Evaluation of Antisseptic Effects on the Eye Surface and the Role of Gentamicin in Microbial Control of Donor Corneas

Research Article

J Bacteriol Mycol. 2017; 4(4): 1056.

Evaluation of Antisseptic Effects on the Eye Surface and the Role of Gentamicin in Microbial Control of Donor Corneas

Ito CRM¹*, de Braga CASB², Carneiro LC², Messias ACMC², Feitosa APP¹, de Moraes AP², de Queiróz PHP2, Monteiro JC1¹ and de Ávila MP²

¹Center of Reference in Ophthalmology, Federal University of Goiás, Brazil

²Institute of Tropical Pathology and Public Health, Federal University of Goiás, Brazil

*Corresponding author: Célia Regina Malveste Ito, Center of Reference in Ophthalmology, Federal University of Goiás, Brazil

Received: October 09, 2017; Accepted: November 14, 2017; Published: November 21, 2017


Decontamination of the surface of the donor eyeballs with antisepsis are effective, ensuring greater transplantation safety. This study evaluated the antiseptic effect in reducing the microbiota of the ocular globe of donors of corneas, with 5% povidone-iodine (right eye) and 0.05% chlorhexidine gluconate (left eye). In the action times of 5, 10 and 15 minutes, and the susceptibility profile of the microbiota isolated from gentamicin. Swabs were collected from the ocular surface before application of the solutions, after and at the time of preservation of the corneal tissue. After identification of the microbiota, an antibiogram test was performed with gentamicin. There was a reduction of 39, 5% in the total of gram-positive bacteria, and of 76, 5% in the gram-negative bacteria, and 1.7% fungi, with no statistically significant difference (p=0.183), which demonstrated that the bacterial elimination capacity of the antiseptics was similar. Both were more effective for gram negative bacteria, with a statistically significant difference (p<0.001). In the third collection, after the residual effect of the antiseptics, there was a reduction of 99.1% of all the microorganisms. In the antibiogram test, 88% of the isolated microorganisms were sensitive to gentamicin. The use of antiseptics is essential for the effective decontamination of donated corneas prior to preservation and the residual time of the antiseptics increased the decontamination power of povidone-iodine and chlorhexidine gluconate, being similar in reducing the microbiota of the ocular globe of the donor of corneas. Gentamicin contained in the cornea preservation medium complements the antisepsis of the donated tissues.

Keywords: Eye bank; Enucleation; Povidone-iodine; Chlorhexidine gluconate; Transplantation


Donated eyes are obtained from cadavers from non-sterile environments such as domicile, public thoroughfare, hospitals and morgues [1], and rigorous screening of the medical and social history, body and eye of the donor is required. For this, preventive strategies are used, such as exclusion of donors with septicemia or endocarditis, antiseptic preparation and decontamination of the donated tissue, as well as preservation in antibiotic containing medium. Some eye bank still performs a microbiological evaluation in order to certify the absence of microbial contamination before the distribution of corneal tissue or at the time of transplantation [2].

On the ocular surface, especially in the human conjunctiva is a resident microbiota that has a fairly uniform pattern, although slight variations of certain micro-organisms occur in some parts of the world. Among the bacteria, species such as coagulase negative Staphylococcus (SCN), Streptococcus viridans group, Corynebacterium spp. and Moraxella spp [3]. Fungi contamination can also occur, with a prevalence of 3 to 28%, especially Candida albicans, Saccharomyces cerevisiae, Cryptococcus neoformans and Aspergillus flavus, among others [4].

Among the antiseptic solutions most used by eye bank or in ophthalmologic surgeries are polyvinylpyrrolidone-iodine and gluconate of chlorhexidine gluconate. Both are broad spectrum microbicides, with rapid action and depending on the concentration used, low corneal toxicity [5].

The povidone-iodine target is the cytoplasmic membrane and its action to kill the micro-organism occurs in a few seconds, since the free iodine released will oxidize and ionize the vital molecules of the cell [6]. A 5mg/mL solution, acting for two minutes, considerably reduces microbial contamination, without damage to the corneal tissue [7].

Gluconate of chlorhexidine, in turn, is a cationic bisbiguanide, which binds electrostatically to negatively charged surfaces, with specific and strong adsorption to the phosphate-containing compounds. Upon contact with the micro-organisms, gluconate of chlorhexidine damages the outer layers of the cell wall, which makes the cell permeable and allows its entry into the cytoplasmic membrane. This cause loss of low molecular weight components, such as ions of potassium [8].

After decontamination of ocular globes using the antiseptics cited, the cornea is storage in preservation media, enriched with nutrients such as glucose, amino acids, minerals and vitamins, whose purpose is to protect cells from the córnea. The medium can have too the gentamicin and streptomycin antibiotics [9,10], allowing prolonged storage. This médium is widely used by eye bank in Brazil, as well as an analogue, but without streptomycin [11].

The objective of this study was to evaluate the antiseptic effect in the reduction of ocular globe microbiota from corneal donors, prior to enucleation with 5% povidone-iodine and 0.05% Gluconate of chlorhexidine at different application times, as well as to analyze the susceptibility of the microbiota alone to gentamicin.


The research was submitted to the Ethics Committee of the Hospital das Clínicas of the Federal University of Goiás (HC / UFG), under the number CAAE: 58444316.3.0000.5078, and was approved. All had the donation term signed by a first or second-degree relative, as well as a witness.

The evaluation of the donor’s medical history was carried out by means of medical records, exams, anamnesis by the coroner and epidemiological interviews with the family. We also documented the location, time of death and time of enucleation of the eyeball. Donors with signs or suspicions of infection were excluded from the study.

Prior to face and ocular surface antisepsis, a swab soaked in 0.9% saline solution was rubbed throughout the conjunctival fornix of the right eye and immediately transferred to a tube containing Brain Heart Infusion (BHI) broth, duly identified with the data of the donor. The same procedure was then performed on the left eye.

After this first harvest, the anti-sepsis of the donor’s face and eyelids was performed, according to the PMS) of the eye bank and it is essential to change gloves. Cleaning was performed with 0.9% saline irrigation, above the eyelids, to remove any impurity. Then, with the aid of sterile gauze, the 10% topical povidone-iodine was applied, always in the same direction. To do so, the eyes were closed tightly and the procedure performed very carefully, so that there was no penetration of the antiseptic on the ocular surface.

The eyes were then opened and the ocular surface was cleaned by irrigating 10mL of 0.9% saline solution. After the excess liquid was withdrawn with sterile gauze and with a sterile glove, the antiseptics were applied to the ocular surface.

On the ocular surface of the right eye were applied 5ml of 5% povidone-iodine diluted solution and in the left eye 5ml of 0.05% dilute Gluconate of chlorhexidine solution. After 5 minutes of antiseptic application, a new cleaning with 10mL of 0.9% saline was performed. Then a swab was passed back into the conjunctival fornix and transferred to a tube containing BHI broth, duly identified with the donor data.

This procedure was also carried out in 10 and 15 minutes, with 10 samples in each group, totaling 30 donors. After the second harvest, enucleation of the ocular globes was performed, which were sent in a humid chamber to the eye bank. In the eye bank, after biomicroscopic evaluation of the eyeball and cornea, the tissue preservation phase and the last sample harvest were initiated. In laminar flow was irrigated using saline 0.9% over the entire surface of the corneal tissue. A swab was then rubbed across the corneal surface and horn-scleral flap and immediately transferred to tube containing BHI broth.

The tubes containing the samples were immediately sent to the Laboratory of Anaerobes, Phenotyping and Molecular Biology (LAFEBIM) of the Institute of Tropical Pathology and Public Health of the Federal University of Goiás, for processing. A 0.1mL aliquot of each tube was seeded on nutrient Agar, which was incubated in aerobic at 37°C for 24 hours for bacterial counting. The tubes containing the samples were also incubated under the same conditions. After the tubes were homogenized and a 0.1mL aliquot of the broth was seeded in the Mac Conkey agar medium, Saline Mannitol agar, base agar supplemented with 5% horse defibrinated blood, which were incubated under the same conditions. A 0.1mL aliquot of the broth was also seeded on Sabouraud agar and kept at room temperature for seven days.

Afterwards, morph colonial, morphotintorial characterization and biochemical tests were performed to identify the microorganisms, as well as antibiotic testing with gentamicin. The option of using gentamicin in the antibiogram test is justified by the fact that the preservation medium used in the BTOC participant of the present study only contains this antimicrobial. To compare proportions, the Chi-square test or Fisher’s exact test, when applicable, was used; to compare continuous variables, the T-test was used; and to evaluate the reduction in the number of colonies, the paired McNemar Test was used. The level of statistical significance of 5% (p <0.05) was considered for all tests.


Microbiological samples were collected from 60 eyes, which corresponded to 30 donors of corneas, and 63% (19/30) were male. Donor age had a median of 54 years, with a minimum of 29 and a maximum of 78 years (Table 1). The mean temperature at the time of collection had a median of 29°C (minimum 24°C, maximum 32°C), but the lowest temperature was observed in the group of donors who received 15-minute antiseptic treatment, whose median was 25.5°C. This lower temperature had a statistically significant analysis, with p = 0.011, for a better antiseptic effect.