Investigating the Effect of Drugs of Chloramphenicol and Gentamicin on Acanthamoeba genotype T3 Causing keratitis Isolated from Environmental samples in vitro

This study is an investigation of the drugs effect on some pathogenic Acanthamoeba isolated from Iraqi waters, where the problem of environmental adaptation that characterizes this organism in addition to being a reservoir for many pathogenic microorganisms that take shelter in it to escape disinfectants and medicines is sometimes difficult to treat it with traditional treatments. Twenty water samples were collected from different water regions in Iraq, namely the Dokan Lake, Tigris River, Euphrates River and Najaf Sea, 5 samples from each source. Acanthamoeba was isolated from water samples on NNA and PYG media, using an inverted microscope with an electron microscope to determine their phenotypic features. PCR and Sequencing were also used to determine their genotype. The isolates were belonged to the T3 genotype that causes corneal infections. Then two types of drugs were used to treat it, which were Chloramphenicol and Gentamicin at three different concentrations, 0.1%, 0.3% and 0.6% and their inhibitory effect was compared with each other and with the positive control of the Chlorohexidine drug at a concentration of 0.2% which was recommended to treat Acanthamoeba keratitis. The results showed that both drugs have an inhibitory effect against Acanthamoeba growth, and that the chloramphenicol had more effect to inhibit compared with gentamicin and chlorhexidine, and it can be used as an alternative treatment instead of chlorohexidine for treating Acanthamoeba keratitis.


Introduction:
Some of free-living amoeba, including Acanthamoeba in water and soil cause serious diseases to humans, such as keratitis, and can also infect the skin, lung, and central nervous system 1 . Genotypes of Acanthameba range from T1 to T20. The genotype T4 is the main cause of infection worldwide. By contrast, the genotypes T3 and T11 show lower prevalence than genotype T4, but they are the most common causative agents of amebic keratitis [1][2][3][4] . Acanthamoeba keratitis is especially common in swimmers and contact lens wearers, and sometimes in people who have immunity deficiencies.
During the two past decades, Acanthamoeba has been increasingly recognized as important medical microbes in the ecosystem, and has a role in the ecosystem being as vectors and reservoirs of prokaryotes such as bacteria 5 . In spite of the studies that reported it, it is still not enough.Many drugs eliminate Acanthamoeba active phase (trophozoite) keratitis, such as antibacterial, antifungal, etc., while there is no drug that eliminates its active and inactive phase (cyst) at one time. 6 used in their study the chlorohexidine to observe its effect on Acanthamoeba strains isolated from environment and from patients infected by keratitis in vitro. They also showed that the drug (chlorhexidine), at a concentration of 0.02% -0.2% (0.2 mg/ ml -2 mg/ ml), inhibited the growth of Acanthamoeba, because it works to undermine the cell wall of trophozoites 6 -9 . Due to the effectiveness of this drug (chlorohexidine) and its low toxicity to the cornea, it is used as a medicine for the treatment of Acanthamoeba keratitis 10,11 .
Gentamicin is an antibiotic isolated from Micromonospora purpureochromogenes. It has broad activity against different types of gramnegative bacteria and some streptococcal strains,thus it is used to treat bacterial infections of the eye 12 . Chemical formula: C 21 H 43 N 5 O 7 . Chloramphenicol is an antibiotic that has broad activity against different types of Gram-negative, anaerobic and aerobic bacteria, therefore it is used to treat bacterial infections of the eye 13 . Chemical formula: C 11 H 12 Cl 2 N 2 O 5 .
There are many studies in the world and neighboring countries have reported the presence of pathogenic Acanthamoeba and its treating in both vitro and vivo, but there is not any study about Acanthamoeba treating in Iraq. The aim of this research is finding suitable drugs, which may be an alternative from of chlorohexidine recommended for treating of Acanthamoeba keratitis.

Material and Methods: Site and period of samples collection
Twenty water samples were collected during October 2018 to April 2019 from different regions in Iraq, with five isolates from each site as follow: Dokan Lake, Tigris River, Euphrates River and Najaf Sea (Fig.1). The collected samples were stored in sterile 250 ml plastic bottles at 4 °C until they reached to the laboratory for examination.

Filtration and cultivation on Non -Nutrient Agar (NNA) medium
The water samples were concentrated by filtering through a wattman filter papers (0.45µm) under vacuum, and placed by overturned form on a nonnutrient agar medium (15 g difco agar -2.5 mM KH 2 PO 4 , 0.5 mM Na 2 HPO 4 , 40 mM CaCl 2 , and 20 mM MgSO 4 ), supplemented with 0.1 ml of heat-inactivated E.coli, then incubated at 30℃ for two week. The positive growth of plates was recultured several times on fresh plates until to obtain pure isolates 14 .

Cultivation in Peptone Yeast extract Glucose (PYG) medium
A suspension of Acanthamoeba was prepared by scraping with a sterile scalpel from the surface of nonnutrient agar (NNA) media and suspending it in 3 ml of buffer phosphate 0.1 M ((PH = 7.2). Then, 0.1 ml of the suspension was transferred to a peptone yeast extract glucose broth (20 g of peptone, 10 g of yeast extract and 10 g of glucose in 1 liter of distilled water),with antibiotics 0.5 ml of penicillin and 0.5 mg/ml streptomycin. Thence, incubated at 30 °C for a week 6 .

Microscopic examination
The growth cultures on NNA media were examined directly by inverting microscope at magnifications of 10x and 40x in order to distinguish the positive growth of Acanthamoeba from the negative. Negative growth cultures were excluded and left again in the incubator for a month to ensure that they are free of Acanthamoeba, whereas the positive were re-cultured in fresh NNA media for cloning, with three replications for each culture 14 .

Examination using an electron microscope
One ml of suspended Acanthamoeba in buffer phosphate was centrifuged at 500 rpm for 5 minutes. Then, it was fixed with 2% of buffered Glutaraldehyde for two hours, followed by washing process with Buffer solution 0.1 M. Thence, it underwent post fixation with 1 % of Osmic acid for one hour. Then, the samples were subjected to the drying process (Critical drying point) by grading concentrations of alcohol starting from 30% -50% -70% -90% -95% -100% for 10 minutes at each concentration (15). Later, it was plated with gold (thickness 30 nanometers) in sputter device, and then photographed with a scanning electron microscope (FEI Inspects 50) at the magnification of 1200x and 2500x in the Faculty of Science / University of Kufa.

Molecular diagnostic DNA extraction
One ml of suspended Acanthamoeba in buffer phosphate was added to 1.5 ml Eppendorf tubes and centrifuged at 2000 g for 5 minutes to remove the supernatant. After that, the extraction procedure was completed according to the kit (DNA extraction -Promega USA) instructions. Primers and polymerase chain reaction protocol In order to molecularly confirm Acanthamoeba isolates, the JDP Primers (Macrogen, Korea) sequences were used to amplify as follow: (JDP Forward GGCCCAGATCGTTTACCGTGAA) and (JDP Reverse TCTCACAAGCTGCTAGGGAGTCA) [12], depending on the program (initial denaturation step at 95 ℃ for 5 minutes, then 30 cycles at 95 ℃ for 40 seconds, 56 ℃ for 45 seconds, and 72 ℃ for 55 seconds, followed by a final extension step at 72 ℃ for 7 minutes).
Electrophoresis were used to confirm the presence of PCR products by using 1% agarose with 100 Volt/ Milliampere at 75 min 14 .

Standard Sequencing and analysis
The PCR product was sequenced by Macrogen Corporation (Korea), using Sanger sequencing (ABI3730XL, automated DNA sequencer). The results were analyzed using geneious software.

Calculation of Acanthamoeba density and vitality
The 0.1 ml of positive growth media of PYG which was incubated for week, and inoculated in 4.9 ml of fresh PYG media prepared for experiment purpose in this study. Then, incubated at 30 °C for 48 hours, after completing 48 hours of incubation, 0.2 ml was taken from last growth to determine the numerical density of Acanthamoeba growth. The density of Acanthamoeba was 7 x 10 5 cells /ml, with a vitality of 100% by using hemocytometer counting, and eosin stain 1% depending on its incapability to penetrate the living cell 16 .

Drugs used in the study Preparing drug solutions and measurement their 2 cytotoxicity on Acanthamoeba growth
The drug solutions were prepared under sterile conditions using Millipore filter for each of Chloramphenicol (50 mg / ml Drops) and Gentamicin (20 mg / 2ml ampoule) at a concentration of 0.1, 0.3 and 0.6 mg /ml. Then, 0.2 ml of each concentration added to sub-culture media incubated 48 hours with three reps of each concentration. In the same way, the drug Chlorohexidine used at concentration of 0.02% (0.2 mg /ml) as a positive control for comparison. On the other hand, the negative control group was left without treatment and kept all in the incubator at 30 °C.
The growth index was calculated to determine the Inhibition Concentration Fifty values every 24, 48, 72, 96 and 120 hours by using the hemocytometer and eosin stain according to the following equation 17 : (Percentage of Growth index = Number of Live Acanthamoeba / Number of Total Acanthamoeba X 100) The counting process was repeat three times for each experiment and the mean was used.

Microscopic examination
After 2-5 days of inoculation and examination of the cultures using invert microscope, trophozoites of Acanthamoeba were observed in the positive samples (Fig 2). Cysts of Acanthamoeba were observed in the positive cultured samples after two weeks of inoculation and examination by SEM microscope (Fig 3).  In Table No. 1 the results of microscopic examination of the Acanthamoeba cultivation show 5 positive growth cultures (25%) of 20 samples from the four regions, where Euphrates River shows the highest pollution percentage (3/5 or 60%), followed Dokan lake and Sea of Najaf show the same percentage of pollution (1/5 or 20%). While, no any pollution was observed in Tigris River.

Molecular identification (PCR & Sequence)
After PCR amplification of the samples and its electrophoresis, the isolates were belong to T3 genotype as Acanthamoeba genotype T3 Iraq (Accession Number MN462973) 14 (Fig 4).

Effect of drugs on Acanthamoeba growth in vitro
The Gentamicin was added to each PYG media at a concentration of (1,3,6) mg /ml and their effects on survival at 24, 48, 72, 96 and 120 hours. (   The Chloramphenicol was added to each PYG media at a concentration of (1,3,6) mg / ml and their effects on survival at 24, 48, 72, 96 and 120 hours. (Table 3, Figs. 7 and 8).   The drugs used in treating diseases caused by Acanthamoeba are very few, and their lack effect because of Acanthamoeba resistance to environmental conditions, in addition to being a reservoir for many pathogens that shelter for escaping from disinfectants and drugs, which is continuous obstacle for researchers 18 . In this study, the drug gentamicin showed an inhibitory effect on Acanthamoeba growth, and this is consistent with the results of researchers studies that continued for ten-years about the effect of antibiotics against bacterial keratitis in vitro 19 . In another study, the researchers showed that gentamicin had an ability to inhibit the cysts of Acantamoeba in vitro and can be used as a treatment for keratitis, they also added that the Acanthamoeba cysts isolated from environment were more resistant to gentamicin than the Acanthamoeba cysts that were clinically isolated 20 , and this is consistent with what 21 also mentioned in their study, that showed the lack effect of gentamicin against Acanthamoeba isolated from environment in vitro 21 . As for the drug chloramphenicol, 19  chloramphenicol was used against Plankton organisms. In another study, the researchers 22 recommended the use of chloramphenicol or neomycin (an antibiotic belonging to the aminoglycoside group and its properties are near to gentamicin) for treatment of Acanthamoeba keratitis in secondary bacterial infection 22 . This is in consistant with the results of the current study. There are no abundant laboratory studies to evaluate the efficacy of gentamycin and chloramphenicol against Acanthamiba, so detailed comparisons cannot be made with the results of the current study. The results of this study show the effect of gentamicin and chloramphenicol against the growth of Acanthamoeba genotype T3 isolated from Iraqi waters. Table No. 2 shows an inverse relationship between different concentrations of gentamicin with Acanthamoeba growth during different time periods, as observed when using concentrations 0.1 and 0.3% A slight decrease in the percentage growth within 24 hours, which amounted to 97.1% and 94.2%, respectively, then this decline gradually increased to 68.5% and 65.7% with the passage of 120 hours. But, in concentration 0.6%, it had a clear inhibitory effect on growth compared to the negative control sample, as the growth factor decreased from 87.1% to 61.4% within 96 hours and then decreased to 51.4% during the 120-hour period, equivalent to approximately 50% (IC50). This means that time also had a clear adverse effect on the growth rate of Acanthamiba in addition to the effect of different drug concentrations.
In Table No. 3 the results showed that the drug chloramphenicol had a higher effect against Acanthamoeba growth compared with gentamicin and positive control (Fig 9), where it was noticed that the growth index decreased when using concentration 0.1% from 92.8% to 62.8% during 96 hours period time of incubation, then continued to decline until it reached 54.2% within 120 hours, which is roughly equivalent to the value (IC50) of gentamicin concentration 0.6 % during the same time. The growth index when using the two concentration 0.3 % and 0.6% decreased from 85.7% to 28.5% and from 78.5% to 14.2% respectively, during the periods 24, 48, 72, 96 and 120 hours, meaning decrease was observed when using concentration 0.3 % to 45.7 at 96 hours. similarly, concentration 0.6 %, where growth decreased within 72 hours to 42.8%, and this result is less than the IC50 values, which are supposed to be within the limits of approximately 50% compared to the positive control which reached at the same period to about 55.7%. However, no inhibition was observed in the negative control group, as shown in Tables 2 and 3.
The mode of gentamicin drug action is aminoglycoside causes codon misread by binding to the ribosomal subunit 30S, stops the peptidyl-tRNA translocation from the recipient to donor area and interrupts protein synthesis 15 . In chloramphenicol, the binding to the 50S ribosome unit of the microbial cell prevents the amino acids from transferring to the growth peptide chains, which also leads to inhibition of protein synthesis in the cell 16 .

Conclusions & Recommendations
In this study, we reach an effect of gentamicin and chloramphenicol against the growth of pathogenic Acanthamoeba, but this effect is diverse at the different concentration during different times. Also, chloramphenicol has a more effective against Acanthamoeba growth compared to gentamicin and chlorhexidine as a positive control and it can be used as an alternative treatment instead of chlorohexidine for treating Acanthamoeba keratitis. In spite of these conclusions about the effect of the two drugs in the treatment of Acanthamoeba genotype T3, it is in vitro and not clinically, we need more researches to be conducted to identify other genotypes and treat them in vitro and in vivo.
Figures and images, which are not mine ours, have been given the permission for re-publication attached with the manuscript. -The author has signed an animal welfare statement.
-Ethical Clearance: The project was approved by the local ethical committee in University of Tikrit.