Introduction
============

*Vibrio cholerae* is a Gram-negative comma-shaped, facultative anaerobic, motile bacterium, belonging to the *Vibrionaceae* family. * V. cholerae* is both a human pathogen and a natural inhabitant of aquatic environments ([@B9]; [@B10]).  Infections are primarily associated with ingestion of contaminated foods and water, with subsequent diarrheal illness.  This species is divided into more than 200 serogroups, of which only O1 and O139 cause cholera epidemics and pandemics ([@B25]; [@B7]). 

*Vibrio cholerae* is a waterborn bacterium and can be found in marine and freshwaters ([@B7]).  It has been associated with various reservoirs in the aquatic environment: chitinous organisms, i.e., crustaceans and especially copepods ([@B44]); chironomids ([@B3]; [@B16], [@B19], [@B18]; [@B40]; [@B20]); phytoplankton and aquatic plants ([@B30]; [@B39]); protozoa ([@B2]); waterfowl, which may be the disseminators of *V. cholerae* between water bodies, both within and between continents ([@B21]); fish, which consume chironomids and copepods, on one hand, and are consumed by waterfowl on the other, carry *V. cholerae* in their intestine and may play an important role in *V. cholerae* dissemination ([@B41]; [@B17]).  Furthermore, in addition to the particle-associated growth strategy of environmental *V. cholerae*, few reports have demonstrated that *V. cholerae* can also grow in water as a free-living organism in the planktonic phase ([@B45]). 

Unlike the O1 and O139 serogroups, *V. cholerae* non-O1/O139 serogroups rarely produce the cholera toxin (CTX), and cause milder gastrointestinal infections, but are associated with extra-intestinal infections, such as septicemia, wound infection, peritonitis, skin infection, cellulitis, necrotizing fasciitis, endophthalmitis, cholecystitis, meningitis and ear infection ([@B27]; [@B4]; [@B6]; [@B24]). 

We report here a case of non-O1/O139 *V. cholerae* otitis media which developed in a young male after a water skiing accident. 

Case Report
===========

Ethics Statement
----------------

This study was carried out in accordance with the recommendations of the guidelines of Helsinki Committee and was approved by the Helsinki Committee of Clalit Health Services, Israel (approval no.  0192-15-COM1).  The subject gave a written informed consent in accordance with the Declaration of Helsinki. 

A 27-years old Caucasian male with acute otitis media presented to an otolaryngologist at a medical clinic in Haifa, Israel, after suffering for a period of three years from a periodic earache accompanied by a malodorous and purulent exudate from his right ear, without fever.  As the patient recalled, the onset of the symptoms began approximately one week after his 2011 water skiing accident on the Murray River near Mildura, VIC, Australia, in which he fell and perforated his right eardrum. 

Prior to his admission, and with the onset of the periodic ear exudates, the patient had undergone a series of ear cleansings and aspirations without any significant improvement.  During his admission, an ear swab was used to sample his exudate, and later was sent to the microbiology laboratory of Clalit Health Services of Haifa and Western Galilee district for further analysis. 

Twelve hours after initial incubation a prominent bacterial growth was noticed on Chocolate agar, Tryptic soy blood agar, and MacConkey agar plates (Novamed, Israel).  The suspected bacterium was identified as *V. cholerae* by means of VITEK-2 bacterial identifier system (BioMerieux, France) and by using Vitek-MS MALDI-TOF technology (BioMerieux, France).  Additionally, five single colonies were picked and streaked on Luria (LB) agar (HiMedia, Mumbai) in five successive days to verify the purity of the isolates; then their identities were verified by a PCR assay in accordance with [@B37].  The results demonstrated that all the strains were positive for *ompW*, a gene of an outer membrane protein, specific to *V. cholerae*.  The isolate was confirmed as *V. cholerae* non-O1/O139, cholera toxin-negative by the reference laboratory of the Israeli Ministry of Health.  Somatic antigen serogrouping identification was employed on the five *V. cholerae* isolates according to [@B42], and all the isolates were identified as *V. cholerae* serogroup O100. 

The isolates underwent complete antibiogram susceptibility testing by the disk diffusion technique (Oxoid, United Kingdom) with interpretation according to the *V. cholerae* CLSI guidelines ([@B8]).  The strains proved resistant to polymyxin B sulfate and susceptible to co-amoxiclav, cefuroxime, co-trimoxazole, gentamicin, and ciprofloxacin.  Based on the results of antibiotics activity against the isolates *in vitro*, ciprofloxacin ear drops were used until the patients symptoms disappeared completely.  Three weeks later; an additional swab from his right ear was sent to our laboratory; it was found negative for *V. cholerae*. 

Furthermore, the presence of virulence genes in the *V. cholerae* isolates was determined.  All the isolates were positive for *hapA* (soluble hemagglutinin/protease), *hlyA* (haemolysin), *ompU* (outer membrane protein) and *toxR* (regulatory protein) genes, but negative for the virulence gene cassette such as the cholera toxin gene (*ctxA*), zonula occludens toxin (*zot*), accessory cholera enterotoxin (*ace*) toxin coregulated pilus (*tcpA tcpI*) and for the genes encoding the Type Three Secretion System (TTSS) (*vcsC2*, *vcsN2*, *vspD*, and *vcsV2*).  The primers and the PCR procedures for the genes detection are described in [@B37], [@B13], [@B19], [@B5]. 

Discussion
==========

*Vibrio cholerae* belongs to the class of environmental pathogens defined as microorganisms that normally spend a substantial part of their life cycle outside the human host but, when introduced into humans, cause disease with measurable frequency ([@B7]).  While serogroups O1 and O139 cause cholera, the non-O1/O139 serogroups can cause a milder diarrhea, and are also associated with various human infections: blood, wound, ear, and other clinical sites ([@B11]). 

The patient described in the current case is an immunocompetent young male with no history of chronic ear infections before the water skiing accident in Australia, during which his eardrum was perforated. * V. cholerae* O1 and non-O1/O139 had been isolated from river and marine water over a wide area in Australia since 1977 ([@B12]), which may link the ear injury in the Australian river to the subsequent *V. cholerae* otitis media.  The water skiing accident happened in January, which is summer in Australia.  Water temperatures and *V. cholerae* population size in the aquatic environment usually are higher during this season.  Summer months are also the most common months of non-O1/O139 *V. cholerae* infections ([@B6]). 

The isolates identified in this study didn't harbor the virulence gene cassette such as the cholera toxin gene and the genes encoding Type Three Secretion System.  Thus, these genes probably have no significant role in causing otitis media.  On the other hand, the isolate harbored other virulence related genes, *hlyA*, *hapA*, *toxR*, and *ompU*.  The pathogenic mechanisms of *V*. * cholerae* non-O1/139 in otitis media remain unclear, but these genes might play a role in the disease process.  Using cytotoxicity and apoptosis assays, showed that *hly*A-positive strains of *V. cholerae* non-O1*/*O139 had significantly higher cytotoxic activity and levels of apoptosis induction than *hly*A-negative strains, and that a mitochondria-dependent apoptosis pathway is involved ([@B32]). 

Furthermore, *V. cholerae* O100 isolate in the current study was positive to *toxR* and *ompU* genes.  ToxR is the master regulator of *V. cholerae* pathogenicity ([@B14]).  Among other pathogenic genes, it regulates OmpU, one of the major outer membrane proteins of *V. cholerae* ([@B36]). * ompU*, which is an outer membrane porin, has a role in the passage of certain substances through the outer membrane.  Also, it has been shown that ToxR controls resistance to various cationic antibacterial proteins, for example, polymyxin B sulfate, through a porin-mediated mechanism ([@B35]).  Indeed, in the current study, the isolates that were positive to *toxR* and *ompU* genes were resistant to polymyxin B sulfate antibiotics. 

Moreover, OmpU is pro-inflammatory in nature.  Interestingly, it can also down-regulate LPS (lipopolysaccharide)-mediated proinflammatory responses.  OmpU causes suppression of LPS-mediated responses by attenuating the LPS-mediated toll-like receptor signaling pathway ([@B38]).  The presence of OmpU protein in the bacterial isolate may explain the phenomenon that was observed in the current case in which the patient suffered from symptoms of earache and purulent exudate for three years.  He was cured only when the bacteria was identified and treated with ciprofloxacin ear drops.  This antibiotic treatment was effective and all the symptoms completely disappeared. 

A few cases of otitis media or otitis externa caused by *V. cholerae* non-O1/O139 are reported in the literature.  Most were children (*n* = 7, aged between 9 and 15 years) two young males and one female in their early twenties, and one 49 year- old-man (**Table [1](#T1){ref-type="table"}**).  The majority of the reported cases were from European countries and only one report was from the United States.  In seven cases it was reported that the patients had been swimming in a swimming pool, a lake or in the Baltic Sea. 

###### 

Case reports from the literature of otitis caused by *Vibrio cholerae* non-O1/O139 strains. 

  Case reported                                     Place                Age years   Gender   Epidemiology                                                                                                Reference
  ------------------------------------------------- -------------------- ----------- -------- ----------------------------------------------------------------------------------------------------------- -------------------
  Protracted middle-ear inflammation                Sweden               10          Male     Bathing in the Baltic archipelago, off Stockholm, *V. cholerae* O107                                        [@B1]
  Middle ear infection                              United States        --          --       Exposure to salt water                                                                                      [@B28]
  Chronic external otitis                           Belgium              12          Female   Unknown                                                                                                     [@B23]
  Middle ear infection                              Romania              --          --       Unknown                                                                                                     [@B15]
  Otitis media                                      Belgium              20          Female   Swimming in a swimming pool                                                                                 [@B43]
  External otitis 1 year after eardrum transplant   Belgium              15          Male     Unknown                                                                                                     [@B43]
  Otitis media                                      Germany              --          --       Unknown                                                                                                     [@B22]
  Chronic recurrent otitis media                    Austria              8           Female   Unknown                                                                                                     [@B29]
  Otitis media                                      Austria              49          Male     Unknown                                                                                                     [@B29]
  Otitis media                                      Austria              9           Male     Lake Neusiedl in August (year 2004)                                                                         [@B29]
  Otitis media                                      Austria              9           Female   Lake Neusiedl in August (year 2005)                                                                         [@B29]
  Otitis externa                                    Austria              14          Male     Lake Neusiedl in August (year 2000)                                                                         [@B29]
  Otitis externa                                    Austria              22          Male     After vacation in an unknown location                                                                       [@B29]
  Ear infection                                     United Kingdom       --          --       Unknown                                                                                                     [@B34]
  Otitis externa                                    Austria              21          Female   Swimming in Lake Neusiedl (August, 2015)                                                                    [@B26]
  Otitis media                                      Israel (Australia)   27          Male     Water skiing accident on the Murray River near Mildura, VIC, Australia (January, 2011) *V. cholerae* O100   The current study

Untreated otitis media can cause complications such as loss of hearing, inner ear and facial nerve problems, including vertigo ([@B33]), and extracranial and intracranial complications ([@B31]).  Fortunately, the patient in the current case had not such complications.  Nevertheless, physicians should be aware that non-O1/O139 *V. cholerae* can cause extra-intestinal infections that can be acquired from exposure to swimming pools and river, lake or marine water.  Thus, *V. cholerae* should be added to the differential diagnosis of otitis infections. 

Author Contributions
====================

PK, YS, and SL-S identified the strains.  EA, serotyped the strains.  SL-S identified the virulence genes.  PK and YS performed the antibiogram tests.  PK and YS wrote the manuscript, SK-D and MH reviewed the manuscript.  MH Edited the manuscript.  MH, Contributed reagents/materials/publication fee. 

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 

This study was supported by a grant from the German Research Foundation (Deutsche Forschungsgemeinschaft \[DFG\] grant GZ: HO 930/5-2). 

[^1]: Edited by: *José Roberto Mineo, Federal University of Uberlandia, Brazil*

[^2]: Reviewed by: *Rudra Bhowmick, Oklahoma State University, United States; Avi Peretz, Poria Medical Center, Israel*

[^3]: This article was submitted to Microbial Immunology, a section of the journal Frontiers in Microbiology
