Ocular Manifestation of Elizabethkingia Meningoseptica

Background: Elizabethkingia is a Gram-negative, obligate aerobic, oxidase positive bacillus that is known to cause a variety of nosocomial infections and has emerged as an important pathogen because of multiple anti-microbial resistance. We present the largest case series of Elizabethkingia ophthalmic infections, with specic emphasis on its clinical spectrum, risk factors, treatment and outcome. Method: Microbiological specimens growing Elizabethkingia were reviewed retrospectively at a regional tertiary hospital from 2005-2019. Clinical manifestation, risk factors, treatment including types of antibiotics, treatment duration and clinical outcome were documented. Results: Ten cases of culture positive Elizabethkingia ophthalmic infection were identied, which all cultured E. meningoseptica. Four cases keratitis, three cases of conjunctivitis, two cases of blepharitis and one case blepharitis-conjunctivitis were observed. Four cases were found to be associated with contact lens use and were found to colonize E. meningoseptica. One case of blepharitis was associated with and ocular prosthesis (scleral shell) an eviscerated eye. Fluoroquinolone and chloramphenicol were most commonly used topical antibiotics for treatment. The mean treatment duration for all infections was averaged at 6.5 weeks. Conclusion: E. meningoseptica is found to be the predominant species that has caused infection at the ocular surface. Contact lens use and ocular prosthesis appears to be a risk factor for infection due to ocular surface barrier disruption and bacteria colonization. Combination of uroquinolone and chloramphenicol is a safe and effective treatment against Elizabethkingia ocular infections. Further studies are required to determine the susceptibility of commonly used topical anti-microbial agents to Elizabethkingia species.


Introduction
Elizabethkingia is a Gram-negative, obligate aerobic, oxidase positive, non-glucose fermenting bacillus that is widely available in nature and is increasingly being identi ed to survive on hospital surfaces and medical equipment [1][2][3][4]. Elizabethkingia is known to cause a variety of nosocomial human infections and has emerged as a clinically important pathogen because of its resistance to multiple anti-microbial agents [4][5][6][7]. Ophthalmological related Elizabethkingia infections are increasingly being reported where use of contact lens, trauma, ocular surgery and ocular surface disorders were associated [8][9][10][11]. Standardized management, choice of anti-microbial treatment and prognosis of Elizabethkingia ocular infections are lacking due to paucity of cases. We present the largest case series of Elizabethkingia induced ophthalmic infections, with speci c emphasis on its clinical spectrum, risk factors, treatment and outcome.

Materials And Methods
Microbiological specimens growing Elizabethkingia were reviewed retrospectively in a regional tertiary hospital from 2005-2019. Positive Elizabethkingia isolates from eye swabs, corneal scrapping and bacterial culture for contact lens in respective cases were retrieved from the hospital microbiological database. All clinical specimens were collected and handled according to standard protocols. Elizabethkingia was rst identi ed by conventional phenotypic method. Gramnegative bacilli were initially recovered from the specimens. These isolates were incubated on sheep blood agar for 24 hours which gave rise to yellowish, semi-translucent, convex shaped small colonies. No growth was observed after incubation on MacConkey agar. Extracted non-motile Gram-negative rods were then examined biochemically that yielded catalase positivity and oxidase positivity, which were further characterized using VITEK GN (Gram-negative) card and matrix-assisted laser-desorption/ionization time-of-ight mass spectrometry (MALDI-TOF-MS) VITEK-2 system (Biomerieux Vitek, Hazelwood, MO, USA). Acanthamoeba and fungi were also tested on all cases using direct microscopy, polymerase chain reaction (PCR) and real-time PCR. Antibiotic susceptibility of the samples was not examined as VITEK-Page 3/9 2 system Gram negative antibiotic testing card at the institution do not cover most of the topical antibiotics used in the cases.
Clinical data and hospital charts were reviewed retrospectively. Patient demographics including age, gender and past medical history were recorded. Diagnosis were clinical and supplemented by microbiological nding. Treatment including types of antibiotics, total number of antibiotics used, treatment duration and clinical outcome were documented. The study has received approval from the hospital ethics committee and adhere to the principles of the Declaration of Helsinki.

Results
Thirteen cases of culture positive Elizabethkingia species in ocular microbiological specimen were identi ed. Two cases were excluded because of lost to follow up. One case was excluded due to incomplete data. The mean age of patients being infected by Elizabethkingia was 57 years old (age range 16 days old-91 years old). The female to male ratio was 6:4. One patient had a history of underlying pancytopenia, bullous pemphigoid and pre-existing type 2 diabetes. Three other patients had co-existing type 2 diabetes and ischemic heart disease. No apparent immunosuppressive states were noted in others.
The ndings were summarized in the  Table 1 Summary of clinical syndrome associated with Elizabethkingia associated ocular infection and treatment outcome.
All four cases of keratitis were associated with contact lens use, with one case of keratitis complicated by development of corneal abscess secondary to trauma to the eye. Three blepharitis cases were recorded, where one case of blepharitis had a history of evisceration and an ocular prosthesis (scleral shell) in situ, one had a history of pancytopenia and bullous pemphigoid, whilst another one developed blepharitis six weeks after same eye cataract operation whilst using topical steroids for prolonged anterior chamber in ammation. Three cases of conjunctivitis were recorded, in which two cases had a background of type 2 diabetes and history of heart disease. No cases of conjunctivitis had reported use of contact lens or trauma prior to infection. The clinical outcome of conjunctivitis and blepharitis were satisfactory with clinical resolution after treatment. All keratitis cases resulted in corneal scar.
Contact lens were also examined microbiologically in all respective cases. Four cases have cultured E. meningoseptica. Tests for fungal infection in all cases were negative.
All cases in the study were treated with topical medications in drop and ointment form. The average number of topical antibiotics class used were two (range 2-4). Fluoroquinolone (levo oxacin, moxi oxacin, o oxacin) and chloramphenicol were most commonly used topical antibiotics for treatment. Topical Fusidic acid, Gentamicin and Neosporin were added in ve other cases. Four cases of ocular infection included systemic oral antibiotics (cipro oxacin, doxycycline and clarithromycin) as part of the treatment regime (three cases of keratitis, one case of blepharitis with ocular prosthesis in situ). The average duration of treatment were 6.5 weeks (range 4-12 weeks).

Discussion
Elizabethkingia is an important saprophyte that has grown much attention in recent years due to its ability to cause clinically signi cant infections and resistance to multiple anti-microbial agents [1][2][3][4]. Limited cases of ocular Elizabethkingia infections have been reported, where contact lens use, trauma, previous ocular surgery and ocular surface disorders appeared to be important risk factors [8][9][10][11][12]. We present the largest series of Elizabethkingia infection that has manifested in eyes. The spectrum of Elizabethkingia ocular infections is wide and mostly con ned to the ocular surface. In our series, 40% (4/10) of cases resulted in keratitis, whereas 30% (3/10) caused conjunctivitis, 20% blepharitis (2/10) and 10% as concurrent blepharitis-conjunctivitis (1/10). The clinical characteristics of Elizabethkingia echoed with previous reports and is found con ned to the ocular surface. Previously reported Elizabethkingia induced endophthalmitis was not found in our series and no intraocular infections were identi ed [13,14]. To date, six species of Elizabethkingia have been identi ed: E. meningioseptica, E. miricola, E. anopheles, E. bruuniana, E. ursingii, and E. occultia [15]. E. anopheles has traditionally been shown to be the predominant strain that causes life threatening bacteremia and neonatal meningitis, where E. meningoseptica and E. miricola are known to cause biliary tract infections [16]. In our series, E. meningoseptica has been established to be the major species that caused ocular infection. The reason for the "ophthalmic strain" and predominance is unknown and yet to be determined.
The association of Elizabethkingia induced keratitis and contact lens is demonstrated in our study. Contact lens predisposes the cornea to microtrauma and hypoxia which disrupted the corneal epithelium and barrier to bacterial binding. In addition, bio lm formation and pathogen colonization on contact lens also potentiated the infection. This is con rmed in our study, where respective contact lens has cultured the same pathogen. The ability of Elizabethkingia to attach on contact lens, cases and water supplies is an important factor for development of ocular infections [3,4]. Mechanisms including substandard contact lens hygiene, extended wear and trauma may also play a role in the potentiating the infection.
The association between orbital surgery and Elizabethkingia infection are also being reported for the rst time. Previous reports have documented Elizabethkingia infections to occur after intraocular surgeries e.g. penetrating and lamellar keratoplasty [17,18]. The authors believed history of previous orbital surgeries may be linked with development of ocular surface Elizabethkingia infections. In our case, an eviscerated eye with an ocular prosthesis (scleral shell) presents as a risk factor for development of blepharitis, as E. meningoseptica may have colonized the foreign body and acted as a reservoir for the pathogen. We speculate that the uneven surface of the acrylic prosthesis allowed bacterial attachment and subsequent bio lm formation, which created a barrier against removal from normal cleansing and sterilization.
The use of topical steroids in the post-operative cataract period may also be linked to E. meningoseptica blepharitis. Prolonged topical steroid was prescribed in our case due to anterior chamber in ammation. We suspect that the treatment aggravated the development of Elizabethkingia blepharitis, as topical steroid has been shown to delay epithelial healing and inhibit neutrophil activation, which subsequently weakened the ocular surface protective barrier [19,20]. While chronic co-morbidities have been shown to cause Elizabethkingia systemic infections, in our series we could not establish a strong relationship, with only 40% (4/10) of cases identi ed to have signi cant past medical histories [21].
Established reports of antibiotics susceptibility on Elizabethkingia species have shown resistance to ceftazidime, imipenem and aminoglycosides and susceptibility to cipro oxacin, cefoperazone-sulbactam and vancomycin treatments [16,22,23]. The multiple anti-microbial resistance in Elizabethkingia is conferred in β-lactams as a result of possession of Ambler class A serine extended-spectrum β-lactamase (ESBL) gene bla CME and Ambler class B metallo-β-lactamase (MBL) genes bla BlaB and bla GOB [24,25]. Resistance is also seen in quinolones secondary to genetic mutations in DNA gyrase and topoisomerase IV [26]. Empirical treatment using uoroquinolone, glycopeptide and third-generation cephalosporin with β-lactamase inhibitor have been advocated for treating systemic infections [16,22,23].
There is currently no consensus on treatment for Elizabethkingia ophthalmic infections. Treatment of ocular infection frequently employ topical route of administration and susceptibility to commonly used topical antibiotics in ophthalmic practice, such as chloramphenicol and fusidic acid, have not been evaluated. Our series has demonstrated that combined use of 2 topical antibiotic, uoroquinolone and chloramphenicol, could lead to safe and successful treatment of E. meningoseptica conjunctivitis, blepharitis and keratitis. The addition of systemic oral antibiotics such as oral quinolones, tetracycline or macrolide have shown to be effective. Levo oxacin, one of the commonly used topical uoroquinolone in ophthalmological practice, contain a C-8 methoxy group that exert stronger antibacterial activity against uoroquinoloneresistant bacteria that harbored QRDR mutations [27,28]. Whether the single agent of topical uroquinolone alone enabled treatment success or combination with topical chloramphenicol is not determined. Further studies are required to determine the susceptibility of chloramphenicol to Elizabethkingia species. A longer period of treatment for Elizabethkingia ocular surface infections was also observed, which averaged 6.5 weeks in our series. We speculate that this could be due to bacterial intracellular invasion, inability to break multicellular bio lm and ineffective ocular immunity [3,29]. This could be re ected in the outcome, where conjunctivitis, blepharitis and conjunctivitis-blepharitis confer excellent clinical recovery where keratitis all resulted in corneal scarring.

Conclusion
In summary, Elizabethkingia is an emerging organism that could manifest in eyes and is clinically challenging to treat due to its multiple anti-microbial resistance. In our series we have demonstrated that E. meningoseptica is the predominant species that causes infection at the ocular surface. Contact lens use and ocular prosthesis in situ appears to be a signi cant risk factor for development of ocular Elizabethkingia infections due to ocular surface barrier disruption and bacteria colonization. Contrary to previous beliefs, immunosuppressive state does not appear to be a signi cant factor for ocular infections. Combination of topical uroquinolone and chloramphenicol is a safe and effective treatment regime for Elizabethkingia ocular infections. Further studies are required to establish the most effective treatment approach.

Declarations
Ethics approval and consent to participate-