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ORIGINAL ARTICLE
Year : 2019  |  Volume : 26  |  Issue : 1  |  Page : 23-26  

Ocular surface microbial flora in patients with chronic limbal stem cell deficiency undergoing cultivated oral mucosal epithelial transplantation


1 Dr. Rajendra Prasad Centre for Ophthalmic Sciences, New Delhi, India
2 Stem Cell Culture Facility, All India Institute of Medical Sciences, New Delhi, India
3 Ocular Microbiology Services, All India Institute of Medical Sciences, New Delhi, India

Date of Web Publication24-Apr-2019

Correspondence Address:
Dr. Murugesan Vanathi
Prof. of Ophthalmology Cornea, Ocular Surface, Cataract and Refractive Services, Dr. Rajendra Prasad Centre for Ophthalmic Sciences, All India Institute of Medical Sciences, New Delhi - 110 029
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/meajo.MEAJO_172_16

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   Abstract 


PURPOSE: The purpose of this study is to analyze the ocular surface microbial flora in patients with chronic limbal stem cell deficiency (LSCD) due to Stevens–Johnson Syndrome (SJS) and ocular chemical injury undergoing cultivated oral mucosal epithelial transplantation (COMET).
METHODS: Patients of SJS and chemical injury who had bilateral total LSCD planned for COMET were studied. Conjunctival swab was taken before surgery. Parameters evaluated were organism cultured, sensitivity pattern, frequency of positive culture, and clinical impact on management strategy.
RESULTS: Thirteen patients were included in which nine were males and four females. All patients had positive conjunctival swab culture. Most common organism isolated was Staphylococcus epidermidis, followed by Staphylococcus aureus and Pseudomonas aeruginosa. The staphylococcal species isolated were sensitive to all the conventional antibiotics while Pseudomonas cultured showed resistance to cefuroxime, ceftriaxone, and ceftazidime. Repeat conjunctival swab sent after a week of topical antibiotic therapy yielded positive culture of the same organism twice in 25% (3/12), thrice in 58.3% (7/12), and four times in 16.6% (2/12) of the patients. One patient had a polymicrobial flora with positive yield of S. aureus (thrice), S. epidermidis (twice), and P. aeruginosa (twice) in consecutive conjunctival swab culture in the absence of clinical infection. Two patients with persistent positive cultures had to undergo repeat oral mucosal harvesting as the transplantation of the cultivated explants had to be deferred.
CONCLUSION: Ocular surface in LSCD patients yielded pathogenic organisms on culture. Poor ocular surface with absent tear film could be the contributing factors. It is important to perform the conjunctival swab culture before COMET surgery.

Keywords: Conjunctival flora, cultivated oral mucosal epithelial transplantation, microbial flora, stem cell deficiency, Stevens–Johnson syndrome


How to cite this article:
Gunasekaran S, Dhiman R, Vanathi M, Mohanty S, Satpathy G, Tandon R. Ocular surface microbial flora in patients with chronic limbal stem cell deficiency undergoing cultivated oral mucosal epithelial transplantation. Middle East Afr J Ophthalmol 2019;26:23-6

How to cite this URL:
Gunasekaran S, Dhiman R, Vanathi M, Mohanty S, Satpathy G, Tandon R. Ocular surface microbial flora in patients with chronic limbal stem cell deficiency undergoing cultivated oral mucosal epithelial transplantation. Middle East Afr J Ophthalmol [serial online] 2019 [cited 2019 May 22];26:23-6. Available from: http://www.meajo.org/text.asp?2019/26/1/23/256963




   Introduction Top


Cultivated oral mucosal epithelial transplantation (COMET) serves in effective ocular surface reconstruction in patients with corneal and ocular surface diseases such as Stevens–Johnson Syndrome (SJS) and ocular cicatricial pemphigoid (OCP).[1] Reconstruction of a stable ocular surface with complete epithelialization of the ocular surface, reduction in fibrovascular tissue invasion of the corneal surface, and establishing a functional fornix remain primary objectives in COMET surgery. Infectious keratitis has been associated with persistent epithelial defects after COMET.[2] The risk of postoperative ocular surface infection remains high in patients with SJS and OCP.

A study of conjunctival bacterial flora in ocular SJS eyes showed a positive bacterial yield in 95% of the eyes with Gram-positive cocci, Gram-positive bacilli, and Gram-negative bacilli accounting for 55.5%, 19%, and 25.5% of the microbial organisms isolated, respectively.[3] Patients with ocular SJS have a diverse pathogenic conjunctival flora distribution with >50% of these patients noted to have multiple bacterial species in their flora.[3]

Changes in the normal ocular surface flora and the continuous monitoring of the conjunctival flora remain an important factor in predicting future eye infections.[4] Emerging trends in resistance patterns to various topical antimicrobial agents are also affected by the pattern of microbial flora of the ocular surface.[4] Symblepharon lysis along with extensive ocular surface reconstruction with COMET surgery remains the procedure of choice in eyes with chronic ocular surface cicatricial disease such as ocular SJS. Therefore, conjunctival microbial flora can play a critical role in postoperative infection following COMET procedure in these eyes. The current study was hence undertaken to evaluate the preoperative conjunctival microbiological profile in patients of bilateral total limbal stem cell deficiency (LSCD) due to SJS and ocular chemical injury patients undergoing COMET.


   Methods Top


The Institute Ethics Committee approval was taken. The statement for the Declaration of Helsinki for clinical studies was followed. Informed consent was obtained from all patients recruited in the study. We performed a prospective study of the conjunctival microbial flora of patients with bilateral total LSCD (due to ocular SJS and chemical injury) who were planned for COMET surgery. These eyes were on intensive topical lubricant therapy with 0.5% carboxymethyl cellulose drops 2 hourly and 1% carboxymethyl cellulose gel at bedtime. Routine preoperative microbiological specimen from the conjunctiva was sent for all patients, before initiating preoperative topical antibiotic therapy with topical moxifloxacin 0.5% 4 times a day. Conjunctival swab was taken from the inferior conjunctival sac before instillation of any eye drop. Care was taken to avoid touching the eyelid with the swab. Direct plating onto blood agar, chocolate agar, and Sabouraud agar was done. All media for bacterial culture were incubated at 37°C and for fungal culture at 25°C–27°C (Biological oxygen demand (BOD) incubator). The parameters evaluated were microorganisms cultured, susceptibility pattern (by agar diffusion plates), frequency of positive culture report, and clinical impact on the management strategy. Bacterial culture positivity was defined as (a) >10 colonies on the site of inoculation on solid media, or (b) the organism seen in Gram-stained smears, or (c) same organism grown in >1 medium. Fungal culture positivity was defined as (a) same fungus isolated in >1 inoculated media, (b) fungal elements seen in primary microscopy and fungus grows on at least in one fungal culture medium, or (c) confluent fungal growth obtained on the specimen inoculated in a single solid media.


   Results Top


Thirteen eyes of 13 patients were included in this study (male – 9 [69.2%] and female – 4 [30.7%]) with a mean age of 24.76 + 12.735 years (range: 12–52 years). All patients (100%) were found to have a positive yield for bacteria in the conjunctival swab culture [Table 1]. The most common microorganism isolated was Staphylococcus epidermidis in 10 eyes (76.9%), followed by Staphylococcus aureus in 3 eyes (30.8%) and Pseudomonas aeruginosa in 1 eye (7.7%). Analysis of antibiotic susceptibility pattern of the staphylococcal species isolated showed that they were susceptible to all the conventional antibiotics that included ciprofloxacin, tobramycin, tetracycline, gentamycin, cloxacillin, vancomycin, cefazolin, gatifloxacin, and moxifloxacin. Pseudomonas cultured from one eye showed susceptibility to the above antibiotics but resistance to cefuroxime, ceftriaxone, and ceftazidime. Surgery was deferred and the conjunctival swab was repeated after a week of antibiotic therapy with moxifloxacin 0.5% four times a day. One patient had a polymicrobial flora with positive yield of S. aureus (thrice), S. epidermidis (twice), and P. aeruginosa (twice) in consecutive conjunctival swab cultures in the absence of any sign of clinical infection and on topical antibiotic therapy with moxifloxacin 0.5% four times a day. Among the remaining 12 patients, repeat conjunctival swab yielded positive culture for the same microorganism twice in 25% (3/12), thrice in 58.3% (7/12), and four times in 16.6% (2/12) of the eyes. The compliance of the patients with respect to the administration of topical antibiotics was ensured with a weekly follow-up with the administered drug bottles or vials. The persistent ocular surface infection of the conjunctival flora necessitated repeat harvesting of the oral mucosa in two patients as the transplant could not be performed within the recommended viability period of the ex vivo cultivated mucosal epithelial stem cell culture and the cultured mucosal epithelial cells had to be discarded in view of persistent swab positivity for pathogenic organisms. All eyes had symblepharon with total LSCD with corneal opacification and ocular surface keratinization (type I ocular surface failure) with lacrimal punctal stenosis.
Table 1: Clinical details of study eyes

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All the eyes underwent COMET procedure with bandage contact lens application at the end of surgery that was removed after 3 weeks. The postoperative outcome in all these 13 eyes included complete epithelialization of the ocular surface at 6-week postoperative period with improvement in ocular surface lubrication. No postoperative ocular surface infection or endophthalmitis was noted in any of the 13 eyes at a 6-month follow-up after surgery for all patients.


   Discussion Top


Data on the prevalence of microbial flora in normal or inflamed conjunctiva and their susceptibility to antibiotics in recent literature are limited.[5],[6],[7],[8],[9],[10] Grasbon et al.[11] studied 100 conjunctival swabs from 99 patients (including 9 HIV-positive patients) for conjunctival microorganisms (healthy eyes (34), chronic nonspecific inflammation (40), outer inflammatory ocular conditions (17), and HIV patients [9], uninfected [n = 6] and infected [n = 3] conjunctivae). They isolated staphylococci in 89% cases, of which coagulase-negative species were obtained in 86% and while coagulase-positive S. aureus was obtained in 12% cases. In the 86 smears positive for coagulase-negative staphylococci, 151 different strains were isolated. Microorganisms from chronic conjunctivitis eyes exhibited a greater range of resistance than those from normal flora. S. aureus and Gram-negative bacteria were significantly higher in chronic conjunctivitis than in healthy eyes, compared to coagulase-negative species of the Micrococcaceae family, which were significantly more prevalent in the healthy eyes.

This study establishes the simultaneous prevalence of multiple stems of coagulase-negative staphylococci in eyes with chronic inflamed ocular surfaces. Our study, in a smaller pool of patients of LSCD, got similar results with coagulase-negative S. epidermidis isolated in 76.9% of the patients followed in frequency by coagulase-positive S. aureus. In our study, Gram-positive species of bacteria were susceptible to commonly used antibiotics including moxifloxacin and gatifloxacin. However, Pseudomonas species did show resistance to the cephalosporin group of antibiotics such as cefuroxime, ceftriaxone, and ceftazidime. In one of the patients, consecutive cultures showed the growth of different species of bacteria despite the antibiotic therapy with 0.5% moxifloxacin. This perhaps points toward an altered balance of microbial flora of the conjunctiva in chronic inflammatory ocular conditions with a further concern to the antibiotic susceptibility of these varied strains to topical antibiotics commonly used.

While it is still unclear if the conjunctival flora can predict the risk of endophthalmitis, it can be agreed upon that they do serve as an important determinant for postoperative infections of the ocular surface, especially in surgeries that involve invasive and extensive manipulations of the conjunctival tissue in conjunction with ex vivo cultured mucosal epithelial amniotic membrane substrate culture transplants as in COMET surgery. The grave risk of postoperative ocular surface infection will further compromise the slim chances of favorable outcomes for ocular surface reconstruction in eyes with morbid ocular surface pathologies. Postoperative infection of the ocular surface transplanted with bioengineered cells can result in grave outcomes with rapid spread of the infection to involve the entire conjunctival cul-de-sac and the conjunctivalized cornea. Lack of normal conjunctival tissue in these compromised ocular surfaces along with reduced immune mechanisms will further compound the damage. The need for repeat harvesting of oral mucosal epithelial stem cells for repeat ex vivo culture is another problem in cases which have persistent ocular surface infection before planned COMET surgery, as crucial time is lost in treating the ocular surface infection which lapses the viability period of the bioengineered cells for transplantation. The small sample size of our study precludes the ability to study the pattern of antibiotic resistance.

Concerns of microbial flora in eyes undergoing keratoprosthesis surgery are on the rise.[12],[13] As most of such eyes undergoing COMET will subsequently be planned for keratoprosthesis for visual rehabilitation, it is of utmost importance for the treating physician to be aware of the changing patterns of microbial flora colonization and the growing concerns of increased antibiotic resistance in these eyes with cicatrized ocular surfaces. Modifications to the treatment and prophylaxis regimen may perhaps be of help in achieving infection free ocular surfaces in these morbid ocular conditions.

Acknowledgments

The authors do not have any financial interests. This study was part of a clinical trial funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India. The authors also acknowledge Ms. Manisha Purvar and Mr. Anil Kumar, Junior Research Fellow, Stem Cell Facility, AIIMS, for their contribution in data retrieval.

Financial support and sponsorship

This study was part of a clinical trial funded by the Department of Biotechnology, Ministry of Science and Technology, Government of India.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

1.
Sen S, Sharma S, Gupta A, Gupta N, Singh H, Roychoudhury A, et al. Molecular characterization of explant cultured human oral mucosal epithelial cells. Invest Ophthalmol Vis Sci 2011;52:9548-54.  Back to cited text no. 1
    
2.
Satake Y, Higa K, Tsubota K, Shimazaki J. Long-term outcome of cultivated oral mucosal epithelial sheet transplantation in treatment of total limbal stem cell deficiency. Ophthalmology 2011;118:1524-30.  Back to cited text no. 2
    
3.
Frizon L, Araújo MC, Andrade L, Yu MC, Wakamatsu TH, Höfling-Lima AL, et al. Evaluation of conjunctival bacterial flora in patients with stevens-johnson syndrome. Clinics (Sao Paulo) 2014;69:168-72.  Back to cited text no. 3
    
4.
Armstrong RA. The microbiology of the eye. Ophthalmic Physiol Opt 2000;20:429-41.  Back to cited text no. 4
    
5.
Capriotti JA, Pelletier JS, Shah M, Caivano DM, Ritterband DC. Normal ocular flora in healthy eyes from a rural population in sierra leone. Int Ophthalmol 2009;29:81-4.  Back to cited text no. 5
    
6.
Boost MV, Cho P. Microbial flora of tears of orthokeratology patients, and microbial contamination of contact lenses and contact lens accessories. Optom Vis Sci 2005;82:451-8.  Back to cited text no. 6
    
7.
Eder M, Fariña N, Sanabria RR, Ta CN, Koss M, Samudio M, et al. Normal ocular flora in newborns delivered in two hospital centers in argentina and paraguay. Graefes Arch Clin Exp Ophthalmol 2005;243:1098-107.  Back to cited text no. 7
    
8.
Yamauchi Y, Minoda H, Yokoi K, Maruyama K, Kumakura S, Usui M, et al. Conjunctival flora in patients with human immunodeficiency virus infection. Ocul Immunol Inflamm 2005;13:301-4.  Back to cited text no. 8
    
9.
Iskeleli G, Bahar H, Eroglu E, Torun MM, Ozkan S. Microbial changes in conjunctival flora with 30-day continuous-wear silicone hydrogel contact lenses. Eye Contact Lens 2005;31:124-6.  Back to cited text no. 9
    
10.
Park SH, Lim JA, Choi JS, Kim KA, Joo CK. The resistance patterns of normal ocular bacterial flora to 4 fluoroquinolone antibiotics. Cornea 2009;28:68-72.  Back to cited text no. 10
    
11.
Grasbon T, Miño de Kaspar H, Klauss V. Coagulase-negative staphylococci in normal and chronically inflamed conjunctiva. Ophthalmologe 1995;92:793-801.  Back to cited text no. 11
    
12.
Robert MC, Eid EP, Saint-Antoine P, Harissi-Dagher M. Microbial colonization and antibacterial resistance patterns after Boston type 1 keratoprosthesis. Ophthalmology 2013;120:1521-8.  Back to cited text no. 12
    
13.
Lee SH, Mannis MJ, Shapiro B, Li JY, Polage C, Smith W, et al. Evaluation of microbial flora in eyes with a boston type 1 keratoprosthesis. Cornea 2013;32:1537-9.  Back to cited text no. 13
    



 
 
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