Characteristic and Otopathogenic Analysis of a Vibrio alginolyticus Strain Responsible for Chronic Otitis Externa in China

Abstract

Vibrio alginolyticus, a Gram-negative rod bacterium found in marine environments, is known to cause opportunistic infections in humans, including ear infections, which can be difficult to diagnose. We investigated the microbiological and otopathogenic characteristics of a V. alginolyticus strain isolated from an ear exudate specimen obtained from a patient with chronic otitis externa to provide a basis for the future diagnosis of V. alginolyticus-associated infections. The identification of V. alginolyticus was accomplished using a combination of matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS), classical biochemical identification methods, and the use of Vibrio-selective media and advanced molecular identification methodologies. Antimicrobial susceptibility testing revealed that the strain was resistant to ampicillin and sensitive to β-lactam, aminoglycosides, fluoroquinolones, and sulfonamide antibiotics. The potential otopathogenic effects of V. alginolyticus were determined through the performance of cell viability, cell apoptosis, and cell death assays in tympanic membrane (TM) keratinocytes and HEI-OC1 cells treated with V. alginolyticus-conditioned medium using cell-counting kit (CCK)-8 assay, a wound-healing migration assay, Annexin V/propidium iodide (PI) flow cytometric analysis, and terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL staining). The results indicated that the identified V. alginolyticus strain exerts cytotoxic effects on keratinocytes and HEI-OC1 cells by inhibiting cell proliferation and migration and inducing apoptosis and cell death. To evaluate the ototoxicity of V. alginolyticus, the cell density and morphological integrity of hair cells (HCs) and spiral ganglion neurons (SGNs) were analyzed after exposing cochlear organotypic explants to the bacterial supernatant, which revealed the pre-dominant susceptibility and vulnerability of HCs and SGNs in the basal cochlear region to the ototoxic insults exerted by V. alginolyticus. Our investigation highlights the challenges associated with the identification and characteristic analysis of the Vibrio strain isolated in this case and ultimately aims to increase the understanding and awareness of clinicians and microbiologists for the improved diagnosis of V. alginolyticus-associated ear infections and the recognition of its potential otopathogenic and ototoxic effects.

Keywords: HEI-OC1 cell; Vibrio alginolyticus; cochlear organotypic culture; otitis externa; ototoxicity; tympanic membrane keratinocyte.

FIGURE 1

Audiometric and otoscopic examination of a patient with a chronic ear infection. The audiometric evaluation indicated mild hearing impairment associated with the chronic ear infection of the patient (A). Otoscopic examination of the right ear of the patient before (B1) and after microbiological specimen collection (B1′) and after antimicrobial treatment (B1^”). Otoscopic view of the uninfected left ear of the patient after antimicrobial treatment for the right ear (B2).

FIGURE 2

Microbiological characteristics of the Vibrio alginolyticus strain responsible for chronic ear infection. Cultures of the specimen obtained from the ear infection drainage revealed the large growth of a Gram-negative bacterium (A). Gram staining of cultured Vibrio colonies showed a Gram-negative, curved, and rod-shaped morphology (B). Sub-culture of Vibrio on sheep blood (C1), chocolate (C2), and MacConkey agar media (C3). For confirmation, the isolate appeared to be creamy, as expected for V. alginolyticus when sub-cultured on a CHROMagar™ Vibrio-selective agar (D1), whereas the control isolates of Vibrio vulnificus (D2), and Vibrio cholera (D3) appeared to be green-blue to turquoise-blue. Biochemical investigation of the tested strain using API 20E (E) and 20NE (F) strips identified the isolate as V. alginolyticus.

FIGURE 3

Cytotoxic effects on tympanic membrane (TM) keratinocytes induced by Vibrio alginolyticus. The keratinocyte outgrowth from the TM explant formed a monolayer of epithelial cells, displaying a cobblestone-like morphology (A). Cytopathic effects in keratinocytes after exposure to the V. alginolyticus culture supernatant for 24 h (B). Quantitative analysis of cell viability measured by cell-count kit-8 (CCK-8) assay after exposure to bacterial supernatant (C). Wound-healing migration assay was performed by analyzing repair rates of wound gaps (D). To detect cell death and apoptosis, the proportion of apoptotic and dead cells was counted by flow cytometric analysis with Annexin V/propidium iodide (PI) double staining (E) and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive stained apoptotic cells was calculated (F). The results are expressed as the mean ± standard deviation (SD) [*p < 0.05, vs. control/Dulbecco’s modified Eagle’s medium (DMEM)]. Scale bars: A, B = 200 μm, D = 500 μm, F = 100 μm.

FIGURE 4

Cytotoxic effects on House Ear Institute-Organ of Corti 1 (HEI-OC1) cells induced by Vibrio alginolyticus. Cytopathic effects of HEI-OC1 cells treated with Vibrio supernatant for 12 h (A). Quantitative analysis of cell viability measured by cell-counting kit (CCK)-8 assay after exposure to bacterial supernatant (B). The proportion of apoptotic and dead cells was counted by flow cytometric analysis with Annexin V/propidium iodide (PI) double staining (C), and the number of TUNEL-positive stained apoptotic cells was calculated (D). The results are expressed as the mean ± standard deviation (SD) [*p < 0.05, vs. control/Dulbecco’s modified Eagle’s medium (DMEM)]. Scale bars: A = 200 μm, D = 100 μm.

FIGURE 5

Ototoxic effects on cochlear organotypic explant cultures induced by Vibrio alginolyticus. Ototoxic damage was observed at the apical (A), middle (B), and basal (C) turns of the cochlear explants. The numbers of Myosin 7a⁺, phalloidin⁺ hair cells (HCs) (D), and β-III Tubulin (TUJ1)⁺ spiral ganglion neurons (SGNs) were quantified (E). The results are expressed as the mean ± standard deviation (SD) (*p < 0.05, vs. control). Scale bars = 100 μm.