Vibrio cholerae non-O1 - the first reported case of keratitis in a healthy patient

Background Vibrio cholerae non-O1 is a virulent pathogen that causes significant morbidity and mortality in humans. Herein, we report a case of corneal ulcer caused by this pathogen. Case presentation A 59-year-old fisherman with no systemic history was struck in the right eye by a marine shrimp and developed keratitis. Corneal scrapping culture revealed the presence of the V. cholerae non-O1, and its identification was confirmed by Analytical Profile Index 20E system and polymerase chain reaction. He was successfully treated with topical levofloxacin (0.3%) and fortified amikacin (12.5 mg/mL) for 2 weeks. The visual acuity recovered to 20/25 after treatment without complications. Conclusions This is the first case report of keratitis caused by V. cholerae non-O1 strain. Ocular injury by marine creatures and contaminated seawater can contribute to severe corneal ulcer. Early diagnosis can be achieved by meticulous history taking and a comprehensive laboratory workup. Simultaneously, an effective antibiotic therapy can lead to a positive outcome.


Introduction
Vibrio cholerae is a facultative anaerobic gram-negative comma-shaped bacillus that exists ubiquitously in marine and estuarine environments. Exposure to contaminated water and ingestion of raw seafood are crucial routes of infection that causes overwhelming morbidity and mortality in humans [1]. V. cholerae serogroup O1 and O139 primarily contribute to pandemic Vibrio gastroenteritis by means of cardinal virulence factors of cholera toxin (CT) and toxin-coregulated pilus (TCP). Similarly, non-cholera Vibrio including Vibrio parahaemolyticus and Vibrio vulnificus (V. vulnificus) can lead to cholera-like diarrhea, open wound infection, necrotizing fasciitis and septicemia via multiple virulence determinants such as capsular polysaccharide, hemolysin, siderophores and metalloproteases [2,3]. These major pathological Vibrio species have been extensively studied for their detrimental effect on public health. However, pestilent V. cholerae non-O1/ non-O139 infection has gained attention over the past decade. A few studies have reported cases of extra-intestinal Vibrio infections caused by V. cholerae non-O1/non-O139 stains, including soft tissue infection, pneumonia, acute cholecystitis, liver abscess, peritonitis, urinary tract infection, septicemia and meningitis in addition to acute enteric illness [4,5]. Nevertheless, the incidence of ocular infection caused by V. cholerae non-O1/non-O139 was rather low. Only one case of endophthalmitis attributed to V. cholerae non-O1/non-O139 strain in a cirrhotic patient with septicemia has been reported [6]. Herein, we reported a case of a healthy patient with V. cholerae non-O1 keratitis and searched the related literature to discuss pathogenesis, diagnosis and management of V. cholerae keratitis.

Case presentation
A 59-year-old healthy fisherman presented with right eye pain, redness, tearing and photophobia for 1 day and the symptoms persisted. According to his statement, his right eye was struck by a marine shrimp while fishing. The patient denied a history of major systemic diseases such as diabetes, hypertension, or liver cirrhosis. The best-corrected visual acuity was 20/60 in the right eye and 20/20 in the left eye. Slit-lamp examination showed moderate conjunctival injection with slight chemosis, and a central stromal infiltration of approximately 1.3 × 1.8mm 2 . Simultaneously, corneal edema with Descemet membrane folding around the lesion was also observed (Fig. 1a). There was no sign of anterior chamber inflammation or endophthalmitis. Initially, under the suspicion of bacterial or fungal infection, the patient was empirically treated with hourly Cravit (levofloxacin oph. Soln, 0.5%, Santen, Japan) and fortified amphotericin B (1 mg/ mL). Two days later, right eye pain improved slightly. The gram staining of the corneal culture showed Vibrio sp., and the yellow colony in thiosulfate-citrate-bile saltssucrose (TBCS) agar was highly identified as V. cholerae. Moreover, the negative result in the agglutination test confirmed the non-O1 strain ( Fig. 2a-c). Thereafter, the identity of the V. cholerae was reconfirmed by laboratory analysis with Analytical Profile Index 20E (API 20E) ( Fig.  2d) and polymerase chain reaction (PCR) (Fig. 3). Following the identification of V. cholerae non-O1, we halted the use of amphotericin B. Meanwhile, antimicrobial minimal inhibitory concentration testing was performed for this pathogen (Table 1). Based on the results of the test, the antibiotic regimen was changed to topical amikacin (12.5 mg/mL) and Cravit four times daily. One week later, the right uncorrected visual acuity rapidly recovered to 20/25 and corneal infiltration improved (Fig.  1b). The frequency of treatment was tapered gradually without any complication.

Discussion and conclusions
This is the first case of infectious keratitis caused by V. cholerae non-O1. Prior to our case, the first published case of ocular infection with V. cholerae non-O1 was an endophthalmitis in a cirrhotic patient with septicemia [6]. Corneal pathogenesis of the V. cholerae non-O1 strain has not been clearly elucidated yet due to its rare occurrence in ocular tissue. V. cholerae non-O1 can potentially bring on pandemic gastroenteritis and extraintestinal diseases without using lethal virulence factors such as CT and TCP. Some studies have indicated that  V. cholerae non-O1 possess unique characteristics to evade immune surveillance and destroy its target precisely [7,8].
Hemolysin, hemagglutinin protease, repeats in toxin (RXT) and type VI secretion system (T6SS) are crucial virulence factors of V. cholerae non-O1 [7]. V. cholerae non-O1 activates the vacuolating process regulated by hemolysin protein to interfere with intracellular homeostasis and ion exchange, and subsequently impels cellular cytolysis [9]. Therefore, hemolysin-induced vacuolation can potentially disrupt corneal epithelial and endothelial cells to trigger cell damage. Furthermore, occludin and zonula occludens-1 (ZO-1) are enriched in the corneal epithelium as first-line defense components [10]. In V. cholerae non-O1, hemagglutinin protease, encoded by the hap gene, can degrade essential tight junction-associated protein occludin and disrupt ZO-1 conformation [11]. It is reasonable to infer that V. cholerae non-O1 can directly destroy the epithelial occludin and ZO-1 via hemagglutinin protease, digest collagens and abolish corneal transparency. In addition, RTX, akin to exotoxin of V. cholerae, was found to be involved in the detachment and round-up of epithelial cells [12]. V. cholerae non-O1 can break down corneal epithelium and stroma using the distinctive cytotoxic property of the RTX. T6SS, found in gram-negative bacteria such as Pseudomonas aeruginosa and Escherichia coli, is a cellpuncturing device that translocates effector proteins into eukaryotic host cells [13]. P. aeruginosa has been shown to invades cornea with the assistance of T6SS [14], Hence, it is possible for V. cholerae non-O1 to adopt a similar strategy to cause keratitis. These presumptions require further microbiological research and laboratory  In the present case, we initially doubted the presence of this pathogenic strain in the corneal culture because V. cholerae has never been reported in keratitis. Hence, it is necessary to validate this pathogen using advanced laboratory methods. The API 20E microtest and real-time PCR are suitable choices. The API 20E is an efficient biochemical kit to discriminate members of gram-negative bacilli in 24 h. Furthermore, the API 20E has a higher detection rate for V. cholerae than analogous biochemical panels [15]. On the other hand, real-time PCR enables the most accurate molecular identification for V. cholera and is faster than the API 20E detection [16]. Target genes including hlyA, tcpI, toxR and ompU are available for the screening of V. choerae serotypes [17]. Both of methods are recommended for the diagnosis of intractable infectious ophthalmic due to their advantages of a short reaction time and enhanced accuracy of detection.
V. cholerae non-O1 is assumed to be an opportunistic pathogen, although it occasionally causes severe illness [5]. Hence, efficient treatment for V. cholerae non-O1 infection has not been determined yet, particularly in ocular diseases. Yang et al. reported the first case of V. cholerae non-O1/non-O139 endophthalmitis following septicemia in a cirrhotic patient [6]. This patient received an intravitreal injection of vancomycin and ceftazidime, and a systemic intravenous injection of ceftriaxone (2 g, q12h). Eventually, the patient died of sepsis and respiratory failure. Moreover, sporadic medical regimens for V. vulnificus keratitis were delineated. DiGaetano et al. (1989) combined medical treatment and surgical debridement for two cases of V. vulnificus keratitis [18]. Massey et al. (2000) used topical therapy with ciprofloxacin and fortified vancomycin for one case of V. vulnificus keratitis [19]. Penland et al. (2000) demonstrated that topical application of ciprofloxacin and cefazolin can treat the V. vulnificus keratitis [2]. You et al. (2008) used surgical debridement and systemic antibiotics (oral and intravenous injection) for V. vulnificus keratitis [20]. Despite the availability of prior case reports (Table 2), therapeutic options for ocular V. V. cholerae non-O1 can cause keratitis after ocular blunt injury by marine creatures and contaminated seawater. A satisfactory outcome can be achieved by comprehensive history taking, early pathogenic diagnosis and compatible antibiotic treatment.