the ototoxic effect of a topical solution of KR-12-a2 (KRIVQRIKKWLR-NH2), a KR-12 analog, on the cochlea after topical application in the middle ear of guinea pigs.

The diagnosis of MRSA otorrhea should be considered in cases of persistent otorrhea in which standard ototopical and/or systemic antibiotic therapy fails. MRSA isolation rates among S. aureus ear infections have consistently increased up to 60.9%.3

Fishman et al. reported that in a large review of patients with culture-positive, persistent tympanostomy tube otorrhea, S. aureus accounted for 52% of the organisms cultured, of which 57% were MRSA.20 Presently, the most commonly used antibiotic against MRSA is vancomycin. Both laboratory experiments and clinical studies did not demonstrate significant ototoxicity after vancomycin treatment.21–24 Although there is no emergence of VRSA in CSOM, new anti-MRSA peptides need to be studied for the prevention of VRSA or VISA. Mandal et al. reported that approximately 15% of MRSA strains were also resistant to vancomycin (VRSA).25 Recently, the topical application of daptomycin, a lipopeptide antibiotic that naturally occurs in the soil saprotroph Streptomyces roseosporus and that is useful in treating MRSA infections, revealed no ototoxicity in guinea pigs.26 However, the emergence of daptomycin-resistant MRSA has been already reported in other disease27 in addition to vancomycin resistance.28 Therefore, new AMP for MRSA otorrhea should be developed. The only peptide of the cathelicidin family that is found in the human body is LL-37, which has been shown to have broad antibacterial effects against both gram-positive and -negative bacteria.29 LL-37 is considered to play an important role in the first line of defense against local and systemic infection and in systemic invasion of pathogens at inflammation sites and wounds.30 Recently, Hou et al.31 reported that the antimicrobial peptide LL-37 and innate defense regulator peptide IDR-1 could ameliorate MRSA-induced pneumonia by exerting an anti-inflammatory property and attenuating pro-inflammatory cytokine release, thereby providing support for the hypothesis that both innate and synthetic peptides could play a role of protection against MRSA in vivo.

In the present study, we observed the antimicrobial activity of KR-12-a2 against MRSA. Inhibition zone and time-kill assays showed its potential antibacterial activity against MRSA. The present study investigated the ototoxicity by using topical application of KR-12-a2, GM, and PBS twice daily for 7 days. Hair cell damage was evaluated via ABR and SEM. There was no significant difference in the hearing threshold shift and hair cell preservation between the PBS and KR-12-a2 groups. However, the GM group showed total outer hair cell loss. Inner ear lesions occur predominantly in the basal turn, following the apical one with greater commitment of the first OHC row, with subsequent extension to IHC, stria vascularis, spiral ligament, and Reissner membrane.32 In this study, we evaluated only hair cells. In the GM group, the loss of OHC in basal turn than apical turn was observed. Gentamicin is highly water-soluble and has a polar nature that prevents it from readily crossing a membrane. A higher absorption rate would present the basal turn of the cochlea with a higher level of aminoglycoside prior to its equilibration into the perilymph.33 To date, ototoxicity induced by gentamicin depends on susceptibility, genetic predisposition, and caspase-dependent apoptotic cell death.34 The toxicity may be a dose-dependent phenomenon in the inner ear with higher levels resulting in marked hearing loss.35 In this study, we used a higher amount of GM than the dosage used by Kimura et al. Since the limitation of the present study is the absence of demonstrating therapeutic effects of KR-12-a2 in animal models, follow-up in vivo studies are considered necessary. Thus, future research will focus on the evaluation of the effects of topical KR-12-a2 by using a MRSA otitis animal model.

In our experiment, topically applied KR-12-a2 solution did not cause hearing loss or cochlear damage in guinea pigs. The KR-12-a2 solution can be used as ototopical drops for treating MRSA otorrhea; however, further evaluations, such as the definition of optimal concentration and combination, are necessary.

The authors declare no conflicts of interest.