Diagnosis of Microbial Keratitis Causatives by Genetic Method and Using Probiotics for Treatment

The study aimed to determine the most effective diagnostic method for detecting the most prevalent causative agents of microbial keratitis and selecting the most effective probiotic microorganism for the treatment of the causatives. For this purpose (52) corneal scraping samples were taken from patients of
microbial keratitis referred to Ibn Al-Haetham Eye Teaching Hospital in Baghdad. Results showed that 22 (78.57%) of these samples were positive for bacterial occurrence and 6 (21.42%) were positive for fungal occurrence after culturing on the related selective media. After the bacterial isolates were identified by cultural, microscopic and biochemical examinations, the following species and percentages were recorded: Pseudomonas aeruginosa 11 (39%), Staphylococcus spp 9 (32.14%) and Bacillus spp 2 (7.14%). While the fungal isolates were belonged to Aspergillus niger 3 (10.71%), Penicillium sp 2 (7.14%), Alternaria sp 1
(3.57%) according to their identification through the cultural and microscopic xaminations. upon such findings, P.aeruginosa was found to be the predominant bacteria among, followed by staphylococcii and A.niger the prevalent fungi among, followed by Penicillium sp. Regarding to the patients gender, it was found that cases of microbial keratitis in Iraq were more abundant in males as 32 (61.5%) were detected
compared to only 20 (38.4%) in females. Moreover, the age group of over 60 years was most subjected to the infection of microbial keratitis in UK, While the infection was most common in the age groups (41-59) years of patients in Iraq. In order to evaluate the efficiency of using Polymerase Chain Reaction (PCR) in the identifications of microorganisms causing microbial keratitis, 20 corneal scraping samples were collected from patients who attended the Eye Casualty Unit at the Southampton General Hospital in the United Kingdom and culturing on blood, chocolate incubated at 37 ͦ C for 24hrs and on sabrouad agar at 28 ͦ C for one week. PCR procedure was performed with the primer paired that targeted to the 16S rRNA for bacterial species and 18S rRNA gene for fungal species, in addition to the species specific primer for the most common microbial keratitis causatives microorganisms. Results in the regards showed that out of the 20 presumed cases of keratitis, PCR showed positivity in 75% of them, from these 55% were due to the fungal infection and 20 % of the cases indicated that the keratitis belonged to bacterial infections: In comparison, only 25% of positivity was obtained by the cultural method. The species specific primer showed that half of the 20% bacterial infection cases were caused by S. aureus and the other 10% referred to S.epidermidis infection. While the candida albicans primer gave a positive result only in 72% of the 55%, the rest 28% may belong to the other fungal infection. Depending on the above results, it can be concluded that PCR not only proved to be an effective rapid method for the diagnosis of bacterial and fungal keratitis, but was also more accurate and sensitive method than the culture methods. To investigate the antagonistic properties of the fungal isolates (Aspergillus.niger, Penicillium sp, Alternaria sp) obtained from inflamed patients eyes against pathogenic bacteria isolated from keratitic patient, agar well method was used, in which the antibacterial activity of fungal isolates was examined by testing the effect of their culture filtrates on growth of the bacterial
pathogens. Results revealed that A.niger possessed the highest inhibitory effect against P.aeruginosa, S.aureus, S.epidermidis, Bacillus sp with inhibition zones of (15, 25, 30, 32) mm in diameter, respectively, followed by Penicillium sp. Adversely, while no inhibitory effect was recorded by the filtrate of Alternaria
sp used. Among on bacteria Bacillus sp was the most effective and sensitive isolate. The suspected antibacterial compound produced by A.niger was characterized and partially purified by using infrared (IR), UV absorbance and thin layer chromatography (TLC). Results show that the compound appeared as
a single dark spot on the TLC plate under UV light that had Rf value of 0.45. From IR and UV analysis it was confirmed that the compound was aromaticnitrogenous in nature. From such achievement, it can be said that the active antibacterial compound might be tensidol which is an alkaloid or a new derivative of alkaloid.
Inhibitory activity was determined for lactic acid bacteria and Saccharomyces boulardii grown (on the solid medium and in the liquid medium) against bacterial isolates obtained from the microbial keratitis patients.
On the solid medium, After incubation LAB an aerobically for (24, 48, 72) hrs. it has been found that the difference between diameters of inhibition zones at different incubation periods was not significant. while in liquid medium, it was found that the unconcentrated filtrates of LAB had very low effect on the pathogenic bacterial isolates. Upon application of three-folded concentrated filtrate, highest inhibitory activity was achieved when the diameters of inhibition zone reached (26mm) for L.gasserri against Bacillus2. The zone diameters of the growth inhibition caused by S.boulardii grown on solid medium were ranged between (7.5 - 20.5). However, no any noticeable difference was recorded in the inhibitory activity upon extending the incubation periods beyond 24 hrs. and in liquid medium, even the three folded concentrated filtrate was used, no inhibitory effect was recorded against P.aeruginosa and S.epidermidis and low effect was recorded against S.aureus and Bacillus sp.